EGF receptor


PROTEIN INTERACTIONS

Interaction with ligands

One model of Egfr signaling posits that in the absence of ligand the receptor exists as a monomer. Ligand is bound with low affinity by the monomer and stimulates receptor dimerization. In dimeric form receptors exhibit a high affinity for ligand. Dimerized receptors phosphorylate each other by a trans-molecular mechanism. Cell substrates consisting of RAS pathway proteins bind phosphotyrosines via their SH2 domains. Bound substrates are phosphorylated by the activated receptor (Clifford, 1994).

Argos is a secreted molecule with an atypical EGF motif. It is an inhibitor of the signaling triggered by the Drosophila EGF receptor. Argos blocks photoreceptor determination in the eye. Argos protein inhibits the activation of Egfr by Spitz. When Egfr is overexpressed in the eye, a significant amount of autophosphorylation occurs, presumably caused by spontaneous dimerization due to the high concentration of Egfr in the plasma membrane. Argos significantly reduces this ligand-independent activation. Does Argos compete directly for Spitz binding to Egfr, or is the effect of Argos indirect, acting through a second receptor specific for Argos? Similar interaction between Argos and Egfr is observed in the wing (Golembo, 1996).

The activation of the Drosophila EGF receptor (DER) by its natural ligand Spitz is inhibited by Argos. Argos and Spitz both have an EGF-like domain, which in the case of Argos differs from that of Spitz and other EGF receptor agonists in that it has an extended B-loop of 20 amino acids instead of 10 amino acids. This B-loop contains an unusual cluster of charged residues. To investigate whether B-loop sequences are an important determinant for receptor activation and play a causal role in the antagonistic activity of Argos, three human (h)EGF mutants were constructed in which amino acids derived from the Argos B-loop were introduced. In one mutant (E3A4E/B10), the replacement of four amino acids in the B-loop of hEGF (123, E24, D27, and K28) by the corresponding Argos residues neither alters the binding affinity of the growth factor for the hEGF receptor nor does it change its ability to induce a mitogenic response. However, the insertion of 2 additional Argos residues (E3A4E/B12) or extension of the B-loop by 10 amino acids (E3A4E/B20) results in a significant loss of binding affinity. In spite of this, both E3A4E/B12 and E3A4E/B20 appear to be strong agonists for the hEGF receptor with similar dose-response curves for mitogenic activity and MAPK activation as wild-type hEGF. These data show that several nonconservative substitutions in the hEGF B-loop are tolerated without affecting receptor binding or activation. They show that receptor binding and receptor signaling efficiency can be uncoupled, which is a prerequisite for the development of receptor antagonists (van de Poll, 1997).

Gurken is likely to be a ligand of Egfr, activating the receptor during oogenesis. In this stage the GRK transcript becomes asymmetrically localized to the anterior dorsal region of the oocyte. The dorsal localization of GRK RNA results in a spatially restricted ligand that asymmetrically activates the receptor (Neuman-Silberberg, 1993).

Anterior-posterior polarity in Drosophila arises from the movement of the oocyte to the posterior of the egg chamber, and the subsequent acquisition of posterior fate by the adjacent somatic follicle cells. Gurken is necessary in the oocyte, as is Egfr in the follicle cells, for the induction of posterior fate. As the Gurken-Egfr pathway also establishes dorsoventral polarity later in oogenesis, Drosophila uses the same germline to soma signaling pathway to determine both embryonic axes (Gonzalez-Reyes, 1995).

When the secreted Spitz protein is expressed in embryos to assess the gene's biological activity, an alteration in cell fates is observed in the ventral midline ectoderm, such that lateral cells acquire the ventral-most fates. Such results indicate that graded activation of the Egfr pathway may normally give rise to a repertoire of discrete cell fates in the ventral ectoderm. Spatially restricted processing of Spitz may be responsible for Egfr graded activation. On the basis of genetic interactions, the Rhomboid and Star proteins are thought to act as modulators of Egfr signaling. No alteration in Egfr autophosphorylation or the pattern of MAP kinase activation by secreted Spitz is observed when the RHO and Star proteins aree coexpressed with Egfr in S2 cells. In embryos mutant for rho or Star the ventralizing effect of secreted Spitz is epistatic, suggesting that RHO and Star may normally facilitate processing of the Spitz precursor (Schweitzer, 1995).

The spitz gene is required for photoreceptor determination. Mosaic analysis suggests that spitz, which encodes a TGF alpha homolog, produces a diffusible signal during ommatidial development. Other members of the spitz group and the EGF receptor also interact with sev-rho, in a pattern that suggests a model in which rhomboid can act as a mediator of a ligand-receptor interaction between Spitz and Egfr in the developing eye. These data suggest that photoreceptors other than R7 use a Ras1 signaling pathway activated by the Spitz/Egfr interaction, in a manner analogous to the Ras1 pathway activated by boss/sevenless in photoreceptor R7 (Freeman, 1994).

Recessive spitz loss-of-function mutations affect compound eye development. Spitz is homologous to the human Transforming growth factor-alpha. In mosaic clones, spitz function is required in the first photoreceptor cells to differentiate for normal ommatidial development. spitz loss-of-function mutations are dominant suppressors of Egfr gain-of-function mutations. These data suggest that the spitz product is a precluster promoting factor. spitz transcription increases abruptly in the morphogenetic furrow, the obverse of Egfr expression (Tio, 1994).

vein interacts with components of the Egfr signaling pathway. The most dramatic and informative of these is the rescue of vein null phenotypes by gain-of-function Egfr alleles. vein null wing discs are tiny, arresting growth at the equivalent of a late second/early third-instar-size disc, however vein null discs that are heterozygous for the Egfr Ellipse allele are rescued and grow to a large size. These discs are not fully wild-type as they have a duplicated wing pouch, which is a phenotype characteristic of vein hypomorphs. Thus the hyperactive Egfr encoded for by the Ellipse allele can override the wing proliferation defects and partially compensate for vein loss. The Ellipse allele also rescues the larval patterning defects found in vein nulls as reflected in the phenotype of the pupal cas. These results have two major implications: (1) Egfr appears to be epistatic to vein, which is consistent with VN acting as a ligand for EGFR. (2) A redundancy in the signaling system is suggested, whereby activation of a hyperactive receptor by another ligand(s) compensates for Vn loss (Schnepp, 1996).

Interaction with Kekkon-1

To test whether the extracellular domain of Kekkon-1, which contains five LRR and one Ig motif, is required for the inhibition of the Egfr activity by Kek1, transgenic lines were generated that contain either UAS-kek1extra or UAS-kek1intra. No phenotype is observed by overexpressing kek1intra in the follicle cells using the T155 or CY2 GAL4 lines. In contrast, overexpression of kek1extra using the same drivers led to eggs with reduced dorsal appendage materials. These observations indicate that the extracellular domain of Kek1 is sufficient to inhibit the activity of the Egfr. Although the phenotypes obtained following kek1extra overexpression are very similar to those obtained with full-length kek1, these overexpression phenotypes are weaker. One possibility is that Kek1extra is expressed at a lower level than the WT protein. Weaker effects were observed with 12 independent transgenic lines, as well as when multiple copies of the transgenes were added. This suggests that Kek1extra is less stable than the full-length Kek1 molecule or that the cytoplasmic domain of Kek1 participates to some extent in the inhibition. Finally, a test was performed to see whether the inhibition of the Egfr activity by kek1extra requires the presence of the endogenous Kek1 protein. When kek1extra is overexpressed in kek1 mutant females, the eggs are ventralized, which excludes a model whereby kek1extra blocks the Egfr by forming a heterodimer with the endogenous Kek1 protein (Ghiglione, 1999).

Having demonstrated that Kek1 acts to inhibit Egfr signaling, it was important gain insight into the mechanism of inhibition of the Egfr by Kek1. A test was performed to see whether Egfr and Kek1 physically associate. Following coexpression of a Myc-tagged version of Kek1 and the Egfr or the Drosophila Torso RTK in Sf9 cells, Kek1 was immunoprecipitated from the cell lysates using an anti-Myc antibody. Coprecipitation of the Egfr is observed by probing the resulting blot with the anti-Egfr antibody, suggesting that Kek1 associates physically with the Egfr and that this interaction is responsible for the inhibitory effect. When both Kek1 and Torso are coexpressed in Sf9 cells, no Torso is coprecipitated by Kek1, suggesting a selectivity of Kek1 for binding to the Egfr. Because the extracellular and transmembrane portions of Kek1 are essential for the inhibitory effect, an examination was carried out to see whether these portions of Kek1 would be able to bind to the Egfr. The extracellular domain, but not the intracellular domain, of Kek1 can bind to the Egfr. Thus, it is proposed that the inhibitory effect of the Egfr by Kek1 is mediated through direct association of the extracellular and transmembrane domains of Kek1 with the Egfr (Ghiglione, 1999).

The transmembrane protein Kekkon 1 (Kek1) acts in a negative feedback loop to downregulate the Drosophila Epidermal growth factor receptor during oogenesis. This protein plays a similar role in other Egfr-mediated developmental processes. Structure-function analysis reveals that the extracellular Leucine-Rich Repeat (LRR) domains of Kek1 are critical for its function through direct association with Egfr, whereas its cytoplasmic domain is required for apical subcellular localization. In addition, the use of chimeric proteins between Kek1 extracellular and transmembrane domains fused to Egfr intracellular domain indicates that Kek1 forms a heterodimer with Egfr in vivo. To characterize more precisely the mechanism underlying the Kek1/Egfr interaction, mammalian ErbB/EGFR cell-based assays were used. Kek1 is capable of physically interacting with each of the known members of the mammalian ErbB receptor family and the Kek1/EGFR interaction inhibits growth factor binding, receptor autophosphorylation and Erk1/2 activation in response to EGF. Finally, in vivo experiments show that Kek1 expression potently suppresses the growth of mouse mammary tumor cells derived from aberrant ErbB receptors activation, but does not interfere with the growth of tumor cells derived from activated Ras. These results underscore the possibility that Kek1 may be used experimentally to inhibit ErbB receptors and point to the possibility that, as yet uncharacterized, mammalian transmembrane LRR proteins might act as modulators of growth factor signalling (Ghiglione, 2003).

Results from both biochemical experiments and in vivo tests reveal that the LRR domains of Kek1 are crucial for the association between DER and Kek1, and DER inhibition. Furthermore, the Kek1 cytoplasmic domain, which has been shown to play a role in the overall efficiency of Egfr inhibition, appears to be critical for the proper apical subcellular localization of Kek1 in epithelial cells. Interestingly, the Kek1 C terminus contains a concensus sequence for a PDZ domain-binding site that can bind the PDZ domains of proteins such as Disc-Large or Scribble. Because these proteins are crucial for the organization of apicobasal cell polarity, it is possible that Kek1 localization depends on these factors or related polarity cues. Interestingly, subcellular localization of Kek1 to the apical side may be coordinated with Egfr/ErbB subcellular localization as well; PDZ-containing proteins have been implicated in ErbB subcellular localization. Further characterization of these interactions will be needed to clarify how subcellular localization of Kek1 and Egfr is regulated (Ghiglione, 2003).

Epistasis studies placed the action of Kek1 upstream of Egfr. Since Kek1 is expressed in the same cell as Egfr, these observations suggest that Kek1 interacts with either the receptor to suppress its signalling function or with the ligand to sequester its activity. Kek1 can be co-immunoprecipitated with Egfr but not with its ligands suggesting that Kek1 interacts directly with receptors to interfere with ligand binding activity (Ghiglione, 2003),

These findings are consistent with the biochemical interaction of Kek1 with all four mammalian ErbB receptor family members. When reconstituted in Sf9 insect cells, Kek1 blocks the binding of radiolabeled EGF to the population of EGFR associated with Kek1, but not the total receptor pool. Likewise, EGF-stimulated autophosphorylation of the Kek1-associated receptor population is blocked, but autophosphorylation of the total receptor pool is not. These observations suggest that Kek1 acts to suppress receptor signalling at least in part by physically interfering with ligand binding. However, other effects on receptor activation cannot be ruled out. Kek1 suppresses the growth properties of the NF-639 mouse mammary tumor cells, obtained from an activating point mutation in the transmembrane region of the ErbB2 receptor. Since this mutation is thought to generate constitutive receptor tyrosine kinase activity via a ligand-independent mechanism, it is likely that Kek1 also acts to interfere with receptor dimerization or other events necessary for its activity (Ghiglione, 2003),

These studies suggest that Kek1 is functionally similar to another Drosophila suppressor of Egfr signalling called Argos. Argos is also a transcriptional target of activated Egfr in developing tissues, and it has been demonstrated that Argos binds directly to Egfr to inhibit the binding of the natural ligand Spitz. However, the sequences of the two inhibitors are very distinct. Although Kek1 contains a series of LRR and Ig domains in its extracellular region, Argos contains an imperfect EGF-like domain. Given that at least two proteins in the Drosophila genome are dedicated to a similar purpose, it seems likely that ErbB antagonists are also present in higher organisms (Ghiglione, 2003),

The combined extracellular and TM regions of Kek1 are sufficient to mediate its biological activity as well as its interaction with Egfr. The present study indicates that the LRR domains of the extracellular region are necessary for the suppression of Egfr-mediated developmental events in flies. These results suggest that Kek1/receptor interactions are mediated by the LRR domains, pointing to LRR-containing extracellular proteins as candidates for mammalian Kek1 homologs. Numerous mammalian LRR proteins have been described and several have arrangements of subdomains similar to Kek1, including the Trk receptor tyrosine kinases, LIG-1 and a number of proteins of unknown function. The role of such proteins in ErbB-mediated developmental processes and tumor cell growth remains to be explored (Ghiglione, 2003),

Particularly noteworthy is the small leucine-rich proteoglycan decorin, which has been shown to directly bind to human EGFR. However, although decorin is also a potent suppressor of tumor cell growth, its mechanism of action appears to differ from that of Kek1. Treatment of cells with soluble decorin induces the immediate tyrosine phosphorylation of the EGFR and subsequent signalling events, and sustained expression of decorin suppresses EGFR levels without affecting ligand binding activity. These results indicate that decorin is not functionally identical to Kek1. However, taken with the current observations, these data suggest that some LRR-containing extracellular proteins are capable of interacting with ErbB receptors to modulate their activities by multiple mechanisms (Ghiglione, 2003),

In a broader context, proteins such as Kek1 and decorin may be thought of as direct modulators of ErbB receptors that could assist in the integration of extracellular events with growth factor signalling. Numerous studies suggest that signalling through integrins, cell adhesion molecules and other cell surface proteins impact ErbB receptor signalling pathways, largely by influencing the extent to which various intracellular signalling pathways respond to receptor activation. These examples represent indirect modulation of growth factor signalling through crosstalk between downstream components. It is proposed that LRR-containing proteins such as Kek1 and decorin are members of a larger functionally related class of glycoproteins that directly modulate growth factor signalling pathways by interacting with and influencing the properties of the receptors themselves (Ghiglione, 2003),

Throughout development, cells utilize feedback inhibition of receptor tyrosine kinase (RTK) signaling as an important means to direct cellular fates. In Drosophila, epidermal growth factor receptor (EGFR) activity is tightly regulated by a complex array of autoregulatory loops, involving an assortment of inhibitory proteins. One inhibitor, the transmembrane protein Kekkon1 (Kek1) functions during oogenesis in a negative feedback loop to directly attenuate EGFR activity. Kek1 contains both leucine-rich repeats (LRRs) and an immunoglobulin (Ig) domain, two of the most prevalent motifs found within metazoan genomes. Kek1 is shown to inhibit EGFR activity during eye development and this role has been used to identify kek1 loss-of-function mutations that implicate the LRRs in directing receptor inhibition. Using a GMR-GAL4, UAS kek1-GFP misexpression phenotype, missense mutations were isolated in the kek1 transgene, affecting its ability to inhibit EGFR signaling. Genetic, molecular, and biochemical characterization of these alleles indicate that they represent two functionally distinct classes. Class I alleles directly diminish Kek1's affinity for EGFR, while class II alleles disrupt Kek1's subcellular localization, thereby indirectly affecting its ability to associate with and inhibit the receptor. All class I alleles map to the first and second LRRs of Kek1, suggesting a primary role for these two repeats in specifying association with and inhibition of EGFR. This analysis implicates glycine 160 of the second LRR in regulating EGFR binding (Alvarado, 2004a).

Typical LRRs consist of stretches of 21-25 amino acids and are defined by repeats of the conserved sequence LxxLxLxxN/CxL, where conservative substitutions of leucine for similar hydrophobic residues are common. However, the rest of the repeat can be highly divergent. Structurally, these motifs are composed of a ß-sheet, defined by the conserved sequence, which is connected to an alpha-helix. The entire set of LRRs is thought to form a horseshoe structure, with the hydrophobic ß-sheets lining the inside of the structure and the alpha-helices exposed to the outer surface (Alvarado, 2004a and references therein).

The class I allele kek196 disrupts a conserved leucine in the first LRR and is a partial suppressor. L136 is conserved in all Kek family members, suggesting that it does not directly dictate EGFR-binding specificity. In addition, the amino acids surrounding L136 at the n - 2, n - 1, and n + 1 positions are highly conserved in all Kek proteins. This reinforces the notion that L136 plays a structural rather than a direct role in association with the receptor. Consistent with a partial loss of activity in vivo, L136F also displays a reduction in EGFR-binding affinity as shown by co-immunoprecipitation. This partial loss-of-function phenotype could be due to the fairly conservative nature of the substitution, where leucine is changed to phenylalanine, a bulkier hydrophobic residue. Alternatively, the first LRR may meet only a small structural requirement for Kek1 function (Alvarado, 2004a).

The three remaining class I alleles change G160 in the second LRR and may represent the most functionally relevant mutations uncovered in the screen. Several lines of evidence support this suggestion: (1) glycine 160 is mutated in three separate suppressors: to serine in kek182 and kek153B and to aspartic acid in kek17C; (2) these alleles display strong suppression of the Kek1-GFP misexpression phenotype, in both the eye and the ovary, while exhibiting correct subcellular localization(3) G160 is conserved in Kek1 orthologs from Drosophila virilis and Anopheles gambiae, but is divergent in the other Kek family members; (4) these changes reduce the affinity of Kek1 for EGFR, but not for itself in co-immunoprecipitation experiments. These lines of evidence demonstrate that G160 is likely to play an instructive rather than a permissive role in mediating EGFR binding and inhibition. Together, the data from class I alleles suggest that the first and second LRRs function together to direct EGFR binding, consistent with recent findings that the LRRs are essential for inhibition of EGFR. The first repeat consequently may be required for the correct positioning of the second repeat in which G160 specifies EGFR binding (Alvarado, 2004a).

Notably, all class II alleles alter Kek1 subcellular localization and involve changes in proline residues that are conserved, with one exception, throughout the Kek family. Whereas EGFR, Kek1-GFP, and class I mutants localize primarily to the apical membrane of polarized follicle cells, class II alleles localize more uniformly throughout the cell and appear cytoplasmic in their distribution. Furthermore, within class II, strong suppressors display higher degrees of mislocalization than do intermediate suppressors. The class II allele kek1137 (P187S) affects the third LRR and is a suppressor with intermediate activity. This proline is conserved in all Kek1 orthologs and Kek family members, with the exception of Kek6. Two alleles of moderate strength, kek165 and kek1118, both mapped to a single residue (P309) located in the C-flank. N-terminal and C-terminal cysteine-rich flanks are capping motifs commonly associated with LRRs and are defined by the conserved positioning of cysteine residues. All Kek family members contain a proline at the same relative position as P309. kek165 (P309L) behaves as a slightly stronger suppressor than kek1118 (P309S), consistent with the higher degree of subcellular mislocalization in kek165. This minor difference in protein localization is likely caused by the nature of the substituting amino acid. The two remaining class II alleles are mutations in the Ig domain. Proteins with Ig domains constitute a superfamily of molecules with varied function in which the Ig domain confers protein-binding properties. kek182A (P329S) is the result of a change in the first amino acid of the Ig domain, which is conserved in all Kek family members. The allele with this change was the strongest suppressor identified in the screen and localizes uniformly throughout the cell. The mutation encoded by kek1176V (P356S) is also within the Ig domain, but it represents a moderate suppressor. Consistent with only a partial LOF in Kek1, this mutant protein, although localized abnormally, displayed a slight bias for the apical surface of follicle cells. Finally, co-immunoprecipitation experiments between class II alleles and EGFR reveals that most class II alleles have the intrinsic ability to bind the receptor with wild-type affinity. This strongly suggests that the suppression observed in vivo is due to reduced apical membrane localization of Kek1, consequently limiting its ability to interact with and inhibit the receptor. Thus, class II alleles define a set of distinct proline residues that promote Kek1 function through effects on subcellular localization (Alvarado, 2004a).

The cytoplasmic region of Kek1 is dispensable for EGFR binding and inhibition. No mutations affecting Kek1 function were recovered in this region. Interestingly, however, the C-terminal tail (48 amino acids) represents the most highly conserved portion of Kek1 between Drosophila and Anopheles. This portion of Kek1, like Kek2 and Kek5, contains a putative type 1 binding site (S/T-X-I/V/G) for proteins containing PDZ domains. Supporting this, deletion of the cytoplasmic domain of Kek1 can disrupt its trafficking. However, the fact that no mutations in the cytoplasmic domain were recovered in the screen suggests that loss of the cytoplasmic domain does not compromise Kek1 function in the eye. This is consistent with observations and suggests that this region may contribute to Kek1's inhibitory function in a more refined or tissue-specific fashion. Alternatively, the basis for this conservation might lie in an EGFR-independent role (Alvarado, 2004a).

In conclusion, inhibition of EGFR signaling by Kek1 occurs in multiple developmental processes and is mediated by the extracellular portion of Kek1. Mutations affecting Kek1's inhibitory activity are spread throughout the extracellular region, but reflect different LOF mechanisms. Specificity for EGFR binding is likely to reside to a large degree in the second LRR at G160. This residue was mutated in three different suppressors, affects the affinity of Kek1 for EGFR, and is unique to Kek1 among Kek family members. On the basis of this finding it is proposed that the second LRR underlies the binding specificity of Kek1 for EGFR and therefore its inhibitory function. Given this and the plethora of secreted and transmembrane molecules containing LRRs within the Drosophila genome, it will be important to determine if this sequence represents an EGFR interaction motif present in additional LRR-containing molecules and to decipher their contributions to EGFR signaling. Likewise, it will be interesting to determine if the analogous region in other Kek family members directs their function and if they act in a related manner on distinct receptors (Alvarado, 2004a).

In Drosophila, signaling by the epidermal growth factor receptor (EGFR) is required for a diverse array of developmental decisions. Essential to these decisions is the precise regulation of the receptor's activity by both stimulatory and inhibitory molecules. To better understand the regulation of EGFR activity inhibition of EGFR by the transmembrane protein Kekkon1 (Kek1) was investigated. Kek1 encodes a molecule containing leucine-rich repeats (LRR) and an immunoglobulin (Ig) domain and is the founding member of the Drosophila Kekkon family. This study demonstrates with a series of Kek1-Kek2 chimeras that while the LRRs suffice for EGFR binding, inhibition in vivo requires the Kek1 juxta/transmembrane region. It is demonstrated directly, and using a series of Kek1-EGFR chimeras, that Kek1 is not a phosphorylation substrate for the receptor in vivo. In addition, EGFR inhibition is shown to be unique to Kek1 among Kek family members, and this function is not ligand or tissue specific. Finally, a unique class of EGFR alleles has been identified that specifically disrupts Kek1 binding and inhibition, but preserves receptor activation. Interestingly, these alleles map to domain V of the Drosophila EGFR, a region absent from the vertebrate receptors. Together, these results support a model in which the LRRs of Kek1 in conjunction with its juxta/transmembrane region direct association and inhibition of the Drosophila EGFR through interactions with receptor domain V (Alvarado, 2004b).

Throughout development, EGFR activity specifies distinct cellular responses. Essential to this ability is the existence of an integrated network of regulatory molecules that direct receptor activity. Kek1, a member of a family of LRR- and Ig-containing molecules, represents a component of this network through its role as a feedback inhibitor of receptor activity. Deletion and mutagenesis studies have now demonstrated that the LRRs of Kek1, specifically LRR2 and G160, are essential for its association with, and consequently inhibition of, the receptor. The Kek1 cytoplasmic domain and associated Kek1 tail (KT) box have also been implicated (Alvarado, 2004b).

While it is clear that the Kek1 LRRs are essential for EGFR binding and inhibition, secreted forms of Kek1 are nonfunctional, indicating that membrane anchoring is likely to be an essential element to the inhibitory mechanism. Directly testing this, Kek1-Kek2 swaps demonstrate that while the Kek1 LRRs are sufficient for binding in vitro, they provide only minimal inhibition in vivo. Indeed, full inhibition is restored only when the entire extracellular and transmembane regions of Kek1 are placed in the context of a Kek2 backbone. This result supports an active role for the Kek1 jt/tm domain in inhibition, as a chimera containing the Kek1 LRRs in a Kek2 backbone is membrane tethered, but a weak inhibitor. This indicates that LRR-mediated binding alone is insufficient for receptor inhibition. Rather, the results suggest that Kek1-mediated inhibition of EGFR signaling is a bipartite process, in which the LRRs dictate EGFR binding and the jt/tm region facilitates inhibition. Phylogenetic analysis has indicated that this region is well conserved in Kek1 orthologs, supporting an important functional role for this region. Given this requirement for the Kek1 jt/tm region in inhibition, it was interesting to note that the SOK alleles identify three amino acids present in domain V of the receptor. Alteration of these three residues renders the receptor refractory to inhibition by Kek1 and activation by KEDeltaCG, respectively. Moreover, two of the changes, R738Q and E718K, represent viable alleles of the receptor, capable of ligand binding and receptor activation. Together with the binding data, these results assign a role for domain V in mediating regulation by Kek1. It is interesting to note that EGFR domain V represents a third cysteine-rich domain in Drosophila, which is absent in the vertebrate ErbBs. This raises intriguing structural and evolutionary questions, as Kek1 has been reported to associate with all human ErbBs. It will be important in the future to define those elements in the receptor that suffice for its inhibition by Kek1 and determine if additional distinctions in the interactions between Kek1 and the different receptor family members exist (Alvarado, 2004b).

Both direct (absence of phosphorylation) and indirect evidence (chimeras) is provided that Kek1 is not a phosphorylation substrate for the receptor. This was somewhat surprising, as structural work with the vertebrate receptor has indicated that the EGFR kinase domain is in a catalytically open configuration. Such a configuration is unique in that receptor tyrosine kinases normally require activation loop phosphorylation to relieve autoinhibitory interactions that prevent substrate binding and phosphorylation. In light of a distinct mechanism for activation of EGFR, one proposal put forth is the rotation twist model, in which ligand binding induces dimerized receptors to pivot in or near the transmembrane domain, thereby reorienting the kinase domains to their substrates. One potential explanation for the inability of the receptor to phosphorylate Kek1 is that the structure of the Kek1 jt/tm region might act to hinder such a rotation (Alvarado, 2004b).

Considering that kek1 knockouts exhibit subtle and dose-dependent phenotypes, one important question remaining is, what is the role of Kek1 in a cellular and developmental context? An initial explanation for the subtle LOF phenotype of kek1 with respect to EGFR inhibition was the possibility of functional redundancy between members of the Kek family. However, data for Kek2, Kek4, Kek5, and Kek6 indicate that EGFR inhibition is not a common feature of the Kek family. Alternatively, Kek1's inhibitory activity might reflect a recently acquired trait and not an ancestral or conserved role. However, analysis of kek1 orthologs in Drosophila virilis, D. pseudoobscura, and Anopheles gambiae argues against such a notion (Alvarado, 2004b)

Although it is not a common feature within the Drosophila Kek family, it is unclear whether inhibition of EGFR by Kek1 represents a more widely conserved regulatory mechanism for receptors. For instance, the LRR-containing transmembrane protein Decorin binds to the human EGFR and has been implicated in the regulation of receptor activity. It has been reported, however, that the motifs in the Decorin LRRs required for binding EGFR differ from those of Kek1, suggesting these two LRR molecules are unlikely to represent comparable regulatory modes (Alvarado, 2004b).

Finally, a role for Kek1 in the nervous system has also been reported. Expression of most kek family members is observed in the nervous system and recently three molecules that are structurally similar to Kek1, AMIGO1-3, have been implicated in neuronal development in vertebrates. It will be interesting to determine if Kek1 functions in neuronal development in an EGFR-independent manner and if such a role underlies its ancestral function (Alvarado, 2004b).

Control of bract formation in Drosophila: poxn, kek1, and the EGF-R pathway

In Drosophila, the sensory organs are formed by cells that derive from a precursor cell through a fixed lineage. One exception to this rule is the bract cell that accompanies some of the adult bristles. The bract cell is derived from the surrounding epidermis and is induced by the bristle cells. On the adult tibia, bracts are associated with all mechanosensory bristles, but not with chemosensory bristles. The differences between chemosensory and mechanosensory lineages are controlled by the selector gene pox-neuro (poxn). This study shows that poxn is also involved in suppressing bract formation near the chemosensory bristles. The gene kek1, described as an inhibitor of the EGF-R signaling pathway, has been identified in a screen for poxn downstream genes. kek1 can suppress bract formation and can interfere with other steps of sensory development, including SMC determination and shaft differentiation (Layalle, 2004).

Misexpression of poxn at a late stage of mechanosensory bristle development has no effect on the morphology of the organ, but results in a suppression of bract formation. Misexpression of poxn and alteration of the EGF-R pathway affect bract formation during the same time window. It is concluded that poxn is responsible for the absence of a bract near the organs where it is expressed (Layalle, 2004).

kek1, a gene defined as an inhibitor of the EGF-R signaling pathway, is represented in a subtractive library enriched in genes that are specifically expressed in the chemosensory lineage. kek1 is not expressed in cells of the mechanosensory lineage at the time when bract induction takes place, and is expressed at a high level by the outer cells of the chemosensory organs. Its presence in the subtractive library, and differential pattern of expression between mechanosensory and chemosensory lineages, make kek1 a putative target of poxn (Layalle, 2004).

This point was confirmed by demonstrating that the expression of kek1 is modified following ectopic expression of poxn. Specifically, the ubiquitous expression of poxn results in the activation of kek1 expression in the outer cells of the mechanosensory lineage, where kek1 is normally silent. The activation of kek1 in mechanosensory cells is not complete in the experimental conditions that were used. It should be noted, however, that the repression of bract formation is also partial, suggesting that the overexpression of poxn is not complete. Altogether, this dataset reveals that kek1 is a target of poxn (although not necessarily a direct one) (Layalle, 2004).

The difference of expression of kek1 in the chemosensory and mechanosensory lineages, and the role of kek1 in modulating the EGF-R pathway, suggest a role for this gene in the control of bract formation. kek1 mutants do not show any abnormality in bract formation or in sensory organ development, however. More generally, the complete viability and wildtype phenotype of flies deleted for kek1 is a surprise, given the importance of the EGF-R pathway in many aspects of development (Layalle, 2004).

One obvious explanation for this absence of phenotype in kek1 mutant flies would be the existence of a functional redundancy between kek1 and another inhibitor of the EGF-R pathway. This possibility is supported by the identification of five kekkon-like genes in the Drosophila genome. Therefore this study relied on a gain-of-function analysis to decide whether kek1 might play a role in the control of bract induction (Layalle, 2004).

The EGF-R pathway has been implicated in the formation of the precursor cells for at least some of the macrochaetae on the notum. Since kek1 acts as an inhibitor of the EGF-R signaling pathway in ovary development, it might also inhibit this pathway in the notum and thereby interfere with the determination of macrochaetae. The overexpression of kek1 eliminates those macrochaetae that are most dependent on EGF-R signaling (Layalle, 2004).

Macrochaetae suppression was observed when the expression of kek1 was forced in the proneural cluster (using the sca-Gal4 driver), but not when its expression was forced after the SMC had been determined (using the neu-Gal4 driver). This shows that kek1 interferes with the formation of the precursor cells but not with subsequent steps of the lineage. It is concluded that, with respect to SMC determination, kek1 acts as an inhibitor of the EGF-R pathway, much as it does in the ovary. kek1 is expressed in the notum region of third instar wing discs. It may be, therefore, that kek1 plays a role in defining the position where SMCs are formed, or in defining the time window during which they are determined. The expression of kek1 in wing discs is very dynamic, however, and it has not been possible to determine whether this expression overlaps that of the proneural genes during normal development (Layalle, 2004).

The overexpression of kek1 induces a loss of mechanosensory shafts in the legs. At the latest step of the lineage the socket cell was found to express kek1 at a high level, whereas the shaft cell does not. The loss of shafts could therefore be due to a transdetermination of shaft towards socket fate. No socket duplication was observed however, and anti-Cut labeling demonstrated the absence of one of the two support cells at a frequency similar to that of shaft disappearance. It is concluded that the shaft cell has been lost rather than transformed (Layalle, 2004).

The ectopic expression of kek1 can prevent bract induction near mechanosensory bristles. This observation is entirely consistent with the idea that the control of kek1 expression contributes to the control of bract formation. In the sca-Gal4 line, bracts may be absent even when a shaft is formed, suggesting a direct effect of kek1 on bract formation. Since in this line kek1 expression is driven not only in the mechanosensory lineage but also in epidermal cells, it may be that this epidermal expression contributes to bract suppression. Whatever the case, the effect demonstrates that kek1 is capable of interfering with bract formation (Layalle, 2004).

The effect of Kek1 on EGF-R signaling has been shown to involve a direct interaction between the extracellular domains of the two proteins. At least part of the effect may be mediated by heterodimerization, implying that the two genes are expressed in the same cell. The observation that kek1 is expressed in the chemosensory support cells and affects bract formation by ectodermal cells suggests that the Kek1 protein may also interfere with the functioning of EGF-R proteins carried by an adjacent cell (Layalle, 2004).

Bract induction involves the activation of the EGF-R pathway in an epidermal cell, presumably through the expression of the EGF-R ligand, Spitz, by the outer cells of the sensory lineage. In the case of chemosensory lineages, or after ectopic expression of poxn at a late stage of the mechanosensory lineage, the presence of Poxn protein activates the expression of kek1 (and presumably of other members of the kek family). The Kek1 protein binds to the EGF-R and prevents the formation of a bract. The expression of a dominant-negative form of the receptor mimics this effect. When the dominant-negative is expressed both in bristle cells and in epidermal cells the inhibition of bract formation could be due to the inactivation of the EFG-R in the cells that receive the Spitz signal, i.e., in the epidermal cells. An absence of bracts is also observed when the dominant-negative form of the EGF-R is overexpressed only in bristles cells. In this case, it is proposed that the supernumerary receptors sequester the ligand and thereby prevent bract induction. Ligand sequestration would also account for the absence of bract cells when the normal EGF-R is overexpressed in the bristle outer cells (Layalle, 2004).

The Drosophila SHC adaptor protein is required for signaling by a subset of receptor tyrosine kinases

Receptor tyrosine kinases (RTKs) transduce signals via cytoplasmic adaptor proteins to downstream signaling components. Loss-of-function mutations in have been identified in the Drosophila homolog of the mammalian adaptor protein SHC. A point mutation in the phosphotyrosine binding (PTB) domain of the Drosophila SHC-adaptor protein (Shc) completely abolishes Shc function and provides in vivo evidence for the function of PTB domains. Unlike other adaptor proteins, Drosophila Shc is involved in signaling by only a subset of RTKs: shc mutants show defects in Torso and DER but not Sevenless signaling, as confirmed by epistasis experiments. By double-mutant analysis, the adaptors DOS, DRK, and Shc act in parallel to transduce the Torso signal. These results suggest that Shc confers specificity to receptor signaling (Luschnig, 2000).

During the development of multicellular organisms, receptor tyrosine kinases (RTKs) play an important role in transducing a variety of extracellular signals that trigger cellular responses, such as proliferation, differentiation, or cell survival. RTKs activate a cytoplasmic signaling cascade, the RAS-MAPK pathway, which has been highly conserved from invertebrates to mammals (Luschnig, 2000).

Three Drosophila RTKs have been studied extensively. Torso (TOR) is required for the specification of terminal cell fate at the embryonic poles. Sevenless (SEV) induces R7 photoreceptor cell fate in the developing eye. In contrast to TOR and SEV, each of which being required only for one specific process, the Drosophila EGF receptor (DER) has multiple functions during development. Most of the known components of the RAS-MAPK pathway (CSW, DOS, DRK, KSR, SOS, GAP1, RAS1, DRAF, DMEK, DMAPK) are required for signaling by all three receptors. It is an intriguing problem how different RTKs, which activate one apparently common cytoplasmic signaling cascade, can trigger specific biological responses (Luschnig, 2000).

Upon ligand binding, RTK monomers phosphorylate each other on specific tyrosine residues, thereby creating docking sites for cytoplasmic adaptor proteins with phosphotyrosine (pY) binding modules, such as SH2 (SRC homology 2) or phospho tyrosine binding (PTB). One such adaptor protein, DRK (GRB2 in mammals, SEM-5 in C. elegans) acts as a link between activated RTKs and the Son of Sevenless (SOS) protein, which in turn activates RAS by catalyzing GDP-GTP exchange. The identification of more factors involved in signaling between RTKs and RAS1 has led to the view of a complex signaling network rather than a simple linear cascade. Such factors include Daughter of Sevenless (DOS), which potentially acts as a docking site for several SH2 and SH3 domain proteins, and Corkscrew (CSW), a protein tyrosine phosphatase containing two SH2 domains (Luschnig, 2000).

The maternally derived TOR pathway in the Drosophila embryo is a particularly useful system to study RTK signaling because it marks the first time point in development when the RAS-MAPK pathway becomes essential. The TOR receptor is activated by a spatially restricted ligand at the embryonic poles and regulates, via the RAS-MAPK pathway, zygotic expression of the terminal gap genes, tailless (tll) and huckebein (hkb). Posterior expression of tll and hkb is exclusively regulated by TOR signaling and can serve as a quantitative measure for the strength of signal, while anterior expression depends on additional input by the morphogen Bicoid (Luschnig, 2000).

The loss-of-function phenotypes of signaling components acting between the TOR receptor and DRAF are weaker than those of mutations in TOR or DRAF themselves, and it was shown that DRAF can be activated in the absence of RAS1. This indicates that downstream of the receptor the signal splits into branches and that a RAS1-independent signal can activate DRAF. An analysis of tyrosine phosphorylation sites in the activated TOR receptor revealed that individual sites have distinct positive or negative regulatory effects on signaling, by acting as targets for specific molecules (Luschnig, 2000).

Another adaptor protein, SHC, was suggested by studies in mammalian cells to act in RTK signaling. SHC contains two pY-binding modules, an N-terminal PTB domain, and a C-terminal SH2 domain, separated by a region with similarity to α-1 collagen. Upon EGF stimulation, SHC binds to EGFR and becomes phosphorylated on a specific tyrosine, which creates a binding site for the SH2 domain of GRB2. Mammalian SHC can act as an adaptor to link RTKs via GRB2 to RAS activation (Luschnig, 2000 and refences therein).

A Drosophila SHC homolog, SHC, has been isolated and characterized biochemically. SHC associates with and becomes tyrosine phosphorylated by activated DER in vivo, and the PTB domain of SHC binds specifically to a NPXpY motif in DER. While the PTB and SH2 domains of SHC show a high degree of conservation between human and Drosophila, SHC lacks the high-affinity GRB2 site present in mammalian SHC, and no binding of SHC to DRK was observed in coimmunoprecipitation experiments. However, the presence of another conserved motif (YYND/S) in all known SHC proteins suggested that SHC could couple RTKs to other downstream targets besides GRB2 and the RAS pathway. Thus far, no functional evidence for the suggested roles of SHC proteins has been shown, since shc loss-of-function mutations have not been reported in any system (Luschnig, 2000).

This work presents functional evidence that a Drosophila SHC homolog is an essential component of distinct RTK signaling pathways in the fly. Mutations affecting either only the PTB domain or eliminating SHC protein cause the same strength of phenotypes in all assays applied. Based on genetic data, it cannot be distinguish whether both domains, PTB and SH2, bind to pY sites on the same activated RTK or on different proteins. DER contains one bona fide binding site for the SHC PTB domain (consensus NPXpY; NPEY1357 in DER), while in activated TOR two sites with a mismatch at the +2 position are phosphorylated on Tyr (NKGY644; NKEY698; Gayko, 1999). No motifs for binding by SH2 domains (consensus YIXI) are found in DER, TOR, or SEV (Luschnig, 2000).

The shc111-40 allele contains a single amino acid exchange (R152 > Y) in the PTB domain. Structural studies have revealed that the corresponding residue (Arg-175) of the human SHC PTB domain is crucial for binding of the PTB domain to pY-containing phosphopeptides: mutating the corresponding Arg-175 of the human Shc PTB domain (to Gln or Lys) completely abolished binding of SHC to the phosphopeptide, while exchanging amino acids at three other structurally relevant positions had less or no significant effect on binding. This work supports the structural data and provides functional evidence for the role of PTB domains in RTK signaling (Luschnig, 2000).

In the absence of SHC protein, TOR and DER signaling is only partially reduced. It has been shown for the TOR pathway that signaling downstream of the receptor does not occur in a simple linear cascade, based on the finding that components acting between TOR and DRAF show weaker loss-of-function phenotypes than TOR or DRAF (Luschnig, 2000).

Double-mutant analysis shows that the TOR signal splits into at least three parallel branches, represented by DOS, DRK, and SHC. Simultaneous removal of DOS and SHC function from the germline does not completely abolish the TOR signal, although it is strongly reduced. Double mutant phenotypes and epistasis experiments suggest that more TOR signal is transmitted via SHC than via DOS or DRK. However, genetic data do not exclude that direct interactions between any of these proteins may exist; for example, DOS contains putative binding sites for the SH2 domains of DRK and SHC (Luschnig, 2000).

It was also shown that DRK and SHC have parallel activities, in agreement with the finding that the unique DRK/GRB2 binding site (YVNV) present in mammalian SHC proteins is missing in Drosophila SHC, and Drosophila SHC does not bind DRK in vitro. While Drosophila SHC lacks the DRK binding site, a central YYND motif is conserved between Drosophila and mammalian SHC proteins and is likely to serve as a docking site for an as yet unknown protein that triggers a RAS-independent signal. It has been shown that DRAF can be activated in the absence of RAS1, but the molecular nature of a RAS1-independent pathway had remained elusive. Based on genetic results and binding studies, Drosophila SHC is a good candidate to activate downstream targets via a DRK-/RAS1-independent pathway. The identification of signaling components acting independently of RAS will be of great interest for understanding the specificity of RTK signaling, and SHC should provide a useful entry point to study such a pathway (Luschnig, 2000).

It remains a puzzling question why a complex network of adaptor proteins has evolved, if all of these proteins were involved in a single common pathway activated by all RTKs. Interestingly, this study has found that SHC, unlike most other components, functions only in a subset of RTK pathways. DOS, DRK, KSR, and all components downstream of RAS1 are required for signaling by the RTKs TOR, DER, and SEV. Also, unlike all other signaling components, flies lacking SHC can survive to adults, which show a specific phenotype, suggesting that SHC plays a more specific role than other components (Luschnig, 2000).

In the Drosophila eye, DER can replace the function of SEV in R7 determination. This has led to the conclusion that the cytoplasmic signaling pathways triggered by DER and SEV were identical. However, these data are based on the analysis of constitutively activated forms of RTKs that might not faithfully reflect the situation during normal signaling. This study found that SEV signaling appears to be normal in flies lacking SHC protein, and the defects seen in shc mutant eyes appear to be due to impaired DER signaling. The expression of phenotypes caused by activated DER in the eye is very sensitive for the gene dosage of shc. In contrast, dosage-sensitive interactions of shc with activated forms of SEV were not seen, whereas the adaptors DOS and DRK were identified on the basis of their dosage sensitivity for signal transduction by activated SEV. Also, binding of SHC to SEV was not observed, and SEV does not contain consensus binding sites for PTB or SH2 domains of SHC, whereas SHC specifically binds via its PTB domain to DER. Based on these arguments, it is suggested that SHC is not involved in SEV signaling. Even in the absence of SHC protein, the effect of activated SEV was not abolished; however, this study found that the reduction of DER signaling by shc mutations does partially affect the efficiency of SEV-induced overrecruitment of photoreceptors in the anterior of the eye. Accordingly, removing one copy of the DER itself causes partial suppression of the activated SEV phenotype, indicating that induction of photoreceptor cell fate by the DER is necessary before SEV can trigger R7 development (Luschnig, 2000).

The anteriorly-posteriorly graded phenotype of shc mutant eyes can be explained by a common feature of DER signaling: the morphogenetic furrow moves from posterior to anterior across the eye disc and leaves in its wake cells that express Spitz (Spi), an activating DER ligand, and Argos (Aos), a presumptive inhibitory DER ligand. Both Spi and Aos are themselves target genes of DER signaling and are thought to act as diffusible ligands for DER. Hyperactivation of DER by overexpressing activating Spi, or by mutating inhibitory Aos, leads to photoreceptor overrecruitment and eye roughness, which is more pronounced in the posterior of the eye. This graded phenotype is presumably due to accumulation of diffusible Spi in the posterior of the eye disc. It is likely that the same effect accounts for the anterior to posterior graded eye phenotype of shc mutants: although DER signaling efficiency is reduced, Spi can accumulate to sufficient levels in the posterior to allow normal differentiation of photoreceptors. Toward the anterior margin, the level of Spi becomes limiting, and DER signaling activity is reduced below the threshold for photoreceptor differentiation, resulting in the loss of photoreceptors (Luschnig, 2000).

Interestingly, planar polarity defects were found in shc mutant eyes. Frizzled, Notch, and Jak-Stat signaling have been implicated in the establishment of planar polarity in the eye, but to date no role in this process has been assigned to any member of the RAS-MAPK signaling pathway (Luschnig, 2000).

How can different RTKs trigger specific responses by using an apparently common signaling pathway? Several models have been put forward to explain this paradox. In a 'quantitative' model, the kinetics (exact level and duration) of the MAPK signal is critical for the specificity of the response. A 'qualitative' model proposes the existence of cytoplasmic signaling components dedicated to a specific receptor or subset of receptors. Recently, the protein DOF has been shown to act specifically in Drosophila FGFR signaling pathways. Interestingly, DOF is exclusively expressed in cells where also FGFRs are expressed, namely mesoderm and the tracheal system. Tissue-specific expression of a signal transduction component might be one way to create specific responses (Luschnig, 2000).

In contrast, SHC is widely expressed throughout the embryo (Lai, 1995). Specificity appears to be mediated by selective binding of SHC to specific receptors. The curren data show that SHC is functionally dedicated to a subset of RTKs and suggest that the cytoplasmic events triggered by different RTKs (e.g., DER and SEV) can be different. This is a new aspect that could provide an additional explanation for the specificity of RTK signals. It is suggested that different receptors use specific combinations of cytoplasmic adaptor proteins, which have parallel activities, and potentially activate different downstream targets. This could result in quantitatively and/or qualitatively different signal properties. It will be of great interest for the understanding of RTK signaling to elucidate what downstream events are activated by SHC (Luschnig, 2000).

Endosomal trafficking of Egfr: The role of Hrs

Signaling through tyrosine kinase receptors (TKRs) is thought to be modulated by receptor-mediated endocytosis and degradation of the receptor in the lysosome. However, factors that regulate endosomal sorting of TKRs are largely unknown. Here, one such factor is Hrs (Hepatocyte growth factor-regulated tyrosine kinase substrate). Electron microscopy studies of hrs mutant larvae reveal an impairment in endosome membrane invagination and formation of multivesicular bodies (MVBs). hrs mutant animals fail to degrade active epidermal growth factor (EGF) and Torso TKRs, leading to enhanced signaling and altered embryonic patterning. These data suggest that Hrs and MVB formation function to downregulate TKR signaling (Lloyd, 2002).

Membrane trafficking events are tightly regulated to ensure proper spatial and temporal delivery of membrane bound cargo. Fusion of intracellular vesicles with their target membrane requires the formation of a highly stable core complex. Regulation of the formation of this complex may modulate vesicle fusion. One proposed regulator of core complex assembly is Hrs (Hepatocyte growth factor-regulated tyrosine kinase substrate), which binds to the plasma membrane t-SNARE SNAP-25 and inhibits core complex formation in vitro. Addition of Hrs to a neuroendocrine cell assay inhibits neurotransmitter release, suggesting that Hrs may regulate Ca2+-triggered exocytosis. Interestingly, Hrs is predominantly localized to early endosomes, and Hrs mutant mice have enlarged endosomes. Furthermore, Hrs interacts with Eps15, a protein implicated in receptor-mediated endocytosis. Thus, Hrs has been proposed to play roles in both exo- and endocytosis (Lloyd, 2002 and references therein).

Hrs is homologous to yeast Vps27p (vacuolar protein sorting), which regulates protein trafficking from a prevacuolar compartment to the vacuole. Vps27p belongs to the Class E subset of VPS proteins, which are implicated in sorting proteins into the vacuole lumen. Hrs and Vps27p contain a FYVE domain that binds specifically to phosphatidyl-inositol-3-phosphate (PI3P), and this domain has been demonstrated to localize many proteins to the early endosome. Several FYVE domain-containing proteins have been implicated in endosomal trafficking, including early endosome autoantigen 1 (EEA1), which is essential for early endosome fusion, and Fab1p, which is required for sorting into MVBs. Thus, the FYVE domain may allow proteins to mediate membrane trafficking from or to the endosome through its interaction with PI3P (Lloyd, 2002 and references therein).

In addition to a role in vesicle trafficking, Hrs has been proposed to play different roles in several signal transduction pathways. Hrs binds to Stam, a protein implicated in cytokine signaling, and Hrs and Stam both contain VHS (Vps27p, Hrs, Stam) domains present in several proteins implicated in membrane trafficking or signal transduction. Overexpression of Hrs inhibits IL-2-mediated cell growth, suggesting that Hrs may function with STAM to negatively regulate cytokine signaling. In contrast to this inhibitory role, Hrs has recently been proposed to play positive roles in both TGF-ß and Egfr signaling. Hrs binds to SMAD-2, and hrs mutant mouse embryos exhibit a reduced response to activin and TGF-ß. Furthermore, overexpression of Hrs in HeLa cells inhibits ligand-induced degradation of Egfr, suggesting that Hrs may normally promote Egfr signaling by inhibiting endosome to lysosome trafficking of the receptor (Lloyd, 2002 and references therein).

Thus, although numerous data suggest Hrs may play a role in vesicle trafficking and signal transduction, the precise function of Hrs in these processes is unclear. To further investigate the function of Hrs, effects of the loss of Hrs in Drosophila were investigated. The data suggest that Hrs regulates inward budding of endosome membrane and MVB formation. More importantly, hrs mutant animals are unable to degrade active Egfr and Torso TKRs leading to enhanced TKR signaling (Lloyd, 2002).

A single Hrs homolog was identified in the Drosophila genome, and sequence analysis of a 2.7 kb hrs cDNA predicts an open reading frame of 760 amino acids with several well-conserved domains. The hrs gene was mapped to cytological band 23A and is removed by deficiency Df(2L)N19 (Df). Alleles of several complementation groups mapping to Df(2L)N19 were previously isolated in an EMS mutagenesis screen for mutations in synaptotagmin. An 11.5 kb genomic DNA fragment containing the hrs gene or the hrs cDNA driven by the hsp70 promoter (hs-hrs) fully rescues the early pupal lethality of l(2)23AdD28/Df and l(2)23AdD28/l(2)23AdD28 animals but not other mutations in this region. These data demonstrate that the l(2)23AdD28 chromosome (hereafter referred to as hrs) contains a mutation in the hrs gene. Sequencing of DNA from mutant animals reveals a nonsense mutation at amino acid Q270 (Lloyd, 2002).

Polyclonal antibodies were generated to the full-length (anti-FL-Hrs) and amino-terminal half (aa 1-376, anti-N-Hrs) of the recombinant protein. Western analysis of fly extracts using the anti-FL-Hrs antibody detects a major band of 110 kDa in wild-type animals, whereas no protein is detected in hrs third-instar larvae (L3) or white prepupae (WPP). However, the anti-N-Hrs antibody recognizes a 30 kDa band in mutant animals in addition to the 110 kDa band in wild-type animals, suggesting that a 270 amino acid truncated protein is expressed in mutant animals. Furthermore, the presence of full-length Hrs protein in late stage 17 Df embryos suggests that maternally deposited Hrs protein is very stable and may compensate for the loss of zygotic Hrs in embryonic development. Indeed, embryos produced by mothers homozygous for hrs in their germline cells (maternal knockout or mKO) lack full-length Hrs protein and die early in embryogenesis. Thus, the effects of loss of Hrs function may be analyzed in zygotic mutant third-instar larvae/early pupae or in germline mutant embryos (Lloyd, 2002).

Analysis of the protein expression of Hrs suggests that Hrs is ubiquitously expressed. To determine the subcellular localization of Hrs, expression was examined in garland cells and muscle cells of third-instar larvae. Anti-Hrs labels vesicles enriched in perinuclear regions of muscle cells, whereas labeled vesicles are predominantly in the periphery of garland cells. These staining patterns are specific, since they are not seen in hrs mutant cells. Interestingly, overexpression of hrs leads to an enlargement or accumulation of Hrs-positive vesicles and a reduction in cell size (Lloyd, 2002).

Both Hrs antibodies also label type I synaptic boutons of the neuromuscular junction (NMJ). There is some colocalization of Hrs with the synaptic vesicle (SV) marker Synaptotagmin, but most staining appears to be outside SV-rich regions. However, electrophysiological analysis of wild-type and mutant neuromuscular junctions suggests that Hrs does not play an important role in regulating synaptic vesicle exocytosis. Furthermore, Hrs is not enriched in the synapse-rich neuropil of the larval brain, even when overexpressed in neurons using the elav-GAL4 driver (Lloyd, 2002).

To determine if Hrs functions in endocytosis, trafficking of internalized tracers was investigated in third-instar larval garland cells, large cells with a high rate of fluid-phase endocytosis. Wild-type garland cells show strong labeling of peripheral vesicles after a 5 min incubation with avidin-Cy3, indicating rapid internalization of dye into endosomes. Mutant cells are much larger than wild-type cells but show strong labeling of peripheral vesicles, suggesting that dye internalization is not significantly impaired. However, many labeled vesicles (endosomes) in mutant cells are much larger than those observed in wild-type cells. Furthermore, analysis of lysosomal markers suggest that while lysosomes are reduced in size in hrs mutant cells, smaller endosomes are capable of delivering avidin to low pH compartments at a rate similar to wild-type cells (Lloyd, 2002).

Next, hrs mutant endosomes were analyzed using transmission electron microscopy (TEM). Garland cells were incubated with HRP for 5 min, fixed, and sectioned for TEM. In wild-type garland cells, HRP labels the lumen and internal membrane of peripherally located endosomes. In hrs mutant larvae, mutant endosomes are dramatically enlarged, but do not show significant HRP labeling, despite their ability to internalize avidin dye. Rather, in mutant cells, HRP labels a vast tubulo-vesicular network at the periphery of mutant cells, which is not seen in wild-type cells. Finally, endosomes in wild-type garland cells undergo invagination of their limiting membrane to eventually collapse upon themselves. In hrs garland cells, there is a strong reduction in the relative number of invaginated endosomes (5-fold, p = 0.008) and collapsed endosomes (10-fold, p = 0.003). These data suggest that endosomes are enlarged in hrs mutant cells due to an inability of endosomes to invaginate their limiting membrane (Lloyd, 2002).

Inward budding of endosome membrane is believed to be the first step of multivesicular body (MVB) formation. To determine if MVB formation is impaired in hrs mutant animals, electron microscopy was performed at the NMJ. In wild-type synapses, large, classical MVBs are occasionally observed, and they are believed to be endosomal intermediates containing synaptic vesicle proteins destined to be delivered to somatic lysosomes. Much more frequently, though, 60-120 nm vesicles are observed that also appear to contain small internal vesicles. Remarkably, in hrs mutant synapses, there is a 5-fold reduction in the number of these small MVBs. These data suggest that Hrs regulates formation of MVBs at the synapse (Lloyd, 2002).

One proposed function of multivesicular bodies is to partition transmembrane proteins and lipids destined for delivery to the lysosome from those destined to be recycled back to the surface of the cell. While most cell surface proteins are recycled from endosomes, activated TKRs such as the Egfr are trafficked inside MVBs for degradation in the lysosome. Because Hrs is phosphorylated in response to TKR activation, the possibility that Hrs regulates TKR degradation and signaling in Drosophila was investigated (Lloyd, 2002).

The effects of loss of Hrs function on the Egfr pathway were analyzed. Egfr signaling is required for a wide variety of cell fate decisions throughout Drosophila development. Secretion of the Egfr ligand, Spitz, in the ventral midline leads to graded activation of Egfr and the ras/MAPK cascade in the ventral ectoderm. dpMAPK staining is present in the ventral neuroectoderm and dorsal cephalic regions of the gastrulating embryo, and this staining pattern requires Egfr activation. dpMAPK staining in these regions is expanded in hrs mKO embryos, demonstrating that Egfr signaling is enhanced in the absence of Hrs (Lloyd, 2002).

Next, whether enhanced MAPK signaling in the ventral ectoderm results in an expansion of ventral fate was examined. The transcription factor ventral nervous system defective (vnd) is a primary target of Egfr signaling in the ventral ectoderm, and genetic manipulations leading to enhanced Egfr signaling lead to an expansion of cells expressing vnd. In hrs mKO embryos, there is an expansion of the number of cells expressing Vnd from 1-2 in wild-type to 3-4 in the mutant. Furthermore, Fas III expression, known to be a downstream target of Egfr activation, is upregulated in hrs mKO embryos. Thus, a dorsal expansion of cells expressing dpMAPK and Vnd and an upregulation of Fas III expression suggest that Egfr signaling is enhanced in hrs mKO embryos (Lloyd, 2002).

To determine if enhanced Egfr signaling is due to increased receptor activity, tyrosine phosphorylation of the receptor was examined by immunoprecipitating Egfr from pupal lysates and Western blotting with a phosphotyrosine-specific antibody. Levels of tyrosine-phosphorylated receptor are increased 2- to 3-fold in hrs/hrs and hrs/Df animals when compared to wild-type (+/+) or heterozygous (hrs/+) animals. When these same membranes are stripped and reprobed with anti-Egfr antibody, total levels of the receptor are actually decreased approximately 2-fold in hrs/hrs and hrs/Df mutant animals. Together, these data indicate that the relative levels of the active form of Egfr are increased approximately 5-fold in hrs mutant animals. Finally, levels of other ubiquitously expressed surface transmembrane proteins examined at this stage are unchanged or slightly decreased in mutant animals, suggesting that there is not a general defect in turnover of plasma membrane proteins (Lloyd, 2002).

Recent evidence suggests that ubiquitination of endosomal TKRs may be a signal for trafficking to the lysosome rather than recycling to the surface. However, factors that bind ubiquitinated TKRs and sort them into MVBs are unknown. Recently, a 20 amino acid ubiquitin-interacting motif (UIM) conserved in family members of the proteosome subunit 5A (S5A) has been found in a large number of proteins, including several proteins implicated in endocytic trafficking. The UIM present in Hrs is highly conserved among all species examined, so it was determined whether or not Hrs interacts with ubiquitin, using GST pull-down assays. GST-ubiquitin but not GST readily pulls down the full-length Hrs protein from pupal extract. This interaction is direct, since GST-ubiquitin also binds purified recombinant N-Hrs (aa 1-376) protein containing the UIM. These data demonstrate that Hrs binds ubiquitin and suggest that Hrs may regulate endosomal sorting of TKRs via a direct interaction of Hrs with ubiquitinated receptors (Lloyd, 2002).

Extracellular signals are communicated to cells with remarkable temporal and spatial resolution. The rapid kinetics of signal amplification and termination are critical to the precision of signal transduction. One mechanism thought to mediate signal downregulation is the internalization and degradation of cell surface receptors. Although internalization of receptors may inhibit ligand binding, many receptors are still active, or in some cases, more active, after internalization. Once inside the early endosome, TKRs may either be recycled back to the surface of the cell or sorted into the multivesicular body (MVB) for degradation in the lysosome (Lloyd, 2002).

It has long been proposed that lysosomal delivery of cell surface receptors is a negative feedback mechanism for downregulation of receptor signaling. However, there is little in vivo evidence for this model, and it remains possible that deactivation of the receptor or downstream components may compensate for a failure to downregulate active receptor. The data suggest that trafficking of TKRs into the MVB plays an important role in signal attenuation. Interestingly, several of the morphological phenotypes observed in hrs mKO embryos are also seen in mutations affecting the torso pathway. For example, posterior cellularization defects are also observed in fs(1)polehole and l(1)polehole/D-raf embryos, and twisted gastrulation phenotypes are also observed in torso embryos (Lloyd, 2002).

Recently, overexpression of Hrs in HeLa cells has been shown to inhibit ligand-mediated degradation of Egfr, suggesting that Hrs may function to prolong Egfr signaling. In contrast, the data in this study suggest the opposite function for Hrs, namely that it functions to attenuate TKR signaling by promoting degradation of the tyrosine-phosphorylated, or active, receptor. Interestingly, although active Egfr is upregulated in hrs mutants, total levels of the receptor are decreased, suggesting that Hrs is specifically required for degradation of active receptors. This reduction in total receptor is likely due to a well-characterized negative feedback mechanism whereby Egfr hyperactivation inhibits receptor transcription (Lloyd, 2002).

In summary, the following model is proposed for Hrs function. (1) Endocytosis of activated tyrosine kinase receptors (2) leads to the phosphorylation of Hrs on the early endosome membrane. Phosphorylation may enhance the activity of Hrs, which then (3) leads to localized invagination of endosomal membrane. Ubiquitinated receptors may be sorted into the invagination directly via an interaction with the UIM of Hrs or indirectly through an interaction with Hrs binding proteins SNX1, Clathrin, or Eps15. Finally, (4) the membrane is pinched off to form a MVB, and (5) the internalized vesicles are trafficked to the lysosome for degradation. This process of MVB formation leads to a reversal of membrane topology such that the cytoplasmic portion of TKRs is now inside the MVB and unable to signal to downstream components. In this model, receptor-mediated activation of Hrs and MVB formation serves a critical role in attenuating tyrosine kinase receptor signaling (Lloyd, 2002).

Echinoid and Egfr signaling

echinoid (ed) encodes a cell-adhesion molecule (CAM) that contains immunoglobulin domains and regulates the Egfr signaling pathway during Drosophila eye development (Bai, 2001). Genetic mosaic and epistatic analysis, has suggested that Ed, via homotypic interactions, activates a novel, as yet unknown pathway that antagonizes Egfr signaling (Bai, 2001). Alternatively, later studies indicate that Ed inhibits Egfr through direct interactions (Rawlins, 2003; Spencer, 2003). Another body of work suggests that Ed functions as a homophilic adhesion molecule, and also engages in a heterophilic trans-interaction with Drosophila Neuroglian (Nrg), an L1-type CAM. Co-expression of ed and nrg in the eye exhibits a strong genetic synergy in inhibiting Egfr signaling. This synergistic effect requires the intracellular domain of Ed, but not that of Nrg (Islam, 2003). A model for this interaction suggest that Nrg acts as a heterophilic ligand and activator of Ed, which in turn antagonizes Egfr signaling (Islam, 2003).

Echinoid is required to downregulate Egfr activity in the developing Drosophila eye, ensuring a normal array of R8 photoreceptor neurons. Echinoid is an L1-type transmembrane molecule that is expressed in all cells of the eye imaginal discs and, unlike many other Egfr inhibitors, does not appear to be regulated transcriptionally. Echinoid co-precipitates with Egfr from cultured cells and eye imaginal discs, and Egfr activity promotes tyrosine phosphorylation of Echinoid. These observations suggest that Echinoid inhibits Egfr through direct interactions (Spencer, 2003; Rawlins, 2003).

Egfr signaling is essential for the correct patterning and specification of all cell types in the Drosophila eye. Loss of echinoid leads to stabilization of Egfr signaling and Rolled ERKA MAP kinase phosphorylation. Activation of ERKa is closely correlated with expression of the R8 specification factor Atonal, and echinoid mutants show commensurate stabilization of Atonal expression, resulting in the formation of multiple R8 cells in many ommatidia. Mutations in echinoid and Egfr show strong mutual genetic interactions, suggesting that they influence R8 differentiation through a common pathway. Consistent with this view, Echinoid and Egfr are found to co-precipitate from cultured cells, and Echinoid is found to be phosphorylated in response to Egfr signaling in vivo. These data suggest that Echinoid is required to downregulate Egfr signaling after a period of activation in order to limit the number of R8 cells, and may do so through direct interactions (Spencer, 2003).

R8 patterning reflects at least two processes: spacing of emerging R8 equivalence groups and selection from these groups of single R8 cells. It has been suggested that expression of Egfr inhibitors is important for setting the spacing between R8 cells, a view supported by mispatterning in loss-of-function Egfr clones. It is found, however, that echinoid plays no role in this process: while loss of echinoid does increase the duration of Egfr signaling, it does not affect the initial pattern of Egfr activity or the position of R8 equivalence groups within the morphogenetic furrow. Rather, Echinoid appears to be essential only for the second step in R8 specification, the selection of a single R8 cell from the 2-3 cell equivalence group. The role of Echinoid is to ensure that Egfr activity is downregulated within the group in a timely fashion; persistent Egfr activation appears to trigger all cells of the equivalence group to differentiate as R8s. Consistent with this, expression of an activated-Ras, activated-Raf or Pointed-P1 (Rawlins, 2003) promotes multiple R8 cells within individual ommatidia (Spencer, 2003).

Interestingly, Echinoid is the second example of a co-factor required for fine-tuning a major signaling pathway during R8 selection. Selection of R8 from the equivalence group also requires scabrous, a modifier of Notch signaling. Egfr and Notch signaling are used in a number of developing tissues. Echinoid and Scabrous appear to fill the need for high precision during resolution of the R8 equivalence group; this precision is almost unique in the developing nervous system. Therefore, Echinoid and Scabrous appear to have evolved to fine-tune these two pathways for the stringent requirements of the retina. It is anticipated that other factors might provide similar fine-tuning to Egfr and Notch signaling in other tissues (Spencer, 2003).

In an echinoid null allele, only 54% of ommatidia contain multiple R8s (fewer by Boss staining), suggesting that another factor may be acting redundantly to downregulate Egfr signaling in some cells. One candidate for a redundant factor is a highly homologous gene distal to echinoid on the second chromosome. Preliminary data indicates that this gene, fred (friend of echinoid), is expressed in the same tissues as echinoid and displays similar interactions with EgfrEllipse. Further examination of the fred phenotype and creation of fred;ed lines will be necessary to determine if fred acts in a manner similar to echinoid (Spencer, 2003).

In its extracellular domain, Echinoid appears similar to other members of the L1 family of proteins: it undergoes homophilic binding and ectodomain shedding, presumably to regulate cell-cell adhesion. Although some L1 cell adhesion proteins have been shown to interact with receptor tyrosine kinases such as Egfr, those that have been described to date lead to activation, not inhibition, of MAP kinase phosphorylation. In addition, Echinoid lacks two intracellular motifs common to many L1 proteins: a clathrin sorting motif (YRSLE), which regulates internalization, and an ankyrin-binding domain (NEDGSFIGQY), which controls association with the cytoskeleton, suggesting that Echinoid acts by a different mechanism from other L1 proteins. Since overexpression of Echinoid in tissue has no effect on the level of phosphorylated MAP kinase, a read-out of Egfr signaling, it appears that Echinoid does not act as a general inhibitor of Egfr. Instead, the prolonged presence of phosphorylated MAP kinase in echinoid mutants suggests that the role of Echinoid is to downregulate Egfr signaling after a period of activation (Spencer, 2003).

The ability of Egfr to signal depends on its localization and its downstream targets. Ligand-induced endocytosis is a well-documented mechanism for downregulating Egfr activity, and the prolonged Egfr signaling observed in echinoid mutants suggests that one possible role for Echinoid is to facilitate Egfr endocytosis after a period of activity. Another notable feature of Echinoid is its unusual intracellular domain, which differs from other members of the L1 superfamily. This domain is likely required for at least some aspects of Echinoid function (Bai, 2001), and suggests that Echinoid may target downstream signaling molecules. Based on the results, this unknown pathway would intersect with Egfr signaling prior to MAPK phosphorylation (Spencer, 2003).

What downstream molecules might be targeted by Echinoid? One potential model for the function of Echinoid is provided by work on the vertebrate SIRPalpha proteins, the only group of Ig-containing proteins shown to inhibit receptor-tyrosine kinase (RTK) signaling. SIRP-alpha proteins are phosphorylated on tyrosine in response to RTK activation; these phosphorylated residues provide binding sites for the SHP2 tyrosine phosphatase. Analysis of the Drosophila genomic sequence uncovered no clear Drosophila orthologs of SIRP-alpha proteins, but the overall structural similarity of Echinoid, its phosphorylation in response to Egfr signaling and its importance in downregulating Egfr signaling suggest that it may function in a manner analogous to the SIRP-alpha proteins. Genetic interactions have been observed between echinoid and corkscrew, the Drosophila homolog of SHP2, and binding between these proteins has been detected in cultured cells. However, the significance of these interactions will require further study in vivo (Spencer, 2003).

Interaction with ras/raf pathway

The mammalian EGF receptor can serve as a model for Drosophila signaling. SHC is the Drosophila homolog of a mammalian proto-oncogene that serves as a docking molecule, binding to activated receptors as a prelude to assembling other molecules at the site of the activated receptor. SHCp52 polypeptide has an amino terminal phosphotyrosine binding domain that binds an intracellular phosphorylated residue of activated EGF receptor. SHC is then phosphorylated by the EGF receptor and serves to regulate the RAS pathway through its ability to interact with SOS guanine nucleotide exchange factor (Lai, 1995).

Antibodies to the human SHC adaptor protein were used to isolate a cDNA encoding a Drosophila SHC protein (DSHC) by screening an expression library. In flies, the DSHC protein physically associates with activated Egfr and is inducibly phosphorylated on tyrosine by Egfr. DSHC contains an N-terminal phosphotyrosine-binding domain, which associates in vitro with the autophosphorylated Egfr receptor tyrosine kinase. A potential binding site for the DSHC phosphotyrosine-binding domain is located at Tyr-1228 of Egfr (Lai, 1995).

In Drosophila, DRK, an SH2 adaptor protein (and homolog of mammalian Grb2), and Sos (a putative activator of Ras1, Raf and Rolled/MAP kinase) have all been shown to be required for signaling from the Sevenless and the Torso receptor tyrosine kinases. Removing each of these components during the development of the adult epidermal structures produces a very similar set of phenotypes. These phenotypes resemble those caused by loss-of-function mutations in the Egfr . It appears that these components form a signaling cassette, which mediates all aspects of Egfr signaling but that is not required for other signaling processes during epidermal development (Diaz-Benjumea, 1994).

Dominant mutations were isolated that suppress the lethality associated with mutation in the GTP- Ras binding region (CR1) of Drosophila raf (D-raf) serine/threonine kinase. Each of the four intragenic mutations contains one compensatory amino acid change located in either the CR1 or the kinase domain of D-raf . The seven extragenic suppressors represent at least four genetic loci whose effects strongly suggest that they participate in both the sevenless and Egfr signaling pathways. One of these mutations is an allele of D-mek which encodes the known signaling molecule MAPK kinase (Lu, 1994)

In Drosophila, as in mammalian cells, the Raf serine/threonine kinase appears to act as a common transducer of signals from several different receptor tyrosine kinases. Raf acts in the somatic follicle cells to specify the dorsoventral polarity of the egg. Targeted expression of activated Raf (Rafgof) within follicle cells is sufficient to dorsalize both the eggshell and the embryo, whereas reduced Raf activity ventralizes the eggshell. Raf functions downstream of the EGF receptor to instruct the dorsal follicle cell fate. In this assay, human and Drosophila Rafgof are functionally similar, since either can induce ventral follicle cells to assume a dorsal fate (Brand, 1994).

Breathless, a Drosophila FGF receptor homolog, is required for the migration of tracheal cells and the posterior midline glial cells during embryonic development. Deregulated receptors containing the cytoplasmic domains of DFGF-R2, Egfr, Torso, and Sevenless were all able to partially rescue the migration defects. Consistent with the notion that these RTKs share a common signaling pathway, constructs containing the activated downstream elements Dras1 and Draf were also able to rescue tracheal migration, demonstrating that these two proteins are key players in the Breathless signaling pathway (Reichman-Fried, 1994).

Addition of a secreted (but not the membrane-associated) form of Spitz, the Egfr ligand to cells expressing Egfr gives rise to a rapid tyrosine autophosphorylation of Egfr. Following autophosphorylation, Egfr associates with the DRK adapter protein. Consequently, activation of MAP kinase is observed. A dose response between the levels of Spitz and MAP kinase activity is observed (Schweitzer, 1995).

Colocalization of Drosophila and mammalian Cbl proteins with mammalian EGF receptor can be detected in cultured cells as early as a few minutes after addition of EGF; by 20 minutes, an intense punctate pattern of Cbl and the EGF receptor is evident. It is thought that Cbl-EGF receptor complexes are internalized into endosomes through a coated pit pathway. In C. elegans, sli-1 gene has been shown to interact genetically with unc-101 in the regulation of the EGF receptor pathway leading to vulval differentiation. unc-101 encodes a clathrin-associated protein homologous to mouse AP47, which constitutes one of the main components of coated pits and vesicles. It is tempting to speculate that Cbl function in downregulating Egfr signaling could involve the degradation of receptor-Cbl complexes during Egfr trafficking within intracellular membranes (Meisner, 1997 and references).

The c-Cbl proto-oncogene encodes a multidomain phosphoprotein that has been demonstrated to interact with a wide range of signaling proteins. The biochemical function of c-Cbl in these complexes is, however, unclear. Recent studies with the C. elegans Cbl homolog, sli-1, have suggested that Cbl proteins may act as negative regulators of Egf receptor signaling. Because the Egfr and other protein tyrosine kinase receptor signaling pathways are highly conserved between insects and vertebrates, a Drosophila homolog of c-Cbl was sought for a detailed genetic analysis. Drosophila has a single gene, Cbl, that is homologous to c-cbl. Drosophila Cbl encodes a 52 kDa protein that has a high degree of similarity to c-Cbl and SLI-1 across novel phosphotyrosine-binding (PTB) and RING finger domains. Surprisingly, however, Drosophila Cbl is C-terminally truncated relative to c-Cbl and SLI-1, and consequently is unable to bind SH3-domain containing adaptor proteins, including the Drosophila Grb2 homolog, Drk. Although the Drosophila Cbl protein lacks Drk binding sites it can nevertheless either associate either with a tyrosine phosphorylated protein, or is itself tyrosine phosphorylated in an Egfr dependent manner and it associates with activated Drosophila Egrf receptors in vivo. Consistent with a role for Drosophila Cbl in Egfr dependent patterning in the embryo and adult, Cbl is expressed at a high level in early embryos and throughout the imaginal discs in third instar larvae. This study forms the basis for future genetic analysis of Cbl, aimed at gaining insights into the role of Cbl proteins in signal transduction (Hime, 1997).

The active state of receptor tyrosine kinases (RTKs) and the RTK signaling cascade pathways were followed in situ. This was achieved by monitoring, with a specific monoclonal antibody, the distribution of the active, dual phosphorylated form of MAP kinase (ERK). A dynamic pattern is observed during embryonic and larval phases of Drosophila development, which can be attributed, to a large extent, to the known RTKs. Torso-dependent, Egfr-dependent, Breathless-dependent, and Heartless dependent activation profiles have all been identified. This specific detection has enabled the determination of the time of receptor activation, the visualization of gradients and boundaries of activation, and has allowed the postulation of the distribution of active ligands. A novel pattern is observed in the visceral mesoderm at stage 11 that is not Heartless dependent, as patches of cells display activated ERK at normal intensity in heartless mutants. Since the antibody was raised against the phosphorylated form of a conserved ERK peptide containing the TEY motif, this approach is applicable to a wide spectrum of multicellular organisms (Gabay, 1997).

DroVav, the Drosophila melanogaster homologue of the mammalian Vav proteins, serves as a signal transducer protein in the Rac and DER pathways

Mammalian Vav signal transducer proteins couple receptor tyrosine kinase signals to the activation of the Rho/Rac GTPases, leading to cell differentiation and/or proliferation. The unique and complex structure of mammalian Vav proteins is preserved in the Drosophila homologue, Vav. Drosophila Vav functions as a guanine-nucleotide exchange factor (GEF) for DRac. Drosophila cells overexpressing wild-type (wt) Vav exhibit a normal morphology. However, overexpression of a truncated Vav mutant (that functions as an oncogene when expressed in NIH3T3 cells) results in striking changes in the actin cytoskeleton, resembling those usually visible following Rac activation. Dominant-negative Rac abrogates these morphological changes, suggesting that the effect of the truncated Vav mutant is mediated by activation of Rac. In Drosophila cells, stimulation of the Drosophila EGF receptor (DER) increases tyrosine phosphorylation of Vav, which in turn associates with tyrosine-phosphorylated DER. In addition, the following results imply that Vav participates in downstream DER signalling, such as ERK phosphorylation: (1) overexpression of Vav induces ERK phosphorylation; and (2) 'knockout' of Vav by RNA interference blocks ERK phosphorylation induced by DER stimulation. Unlike mammalian Vav proteins, Drosophila Vav was not found to induce Jnk phosphorylation under the experimental circumstances tested in fly cells. These results establish the role of Vav as a signal transducer that participates in receptor tyrosine kinase pathways and functions as a GEF for the small RhoGTPase, Rac (Hornstein, 2003).

The receptor tyrosine kinases (RTKs) play an important role in the control of most fundamental cellular processes including the cell cycle, cell migration, cell metabolism and survival, as well as cell proliferation and differentiation. RTK stimulation leads to the deployment of signalling proteins that relay the appropriate specific signals, resulting in the desired cell fate. Many of the signal transducing proteins, including RTKs, are conserved throughout evolution. In the past few years, genetic and biochemical studies in Drosophila have revealed the identity and function of many signalling cascade molecules that are also shared by mammals. However, there are still many signalling proteins whose roles are still unknown. One such signal transducer protein, the Drosophila melanogaster homologue of mammalian Vav proteins, has been isolated and partially characterized (Hornstein, 2003 and references therein).

Vav proteins represent a novel family of signal transducers that couple tyrosine kinase signals with the activation of the Rho/Rac GTPases and are likely to play an integral role in the regulation of cell differentiation in many tissues. The first member of the mammalian Vav family of cytoplasmic signal transducer proteins to be identified, Vav1, was isolated as an oncogene. Removal of its amino terminus activates Vav1 as a transforming protein. Likewise, the corresponding molecular lesions in Vav2 and Vav3, two other members of the mammalian Vav family, render these proteins transforming. Unlike Vav1, which is exclusively expressed in hematopoietic cells, Vav2 and Vav3 are expressed in both hematopoietic cells and in many cells of nonhematopoietic origin. Numerous biochemical and overexpression experiments revealed that tyrosine phosphorylation of Vav1 in response to activation by one of several cytokines, growth factors or antigen receptors regulates its activity as a GEF for the Rho/Rac family of GTPases, RhoA, Rac1 and RhoG. Activation of these GTPases leads to cytoskeletal reorganization and activation of stress-activated protein kinases (SAPK/JNKs) in T cells. Vav2 and Vav3 function in a similar but not identical fashion. While both Vav2 and Vav3 also act as GEFs, there are conflicting reports regarding which GTPases are activated by them, and whether these GTPases are distinct from those activated by Vav1. Knockout experiments revealed that in T cells, Vav1 integrates signals from lymphocyte antigen receptors and costimulatory receptors to control differentiation, proliferation and the response to activation. Thus, mice deficient in Vav1 exhibit defects in numerous responses to T-cell stimulation, including capping of the T-cell receptor (TCR) postactivation, recruitment of the actin cytoskeleton to the CD3 chain of the TCR, interleukin-2 (IL-2) production and proliferation, cell cycle progression, activity of NF-AT, phosphorylation of SLP-76 and increase in Ca2+ influx. Mice deficient in Vav2 display no obvious defects in T-cell development yet exhibited some defects in B-cell function. Mice lacking both Vav1 and Vav2 displayed major defects in B-cell function that are as dramatic as the defects in T-cell development and activation observed in Vav1-/- mice. Since there are no reports regarding Vav3-/- mice at the present time, the picture of the intricate signalling network induced by the Vav proteins is incomplete. However, it is obvious that the redundancy and complexity of the mammalian Vav proteins even in hematopoietic cells together with the possibility that they differ both in their protein-protein interactions and in their activation of various GTPases, makes it difficult to clearly interpret the results of knockout and other experiments in mammals (Hornstein, 2003 and references therein).

In Drosophila, only one Vav homologue is present (Dekel, 2000). The highly conserved and unique structure of Vav suggests that the vav genes probably evolved from one ancestral gene and that they are important regulatory molecules in flies as well as in mammals. Drosophila Vav encodes a protein whose similarity with hVav1 is 47% and with hVav2 and hVav3 is 45%. Like mammalian Vav proteins, Droosophila Vav encodes a 'calponin-homology' (CH) region, a dbl homology (DH) domain, a pleckstrin homology (PH) domain and both an Src Homology 2 (SH2) and an Src Homology 3 (SH3) domains. However, unlike mammalian Vav proteins, Drosophila Vav lacks an amino-SH3 region. Vav is the only known Drosophila Rho GEF that encodes in addition to a DH region, both SH2 and SH3 domains, attesting that it may be a uniquely versatile signal transducer. The fact that only one homologue of Vav is present in flies, combined with the unique and highly conserved structure of the protein, promises that any study of Vav in flies should be highly beneficial and instructive (Hornstein, 2003).

A hallmark of Vav signal transducer proteins is that their tyrosine residues become phosphorylated on tyrosine residues in response to EGF stimulation. Furthermore, Vav proteins are known to bind to the stimulated EGFR through their SH2 region. In mammalian cells, Drosophila Vav is tyrosine phosphorylated in response to EGFR induction; in vitro, the Drosophila Vav SH2 region is associated with tyrosine-phosphorylated EGFR (Dekel, 2000). These results combined with the encoded domain structure of Vav strongly suggest that Drosophila Vav may function as a signal transducer protein in the Drosophila EGF receptor (DER) signalling cascades. This study investigated the role of Vav in downstream signalling from the DER, its interaction with the Drosophila Rac pathway, and its ability to effect cytoskeletal changes in Drosophila cells (Hornstein, 2003).

This study demonstrates that Vav can activate Rac in vivo. Rac is involved in Drosophila in various cellular processes including cell shape, cell adhesion, gene transcription, protein trafficking and cell cycle progression, as well as numerous developmental processes. One of the best known characteristics of Rac is that it is involved in actin cytoskeletal organization. Indeed, the expression of a constitutively activated form of DRac (V12DRac) in cultured S2 cells causes marked changes in the morphology of the cells, leading to lamellipodia and microspikes. These results indicate that overexpression of oncVav (Drosophila Vav that lacks 214 residues of its amino-terminus), but not wild-type Vav, can induce a morphology similar to that obtained with V12DRac (constitutively active mutant Rac). The fact that N17DRac inhibits the changes in the morphology obtained with oncVav further substantiates the conclusion that the activation of Rac by Vav is responsible for the observed cytoskeletal reorganization. Correspondingly, an inactive hVav1 variant defective in its ability to activate Rac inhibited the ability to induce actin cytoskeletal organization, thus further supporting the tight association between Vav, Rac and actin organization. Notably, coexpression of oncVav and V12DRac leads to more profound changes in cytoskeleton organization compared to those observed in cells overexpressing each protein alone. This result could be explained by the fact that the sum of activation reached by both the endogenous Rac activated by oncVav as well as the constitutively activated Rac yields a more striking morphology. Consistent with these results with wild-type Vav, wt hVav1 does not cause any change in morphology of NIH3T3 cells and COS cells. Conversely, a remarkable change in actin organization has been observed following overexpression of hVav1 in T cells. These conflicting results obtained with mammalian wtVav1 could stem from the use of different experimental systems, including the strong possibility that different levels of endogenous Vav2 and Vav3 exist in these systems. The cytoskeletal changes in S2 cells transfected with oncVav are compatible with a previous study demonstrating that an amino-terminus-truncated hVav1 caused depolarization of fibroblasts and triggered the bundling of actin stress fibers in NIH3T3 cells. Taken together, these results support a pathway in which Drosophila Vav serves as a GEF for Rac, thereby triggering the reorganization of the actin cytoskeleton (Hornstein, 2003).

Drosophila Vav is tyrosine phosphorylated in response to stimulation of DER and it also associates with the stimulated receptor. This result is compatible with the known characteristics of mammalian Vav proteins. For example, when ectopically expressed in nonhematopoietic cells, Vav1 associates with the EGFR through its SH2 region and becomes tyrosine phosphorylated upon induction with EGF. A similar result was reported for the ubiquitously expressed Vav2 and Vav3 proteins. Although it is well established that the Vav proteins are tyrosine phosphorylated upon EGFR stimulation, the exact contribution of the various mammalian Vav proteins to the EGFR signalling pathway is not understood. These studies with Drosophila Vav shed some light on these events. Thus, this study demonstrates that overexpression of Vav in D2F cells leads to increased ERK phosphorylation. Moreover, its elimination by the use of dsRNAi blocks phosphorylation of ERK even following stimulation of DER. There are opposing results regarding the link between Vav and ERK activation in mammals. Overexpression of Vav1 in Jurkat T cells induced 3-4-fold activation of ERK activation. Vav1 activates ERK when ectopically expressed in NIH3T3 or CHO cells. Furthermore, Vav1 coimmunoprecipitates with ERK in a human myeloma cell line stimulated with interleukin-6. Finally, T cells deficient in Vav1 exhibit defects in ERK phosphorylation. Conversely, the activation of Ras and ERK following stimulation of the TCR in Vav1 null Jurkat T cells appears normal. Without the complication of multiple homologues, these studies strongly suggest a role for Drosophila Vav in the ERK pathway in S2 cells (Hornstein, 2003).

Despite the existence of several studies that point to a link between hVav1 and Ras, it is still unclear how Vav proteins can affect ERK. Drosophila Vav may affect the Ras/ERK pathway through its function as a GEF towards DRac. Cross talk between the Rac and Ras pathways has been shown to exist in mammals. Rho family small GTPases were found to play an important role in mediating the activation of Raf by Ras. Thus, a dominant-negative mutant of Rac can block Raf activation by Ras. Additionally, the effect of Rac can be substituted by the PAK kinase, which is a direct downstream target of Rac. Moreover, PAK directly associates with Raf-1 under both physiological and overexpressed conditions. The extent of interaction between PAK and Raf-1 is correlated with the ability of PAK to phosphorylate Raf and induce mitogen-activated protein kinase activation. These studies strongly suggest that cross talk between Rac and Ras exist and it is mediated through activation of downstream effectors of Rac, such as PAK. Furthermore, it was demonstrated that MEK kinases are regulated by EGF and selectively interact with Rac/Cdc42. A novel Drosophila gene, DRacGAP, has been identified which behaves as a negative regulator of the GTPases, DRac1 and DCdc42. Reduced function of DRacGAP or increased expression of DRac1 in the wing imaginal disk causes effects on vein and sensory organ development and cell proliferation as a result of enhanced activity of the EGFR/Ras signalling pathway. Thus, DRac and DRas are involved in cross-talk mechanisms that modulate Drosophila development (Hornstein, 2003 and references therein).

Drosophila Vav might also activate ERK in a GEF-independent manner. For instance, Vav might stimulate the Ras/ERK pathway via PLC activation, just as Vav1 was shown to activate PLC. PLC contributes to the activation of Ras, probably by stimulating the activity of the diacylglycerol-dependent exchange factor, Ras GRP. It is highly conceivable that the activity of hVav1 towards PLC is mediated in a GEF-independent mode. Whether such a mechanism is also elicited in flies remains to be determined. Drosophila Vav contains several protein-binding domains (SH2, SH3) that might participate in various pathways that result in activation of the Ras/ERK pathway. For instance, Vav may bind to the adapter molecule DShc, that was shown to be associated with the Grb2/Drk proteins leading to DRas activation. Indeed, mammalian Shc binds mammalian Vav proteins. Collectively, the current results clearly illustrate that Vav influences both the DRac and ERK pathways. However, it is not clear yet whether it exerts its influence on ERK by an exclusive inducement of the DRac pathway and/or through a GEF-independent activity (Hornstein, 2003).

The involvement of mammalian Vav proteins, by functioning as GEFs towards Rac in the JNK signalling cascade, is well establishe. Moreover, Vav proteins mediate this response through their function as GEFs towards Rac. This pathway is highly conserved between mammals and flies. In Drosophila, it can transduce signals of a diversified nature, leading to changes in cell polarity and mediating immunity in the adult. It is also required for dorsal closure during embryonic development. Genetic studies focusing on these processes placed the Rho family small GTPases in the JNK signalling cascade. However, although this study demonstrated that Vav functions as a GEF towards DRac in Drosophila, Vav does not seem to be involved in the sorbitol-induced JNK activation. In accordance, no effect has been detected of Rho family small GTPases on sorbitol-induced JNK activation in S2 cells. It is therefore conceivable that in S2 cells, the sorbitol-induced activation of JNK is not mediated through activation of DRac, and therefore does not require Vav. The possibility that Vav is involved in JNK activation under other physiological pathways in Drosophila, such as dorsal closure, still exists; however, this question merits further investigation (Hornstein, 2003).

In summary, these results show that, in fly cells, Vav functions in various signalling cascades in which it can play a role as a GEF or participate as an adapter protein. A P-element insertion has recently been reported to inactivate Vav, leading to lethality of flies (Bourbon, 2000). Further genetic experiments will be required to better understand the physiological function of Vav in developmental systems (Hornstein, 2003).

Antagonistic roles of Rac and Rho in organizing the germ cell microenvironment

The capacity of stem cells to self renew and the ability of stem cell daughters to differentiate into highly specialized cells depend on external cues provided by their cellular microenvironments. However, how microenvironments are shaped is poorly understood. In testes of Drosophila, germ cells are enclosed by somatic support cells. This physical interrelationship depends on signaling from germ cells to the Epidermal growth factor receptor (Egfr) on somatic support cells. Germ cells signal via the Egf class ligand Spitz (Spi), and evidence is provided that the Egfr associates with and acts through the guanine nucleotide exchange factor Vav to regulate activities of Rac1. Reducing activity of the Egfr, Vav, or Rac1 from somatic support cells enhanced the germ cell enclosure defects of a conditional spi allele. Conversely, reducing activity of Rho1 from somatic support cells suppresses the germ cell enclosure defects of the conditional spi allele. It is proposed that a differential in Rac and Rho activities across somatic support cells guides their growth around the germ cells. These novel findings reveal how signals from one cell type regulate cell-shape changes in another to establish a critical partnership required for proper differentiation of a stem cell lineage (Sarkar, 2007).

In the male gonad of Drosophila, germ cells are surrounded by somatic cells that define their cellular microenvironmen. Germline stem cells (GSCs) are attached to a cluster of nondividing cells at the apical tip, called hub cells, and associated with cyst progenitor cells (CPCs) that act as stem cells for the somatic support cell lineage. Two CPCs extend their cytoplasm around one GSC, toward the hub, and toward each other such that each GSC appears to be completely enclosed in its cellular microenvironment. GSCc and CPCs generate differentiating daughters, called gonialblasts and cyst cells, respectively. The gonialblasts undergo transit amplification divisions to produce 16 spermatogonia, which become spermatocytes, grow in size, undergo the meiotic divisions, and differentiate into sperm. Two cyst cells grow cytoplasmic extensions around one gonialblast to form the germ cell cellular microenvironment that controls various aspects of germ cell differentiation (Sarkar, 2007).

Germ cells associated with somatic cells mutant for the Map-Kinase Raf fail to differentiate and accumulated as early-stage germ cells instead. A similar accumulation of early-stage germ cells was observed in Egfrts mutant testes shifted to nonpermissive temperature, and in testes from animals mutant for Stem cell tumor (Stet; Rhomboid2), a protease that cleaves Egfr ligands. However, stet mutant germ cells in addition fail to associate with somatic support cells, suggesting that the Egfr pathway is required for setting up the critical cellular microenvironment (Sarkar, 2007).

Loss of spi results in a failure of germ cells to differentiate, similar to the effects of loss of stet or the Egfr. Wild-type testes are long (~2 mm) tubular structures that contain germ cells in a spatio-temporal order along the apical-to-basal axis. Early germ cells (GSCs, gonialblasts, and spermatogonia) are small and have small, densely packed nuclei in DAPI-stained preparations. Spermatocytes are located basal to the spermatogonia, and differentiating spermatids fill the distal part of the testis (Sarkar, 2007).

Animals carrying a temperature-sensitive allele of spi, spi77-20, die when raised at 29°C. However, spi77-20 animals raised at a slightly permissive temperature (27°C) survive and have tiny testes. Most of these testes (40 of 50) contain only small cells, as seen at the tip of wild-type testes, and do not have spermatocytes or differentiating spermatids. Staining with molecular markers revealed that the testes contains increased numbers of GSCs, gonialblasts, and spermatogonia compared to wild-type. The remaining testes (10 of 50) have high numbers of early germ cells and a few spermatocytes, but no differentiating spermatids (Sarkar, 2007).

Testes from spi77-20 animals raised at an intermediate permissive temperature (25°C) are longer than testes from animals raised at 27°C, but significantly shorter (500 μm-1.5 mm) than wild-type testes. A substantial part of the testes is occupied by tumor-like aggregates of early-stage germ cells. However, spermatocytes and differentiating spermatids are also present (Sarkar, 2007).

spi activity is both sufficient and required within the germ cells. Expression of a cleaved version of Spi (sSpi) in germ cells but not in somatic support cells of spi77-20 testes restores the wild-type phenotype, and germ cell clones mutant for spi accumulate at early stages based on phase-contrast microscopy and DAPI-stained preparations) (Sarkar, 2007).

spi was also required for somatic support cells to associate with and enclose the germ cells. Germ cell clones mutant for the conditional spi77-20 allele from animals raised at 27°C either do not associate with somatic support cells or associated with only one somatic support cell (4 of 20 clones), based on staining with soma-specific antibodies, such as the transcription factor Traffic Jam (Tj). Tj labels the nuclei of somatic support cells that are normally associated with early-stage germ cells (Sarkar, 2007).

Germ cell enclosure can be investigated by labeling testes with molecular markers such as antibodies against the membrane-bound β-catenin Armadillo (Arm) that labels the cell membranes of somatic support cells as they surround the germ cells. In wild-type testes, each GSC, gonialblast, and cluster of developing germ cells is associated with and surrounded by two somatic support cells. In testes from spi77-20 animals raised at 27°C, Tj-positive cells did not form cytoplasmic extensions around the germ cells. Similar results were obtained with other markers, including a cytoplasmic UAS-Green Fluorescent Protein (UAS-GFP) expressed in somatic support cells under control of a soma-specific Gal4-driver. In control testes, GFP is detected in the cell bodies of the somatic cells surrounding the germ cells. In contrast, in spi77-20 testes from animals grown at 27°C, GFP is detected in balls, most likely small round cell bodies of somatic support cells. Occasionally, cytoplasmic extensions emerged from somatic support cells, but they remained short and did not enclose the germ cells (Sarkar, 2007).

The lack of cytoplasmic extensions from Tj-positive cells in spi77-20 mutant testes is similar to the phenotype observed in stet mutants. This suggests that the Egf class ligand Spi, expressed in germ cells and processed by Stet, stimulates the Egfr on somatic support cells, inducing them to send out cytoplasmic extensions to enclose the neighboring germ cells (Sarkar, 2007).

Association of germ cells with somatic support cells is sensitive to the level of Spi. Germ cell clones from spi77-20 animals raised at 25°C and germ cell clones from animals mutant for the spi2 allele often associated with more than two somatic support cells (Sarkar, 2007).

The growth of cytoplasmic extensions around the germ cells is also sensitive to the level of Spi. When spi77-20 animals are raised at 25°C, many Tj-positive cells form cytoplasmic extensions directed toward and/or around the germ cells. However, not every germ cell cluster appear to be associated with and/or surrounded by somatic support cells. Furthermore, many of the Tj-positive cells forme cytoplasmic extensions toward each other, suggesting that multiple somatic support cells may surround one tumor-like aggregate of germ cells. Similar abnormal associations of somatic support cells with germ cells are also seen in Egfrts mutants shifted to nonpermissive temperature. One possible explanation for the different phenotypes of loss compared to reduction of Egfr signaling is that different levels of Egfr stimulation may affect different cellular properties of somatic support cells, such as cell adhesiveness and/or growth (Sarkar, 2007).

To identify novel players in germ cell enclosure, the sensitized background of the spi77-20 allele was used to search for genetic modifiers. It was found that impaired activity of the small monomeric GTPase (small GTPase) Rac1 enhances the spi77-20 testes phenotype. Activity of Rac1 was impaired by two strategies-either by removing one copy of the rac1 gene or by expressing a dominant-negative version of Rac1 (dnRac1) in somatic support cells of testes from spi77-20 animals raised at 25°C. In either case, the enhanced testes are shorter than testes from spi77-20 animals raised at 25°C. In 12 of 20 enhanced testes, Tj-positive cells do not enclose the germ cells, and early-stage germ cells accumulate (Sarkar, 2007).

Reducing activity of Vav, a guanine nucleotide exchange factor for Rac-type small GTPases, from somatic support cells by antisense expression also enhances the spi77-20 testes phenotype from animals raised at 25°C. 11 of 20 enhanced testes were tiny and contained mostly early-stage germ cells that were not surrounded by somatic support cells. The enhanced phenotypes caused by impairing Rac or Vav raises the possibility that Rac1 and Vav act downstream of the Egfr in somatic support cells and that Vav plays a role in regulating somatic support cell-shape changes associated with germ cell enclosure (Sarkar, 2007).

In mammalian cells, autophosphorylation of specific Vav-binding motifs within the cytoplamic tail of the Egfr allows for binding and phosphorylation of mammalian Vav2. Phosphorylation of Drosophila Vav has been shown to depend on Egfr stimulation in both mammalian and Drosophila cultured cells, and Drosophila Vav bound to mammalian Egfr (Sarkar, 2007 and references therein).

Consistent with a role for Drosophila Vav in Egfr signaling in testes, Vav protein immunoprecipitates from testis extracts with an antibody against the Egfr. Vav does not immunoprecipitate from testis extracts that had been pretreated with phosphatase, suggesting that the interaction between Vav and the Egfr is phosphorylation dependent. The immunoprecipitated Vav band comigrates with a band detected by antibodies against phospho-tyrosine, suggesting that Vav is phosphorylated when in a complex with the Egfr (Sarkar, 2007).

In the classical view of the Drosophila Egfr pathway, only one docking protein-Downstream receptor kinase (Drk)-binds to the stimulated Egfr and activates a MAP-Kinase cascade for transcription of target genes. However, genetic and biochemical data suggest that the Egfr pathway is branched at the level of docking proteins and that the adaptor protein Vav binds to the Egfr to activate the small GTPase Rac1. These data suggest that Rac regulates cell-shape changes associated with germ cell enclosure, and studies on Raf suggested that it regulates the transcription of target genes. However, the possibility of crosstalk between the two branches cannot be excluded: Vav may contribute to transcriptional regulation and Map-Kinases may contribute to germ cell enclosure. A possible crosstalk is consistent with findings that in cultured Drosophila cells (Hornstein, 2003), Vav can contribute to Erk phosphorylation (Sarkar, 2007).

Surprisingly, impairing activity of the Rho-type small GTPase Rho1 has the opposite effect to impairing Rac1. Testes from spi77-20 animals raised at 27°C that express dominant-negative Rho1 (dnRho1) in somatic support cells are long and appear almost wild-type. In contrast to somatic support cells in spi77-20 testes from animals raised at 27°C without dnRho1 expression, the somatic support cells expressing dnRho1 enclose the germ cells. The same dominant suppression is observed in spi77-20, rho1/+ testes, indicating that expression of dnRho1 reflects loss of Rho1 activity (Sarkar, 2007).

These data raise the possibility that Rac and Rho have antagonistic effects on germ cell enclosure. Rac appears to be required for somatic support cells to grow cytoplasmic extensions around the germ cells, and Rho appears to suppress this growth. Antagonistic roles for Rac and Rho have also been reported in cultured mammalian cells, where Rac and Rho regulate cell-shape changes and growth via different effects on the actin cytoskeleton. Prominent readouts for small GTPase activities on the actin cytoskeleton are the appearances of ruffles and lamellipodia in the cell membranes (Sarkar, 2007).

To address a potential role of Rac and Rho in shape changes of somatic support cells, dominant-negative Rac or Rho were expressed in somatic support cells of otherwise wild-type testes, and transmission electron microscopy (TEM) was used to investigate changes in the membranes of somatic support cells surrounding single germ cells and spermatogonia at the apical tip of the testes. Germ cells and somatic support cells can be identified based on their different shapes and density of staining in TEM. In wild-type, the somatic support cells surrounding single germ cells and spermatogonia exhibit wavy plasma membranes, possibly analogous to membrane ruffles accompanying cellular growth and rearrangements of the actin cytoskeleton in cultured cells (Sarkar, 2007).

Somatic support cells expressing dnRac1 have much smoother plasma membranes than do wild-type somatic support cells. Conversely, somatic support cells expressing dnRho1 have lamellipodia-like extensions in their membranes. Lamellipodia-like extensions were not detected in somatic support cell membranes in serial sections of wild-type testes or in testes expressing dnRac1. In mammalian cells, formation of lamellipodia depends on Rac-type small GTPases. The presence of lamellipodia-like extensions in somatic support cells expressing dnRho1 suggests that Rac may become hyperactive in the absence of Rho. Based on these TEM data, it is hypothesized that, just as their mammalian counterparts do in cultured cells, Drosophila small GTPases may act on the cytoskeleton of somatic support cells to mediate cell-shape changes and growth of cellular extensions and that the effects of Rac and Rho are antagonistic (Sarkar, 2007).

This model predicts that expression of a constitutively active Egfr ligand in somatic support cells might compromise the differential in smGTPase activities. Indeed, forced expression of cleaved ligand in somatic support cells, but not in germ cells, closely mimics the effect of dnRho expression: the somatic support cells formed lamellipodia-like structures in their membranes (Sarkar, 2007).

This research on the Drosophila gonad provides a striking example how one cell type in tissue communicates with another cell type to induce and direct the formation of a proper cellular microenvironment: a signal from one cell induces subcellular changes throughout the body of another cells. This mechanism underlying the formation of a cellular microenvironment may be conserved across species (Sarkar, 2007).

Cbl and Sprint regulate early steps of RTK endocytosis

Guidance receptors detect extracellular cues and instruct migrating cells how to orient in space. Border cells perform a directional invasive migration during Drosophila oogenesis and use two receptor tyrosine kinases (RTKs), EGFR and PVR (PDGF/VEGF Receptor), to read guidance cues. Spatial localization of RTK signaling within these migrating cells is actively controlled. Border cells lacking Cbl, an RTK-associated E3 ubiquitin ligase, have delocalized guidance signaling, resulting in severe migration defects. Absence of Sprint, a receptor-recruited, Ras-activated Rab5 guanine exchange factor, gives related defects. In contrast, increasing the level of RTK signaling by receptor overexpression or removing Hrs, an endosome-associated, ubiquitin binding protein required for multivesicular body formation and degradation of RTKs, and thereby decreasing RTK degradation, does not perturb migration. Cbl and Sprint both regulate early steps of RTK endocytosis. Thus, a physiological role of RTK endocytosis is to ensure localized intracellular response to guidance cues by stimulating spatial restriction of signaling (Jekely, 2005).

It was reasoned that regulation of RTK turnover might be important to maintain a directional response in border cells, and the effects of mutations likely to affect this process were analyzed. Cbl is a ubiquitin ligase with a conserved role in regulating RTK signaling. Clones of border cells mutant for Cbl are correctly specified, as judged by staining for Slbo, a marker specific for differentiated border cells, but have severe migration defects. To explore the relationship between Cbl and RTK signaling, RTK signaling was manipulated in Cbl mutant border cells. The migration defect in Cbl mutant cells was suppressed by reducing the level of an EGFR ligand (grk/+) and was enhanced by overexpression of either receptor in border cells (UAS-PVR or UAS-EGFR. Note that overexpression of either PVR or EGFR alone has no effect. This indicates that the Cbl phenotype is not due to lack of guidance signaling but instead due to excessive, misregulated RTK signaling (Jekely, 2005).

Consistent with the interpretation that Cbl is required to restrict signaling, the complete failure of many Cbl mutant border cell clusters to migrate resembles the effect of increased guidance receptor signaling due to expression of constitutively active receptors or a strong ligand. In contrast, border cells lacking Pvr and Egfr all eventually initiate migration but never make it to the oocyte. Similar phenotypic effects of manipulating guidance cues have been observed by live imaging of germ cell migration in the zebrafish embryo, a migration guided by a G protein-coupled receptor: migratory cells lacking guidance cues migrate, but randomly, whereas the same cells subject to high uniform guidance cues did not migrate. Thus, border cells lacking Cbl responded as if they were receiving high uniform signaling, a situation that mimics the endpoint of migration (Jekely, 2005).

Mammalian Cbl proteins negatively regulate multiple RTKs by stimulating their ubiquitination and lysosomal degradation. The N-terminal phospho-tyrosine binding domain of Cbl directly binds to activated receptors, and ubiquitin-conjugating enzymes are recruited via the E3 type RING finger. The N-terminal part of Drosophila Cbl also physically interacts with autophosphorylated Pvr. In some assays, these conserved domains are sufficient for mammalian Cbl to regulate Egfr. Cbl can also interact with proteins regulating endocytosis as well as other signaling molecules through its less well conserved C-terminal region. Drosophila Cbl is expressed as two isoforms. Ubiquitous expression of either Cbl-L or Cbl-S, which lacks the C-terminal tail, rescues lethality associated with the Cbl null mutation as well as the migration phenotype. To determine whether E3 ligase activity of Cbl is required, a single cysteine residue essential for this activity was mutated to alanine (Cys-369, corresponds to Cys-381 in human Cbl). The Cbl ring finger mutant was unable to rescue viability of the Cbl mutant or migration of Cbl mutant border cell clones, showing that this function is essential for Cbl activity during migration (Jekely, 2005).

Overall level of RTK activity, in the cell or at the cell cortex, does not need to be precisely controlled to allow migration. Yet Cbl is apparently required to restrict RTK activity. This prompted examination of whether Cbl might affect subcellular localization of RTK signaling at a more refined level. Experiments with Hrs mutants and RTK overexpression suggest that total phospho-tyrosine might be used as a local indicator of RTK signaling. Many proteins are tyrosine-phosphorylated in cells by a number of kinases, but the RTKs have a quantitatively significant effect (direct and indirect). Remarkably, wild-type border cells initiating migration show a clear localization of phospho-tyrosine signal to the front. The front is the side facing the direction of subsequent migration and the source of the ligands, namely the oocyte. Since border cells migrate as a tight cluster of cells, several cells contribute to the front. To further test whether the signal reflected stimulation of endogenous RTKs, a strong Egfr ligand (secreted Spitz) was expressed in border cells to stimulate the endogenous receptor uniformly. This resulted in delocalized phospho-tyrosine signal all over the cortex of the border cells, and, as expected, a block in directed migration (70% nonmigrating clusters). This validated the use of phospho-tyrosine as a reasonable local readout of endogenous RTK activation (Jekely, 2005).

Endogenous Pvr and Egfr are detected at low uniform levels in border cells. Overexpression of Egfr or Pvr results in high level of receptor throughout the cluster; however, the phospho-tyrosine signal remains localized. Thus, local activation is maintained despite RTK overexpression. Consistent with signal location being the critical parameter for guidance signaling, directed migration also proceeds normally upon RTK overexpression. In contrast, border cells mutant for Cbl show a high frequency of delocalized phospho-tyrosine signal. These results indicate that Cbl is important in migrating cells because it is required to restrict RTK signaling spatially within the cell; without Cbl, signaling becomes delocalized. Since Cbl affects RTK endocytosis, whether perturbing endocytosis more generally would have the same effect was tested. Expression of a dominant-negative form of Shibire (dynamin) in border cells initiating migration also caused efficient delocalization of the phospho-tyrosine signal (Jekely, 2005).

The incomplete penetrance of the Cbl phenotype suggests that other molecules might partially compensate for the loss of Cbl. Indirect evidence is available that another potential RTK binding endocytosis regulator called Sprint might have a role in border cells. The mammalian counterpart of Sprint, called RIN1, displays Ras-activated Rab5 guanine nucleotide exchange factor (GEF) activity and can bind Egfr and stimulate Egfr activity. RIN1 also binds and activates the Abelson tyrosine kinase. To analyze the function of Drosophila sprint in vivo, sprint mutants, including a complete loss-of-function mutant, were generated. Despite sprint being the only rin1-related gene in Drosophila, homozygous sprint mutant flies are completely viable and fertile with normal oogenesis. To determine whether Sprint might contribute to regulating RTKs during border cell migration, the cells were challenged by overexpressing Pvr or Egfr in the mutant background. By itself, this overexpression has no effect on migration. In the sprint mutant background, however, RTK overexpression results in significant migration defects and, as for Cbl mutants, a corresponding increase in delocalized phospho-tyrosine signal. This suggests that Sprint might play a role similar to Cbl. Sprint might not be essential under normal conditions due to overlap in function with Cbl. To test this further, border cells mutant for both sprint and Cbl were analyzed. These cells have very severe migration defects and rarely reach the oocyte. For comparison, almost half the Cbl single mutant clusters reach the oocyte. Since sprint has barely any defect on its own, this strong enhancement of the Cbl phenotype is significant. Such a synergistic effect of two null mutants indicates that the gene products function in parallel to regulate the same process (Jekely, 2005).

To understand more about how Sprint might function in vivo, an antibody was generated that detects endogenous Sprint. In a pattern strikingly similar to the wild-type polarized phospho-tyrosine signal, endogenous Sprint was detected at the front of border cells initiating migration. This is consistent with Sprint being recruited to active RTKs. This was confirmed by the ability of overexpressed Egfr or Pvr to recruit endogenous Sprint. In overexpression experiments, it was also found that Sprint has the characteristics expected from its homology to RIN1: Sprint binds Ras-GTP recruited Abelson kinase to the cell cortex and associates with endocytic vesicles. Finally, endogenous Sprint accumulates at the apical cortex of follicle cells, contacting the oocyte, upon transient block of endocytosis. Since endogenous Egfr and Pvr ligands come from the oocyte, this supports the idea that Sprint dynamically associates with early endocytosis of RTKs at the cell cortex. Taken together with the genetic analysis, it is concluded that Cbl and Sprint both serve to maintain RTK signaling localized for guidance, although they stimulate early endocytosis events in molecularly unrelated ways (Jekely, 2005).

Regulators such as Cbl and Sprint might be recruited directly to activated and autophosphorylated RTKs or might bind indirectly, via phosphorylated adaptor proteins. A yeast two-hybrid assay was set up to detect possible direct, phosphorylation-dependent binding. The intracellular domain of Pvr is able to autophosphorylate in yeast and bind SH2 and PTB domain proteins. Binding was detected of both Cbl and Sprint to Pvr, but not a kinase-dead Pvr mutant. Potential docking tyrosines in Pvr were systematically mutated. Mutation of 16 or 14 (YF14) tyrosines results in strong decrease in binding of Cbl and Sprint. To map the binding sites, 5 tyrosines at a time were 'added back' to the YF14 mutant. Although there was no overlap in tyrosines, two of the resulting constructs regained full binding to Cbl and Sprint, indicating that both proteins have more than one direct binding site on Pvr. One construct (YFc) did not bind either Cbl or Sprint directly and therefore seemed to be a potentially useful tool to study the role of their direct binding to Pvr in vivo (Jekely, 2005).

To determine the signaling potential of each Pvr mutant in vivo, the mutations were placed in the context of a constitutively active form of Pvr (λ-Pvr) to induce full, unregulated activation. The ability to block border cell migration and induce F-actin accumulation was monitored. The activity of YF14 was strongly reduced, but each add back mutant had only slightly reduced activity relative to wild-type despite missing nine potential docking tyrosines. Thus, each of the add back mutants was still capable of signaling to affect migration and guidance when artificially activated (Jekely, 2005).

To determine whether the YFc mutations affected receptor regulation, they were placed in the context of full-length Pvr. From transgenes, Pvr and Pvr-YFc were expressed in the ovary at similar levels. As expected, the ability to activate signaling in border cells (measured by anti-dpERK staining) was quantitatively reduced in Pvr-YFc compared to Pvr. This result was confirmed using the sensitive MAP kinase-activated reporter gene kekkon-lacZ. However, expression of Pvr-YFc but not Pvr caused border cell migration defects. Although the frequency of defects was low, finding a gain-of-function activity at all was significant, given that the signaling strength of Pvr-YFc was reduced compared to Pvr. The migration defects were qualitatively similar to those of Cbl mutants and distinct from dominant-negative effects, which even in their strongest form cause migration delays but not arrest. Uniform expression of the ligand PVF1 did not further affect the phenotype of Pvr-YFc, indicating that this form of Pvr that cannot bind Cbl and Sprint had already lost its spatial information. Consistent with this, expression of Pvr-YFc also induced a delocalized phospho-tyrosine signal at a frequency corresponding to the migration defects. This analysis of Pvr itself further indicates that the phenotypes of Cbl and sprint mutant border cells are due to their effects on RTK signaling: recruitment of Cbl and Sprint to Pvr serves to regulate Pvr guidance signaling, specifically to keep it localized (Jekely, 2005).

Thus, Cbl and Sprint are required to keep RTK signaling properly localized. To show this, phospho-tyrosine was used as a read-out of local RTK signaling. Although this reagent is not uniquely specific for the active receptor, the visualized effects of Pvr or Egfr overexpression, Hrs mutation, ligand misexpression, as well as Sprint colocalization, validates its utility in visualizing the high level of local receptor activity found at the leading edge of migrating border cells. The requirement for Cbl and Sprint suggests that the cellular activity required for signal restriction is receptor endocytosis, which is supported by experiments with dominant-negative dynamin. Thus, in this physiological context of guidance by RTKs, receptor endocytosis serves not to downregulate active receptors, but to ensure their correct spatial localization (Jekely, 2005).

The proposed role of RTK endocytosis regulators should be seen in the context of what is already know about RTK signaling and regulation. Signaling from RTKs is initiated upon transphosphorylation of activating tyrosines and docking tyrosines, the latter generating binding sites for PTB and SH2 domain proteins. Receptor activation is elicited by binding of activating ligand but can also occur if two receptor molecules contact each other productively for other reasons. The likelihood of ligand-independent activation depends on receptor density, and hence overexpressed receptors may have ligand-independent activity in addition to responding more strongly to ligands. Inactivation of receptors is therefore critical for proper signaling in the cell. Phosphatases inactivate receptors by catalyzing the reverse reaction of the activation. Phosphatases are very abundant in cells and may be constitutively active. Local inactivation of phosphatases is one mechanism that can lead to spreading of an initially localized RTK signal. In addition, signaling can be inactivated by endocytosis, which leads to degradation of activated receptors, stimulated by molecules such as Cbl and Sprint/RIN1 and at a later step by Hrs (Jekely, 2005).

Most studies of induced endocytosis, in order to give maximal experimental resolution, have been done in tissue culture cells with acute stimulation by high levels of ligand. In tissues, which have steady and modest levels of ligand and a complex, multicellular environment, the role of endocytosis in RTK regulation is less well understood. For example, Egfr signaling is mildly increased in Cbl mutant follicle cell clones, but so mild that even in Cbl, sprint double mutant follicle cells, there are no detectable changes in levels of Egfr, Pvr, or phospho-tyrosine. However, the effects on border cell migration are striking. Receptor proteins do turn over in the tissue and at least some of this turnover is blocked in Hrs mutant cells. However, under physiological conditions in the ovary, the Hrs-dependent degradation is not dependent on ligand and is not required for guided migration. These results show that the physiological role of Cbl and Sprint in border cell guidance is not to control receptor degradation and/or to turn off signaling, but instead to keep the signal localized (Jekely, 2005).

It is becoming appreciated that endocytosis of signaling receptors is not simply a matter of signal attenuation and receptor removal. It was found that RTK endocytosis differentially affected signaling through different pathways, suggesting (1) that signaling can happen in different compartments and (2) that the process of endocytosis could be used to differentially regulate signaling outcomes. For TGF-β signaling, the process of endocytosis actively brings receptors to internal signaling. This study suggests a third role for early aspects of receptor endocytosis in signaling: to keep active signaling complexes localized in the plane of the membrane. This activity prevents signaling from becoming uniform and therefore uninformative about the spatial distribution of the ligand (Jekely, 2005).

How do Cbl and Sprint spatially restrict signaling? They may prevent signaling from becoming delocalized by restricting lateral movement of activated receptors or lateral spread of RTK activation. Microdomains of active RTKs on the plasma membrane or in endocytic pits could maintain activity, whereas they would be inactivated at other places by ubiquitous phosphatases. Alternatively, recycling of activated receptors to new regions of the cell membrane could delocalize signaling. Normally, this recycling might be prevented by Cbl and Sprint activity by routing active RTKs to degradation via the proper endosome compartment (without requiring Hrs). For these two scenarios, however, it is not obvious why physically blocking endocytosis (Shibire dominant-negative) should also delocalize signaling. Shibire/dynamin is a general effector of endocytosis (required for cell viability), and interfering with it therefore is a more blunt tool than manipulating Cbl and Sprint or mutating Pvr. But the effects are unambiguous. This leads to a third hypothesis, whereby endocytosis of active RTKs allows their redelivery or recycling to regions of higher signaling. Endocytosis and plasma membrane redelivery of active proteins contributes to polarization in yeast, another case of controlling spatial information. Obviously, further analysis will be needed to fully explore these cellular mechanisms in vivo. In any case, at sufficiently high level of receptor expression and activation, the regulatory mechanism may collapse. Indeed, when a C-terminally tagged Egfr was expressed at extremely high levels in border cells, migration and phospho-tyrosine staining were perturbed in a manner similar to what was observed for Cbl mutant clones. Like many regulatory mechanisms, the spatial restriction imposed by Cbl and Sprint works effectively within a certain range of input, emphasizing the need for understanding the mechanism of regulation at a physiological range in vivo (Jekely, 2005).

The role of early endocytosis regulators in spatial signal regulation described in this study is clearly physiologically relevant. But how general might it be? The regulation is likely to be relevant when RTKs are used for spatially resolved signaling. RTKs can act as canonical guidance receptors to detect specific ligands. In addition, mammalian Cbl is required for integrin-dependent migration of macrophages and osteoclasts in vitro and in vivo. This requirement was suggested to reflect an active signaling role of Cbl downstream of integrins, but it could also reflect a role for Cbl in localizing signaling analogous to what was seen in border cells. There is ample evidence for cross-talk between integrins and RTKs, which in turn are regulated by Cbl. That RTKs can be activated in the absence of cognate ligand by high receptor density or by cross-talk from other pathways such as integrins might seem at odds with their serving as guidance receptors. But with effective regulatory mechanisms to maintain localized signaling, this excitable signaling system may help migrating cells obtain sufficient sensitivity to read guidance cues over a large dynamic range (Jekely, 2005).

A key issue in guidance signaling is that migrating cells must achieve a polarized output despite having to respond, over a large dynamic range, to subtle concentration differences of an attractant or repellant from one end of the cell to the other. One way to achieve this is to amplify the initial signal difference between stimulation of receptors at the front and back of the cell. The use of PI3 kinase and PTEN phosphatase, two antagonistic enzymes that are reciprocally regulated in Dictyostelium chemotaxis, may be an example of this. Alternatively, guidance cues may simply bias a separate, preexisting intrinsic polarity in the migrating cells. Finally, guidance signaling and intrinsic polarity may interact dynamically to reinforce one another during directed migration. The net outcome is a robust difference between the front and the back of the cell, allowing migration. It is interesting to consider that RTK endocytosis may also function to enhance the difference between signaling in the front and back of migratory border cells. Although the gradient of RTK ligands around border cells can not be measured, it is very unlikely to be as steep as the observed difference in phospho-tyrosine staining. Enhancement of a signaling differential can occur at different levels. Binding of Cbl and Sprint to activated RTKs and recruitment to membrane subdomains may effectively concentrate activated receptors. Due to the density dependence and positive feedback in RTK activation, local activity will then be increased, whereas global inactivation by phosphatases could ensure that signaling is reduced elsewhere. It is suggested that spatial organization of signaling may be controlled by endocytosis and redelivery of active receptors. Such active turnover processes can be used as an effective mechanism to increase signaling differentials within a cell, leading even to spontaneous, or self-organized, polarity. A distinct cell front and cell rear and hence short-term productive migration is often seen in migrating cells even without perception of localized guidance cues, indicating intrinsic polarity. Most migrating cells may need to integrate intrinsic polarity and external guidance. Using a regulatory principle that can produce both intrinsic polarity and local response to guidance cues would provide an elegant means to achieve this (Jekely, 2005).

The tumor suppressors Merlin and Expanded function cooperatively to modulate EGFR and Notch endocytosis and signaling

The precise coordination of signals that control proliferation is a key feature of growth regulation in developing tissues. While much has been learned about the basic components of signal transduction pathways, less is known about how receptor localization, compartmentalization, and trafficking affect signaling in developing tissues. This paper examines the mechanism by which the Drosophila Neurofibromatosis 2 (NF2) tumor suppressor ortholog Merlin (Mer) and the related tumor suppressor expanded (ex) regulate proliferation and differentiation in imaginal epithelia. Merlin and Expanded are members of the FERM (Four-point one, Ezrin, Radixin, Moesin) domain superfamily, which consists of membrane-associated cytoplasmic proteins that interact with transmembrane proteins and may function as adapters that link to protein complexes and/or the cytoskeleton. Merlin and Expanded function to regulate the steady-state levels of signaling and adhesion receptors, and loss of these proteins can cause hyperactivation of associated signaling pathways. In addition, pulse-chase labeling of Notch in living tissues indicates that receptor levels are upregulated at the plasma membrane in Mer; ex double mutant cells due to a defect in receptor clearance from the cell surface. It is proposed that these proteins control proliferation by regulating the abundance, localization, and turnover of cell-surface receptors and that misregulation of these processes may be a key component of tumorigenesis (Maitra, 2006).

Merlin's tumor suppressor function is conserved from humans to flies, but the cellular basis for this function remains unclear. Genetic studies in Drosophila suggest that Mer regulates signaling pathways that control proliferation, and cell biological experiments indicate that Merlin may play a role in endocytic processes. In addition, Merlin physically interacts with Expanded, a distantly related member of the FERM superfamily, and these proteins colocalize in the apical junctional region of epithelial cells. Furthermore, genetic studies have shown that while mutations of each gene produce modest overproliferation phenotypes in the eye and wing, double mutant Mer; ex cells display severe overgrowth and differentiation defects that are not seen in either mutation alone. Thus, Mer and ex are partially redundant in regulating proliferation and differentiation (Maitra, 2006).

Given these observations, it was reasoned that the difficulty in identifying precise cellular functions for Merlin might stem from its redundancy with Expanded and that this difficulty could be overcome by examining tissues from double mutant animals and double mutant cell clones generated by somatic recombination. Overproliferation of Mer; ex wing imaginal discs is more extreme than that observed with either mutation alone. Surprisingly, however, Mer4; ex697 eye-antennal imaginal discs have severely reduced eye primordia with a substantial reduction in or total absence of photoreceptors, although the antennal portion is normal or slightly larger than normal and occasionally is duplicated. Apoptosis does not appear to be enhanced in double mutant eye-antennal discs, suggesting that loss of the eye primordium is not due to cell death. Thus, loss of Mer and ex function has a tissue-specific defect in the developing eye that is very different from its effects on proliferation in the wing imaginal disc (Maitra, 2006).

Why does the combined loss of two tumor suppressors cause reduction rather than hypertrophy of eye tissue? Previous studies have shown that initiation of the morphogenetic furrow, which organizes development of the eye, is regulated by a complex network of signals at the posterior and lateral margins of the eye-antennal disc. Mutations that affect these signals not only block furrow initiation, but also may significantly reduce the size of the eye field and disrupt photoreceptor differentiation. For example, ectopic Wingless expression either at the posterior and lateral margins or throughout the eye primordium results in dramatic losses of eye tissue that closely resemble the Mer; ex phenotype just described. Similar effects are seen from reduction in Decapentaplegic (DPP) or Hedgehog signaling in the same cells (Maitra, 2006).

If Merlin and Expanded affect initiation of the morphogenetic furrow rather than differentiation of photoreceptors, then Mer; ex double mutant somatic clones should block ommatidial development only when present at the posterior or lateral margins of the eye field. Indeed, Mer; ex clones could differentiate photoreceptors, but only when located in the middle of the eye field. In contrast, clones in contact with the posterior or lateral margin of the eye fail to produce photoreceptors. It is inferred from these observations that one or more of the signaling pathways that control initiation of the morphogenetic furrow are likely disrupted in Mer; ex double mutant cells (Maitra, 2006).

Given that Merlin is associated with the plasma membrane and may function in endocytic processes, it was asked if Merlin and Expanded play a role in regulating localization and/or abundance of transmembrane receptors that function in eye development. For these studies, Mer; ex somatic mosaic cell clones were examined to allow side-by-side comparisons of wild-type and mutant cells in the wing and eye imaginal discs. Immunofluorescence staining with specific antibodies then allowed comparison of the steady-state levels of receptors between adjacent wild-type and mutant cells. Intriguingly, Notch, the EGF receptor, Patched, and Smoothened all displayed increased antibody staining in double mutant cells relative to their wild-type neighbors. Notch, which is primarily localized to the apical junctional domain in wild-type cells, showed not only increased junctional staining in mutant cells, but also more diffuse staining. Similarly, preparations with anti-EGFR display more abundant membrane-associated and cytoplasmic staining in mutant than in wild-type cells. Patched staining, which is less obviously junctional than Notch or EGFR, appeared more punctate in Mer; ex cells. Thus, simultaneous loss of Merlin and Expanded results in increased abundance of receptors for multiple signaling pathways, though the precise localization defect seems to be specific to each receptor. Two adhesion-related receptors, E-cadherin and Fat, a cadherin superfamily member, were examined; both are similarly upregulated in Mer; ex cells. However, Coracle, a membrane-associated cytoplasmic protein, is not affected. In addition, the localization of markers for apical-basal polarity, including DLG, PATJ, and aPKC, was unaffected in the double mutant cells, indicating that epithelial polarity is not disrupted. In contrast to the double mutant cells, clones lacking just Merlin show no apparent difference in receptor localization or abundance, and exe1 cells display only a slight increase in staining. Taken together, these results indicate that Merlin and Expanded are required to reduce the steady-state abundance of a variety of signaling and adhesion receptors in developing epithelia (Maitra, 2006).

Membrane trafficking was examined in Mer; ex double mutant cells. Antibodies were used against the extracellular domain of Notch (anti-ECN) to label protein on the surface of living cells in imaginal discs bearing somatic mosaic clones. Side-by-side comparisons of wild-type and Mer; ex mutant cells show increased cell-surface Notch labeling, consistent with what was observed with fixed tissue and indicating that there are increased levels of receptor at the plasma membrane in mutant cells. In addition, in double mutant cells, the junctional band of Notch staining is broader, indicating that Notch localization to the junctional region also may be affected. Similar differences in junctional staining were observed with the same antibody on fixed and permeabilized tissues, indicating that surface labeling of live cells does not affect Notch localization (Maitra, 2006).

To ask if the increased abundance is due to a defect in turnover, a pulse-chase approach was used to label Notch receptor at the plasma membrane and then its removal from the cell surface was followed. To restrict analysis to Notch that remains at the cell surface, tissues were fixed but not permeabilized at the end of the chase period. A progressive loss was observed of Notch staining at the cell surface during the chase period that appeared more rapid in wild-type than in mutant cells, suggesting a defect in trafficking off the plasma membrane. Quantitative fluorescence analysis was used to determine the relative quantities of Notch on wild-type and mutant cells at the various chase time points. The results indicate that the ratio of cell-surface Notch fluorescence in mutant versus wild-type cells increases significantly between 0 and 10, 30, or 60 min postlabeling. Therefore, Notch protein is cleared more rapidly from the surface of wild-type than mutant cells (Maitra, 2006).

It is worth noting that current models for Notch receptor activation require cleavage and release of its extracellular domain in response to ligand binding. Because an antibody was used that recognizes this domain, it follows that these studies examined only ligand-independent trafficking of the receptor. In support of this inference, the pattern of Notch internalization in pulse-chase experiments was unaffected in Delta clones. These observations suggest that Merlin and Expanded function in steady-state, ligand-independent clearance of receptors from the plasma membrane, rather than internalization and degradation that occurs in response to ligand binding (Maitra, 2006).

Increased receptor abundance may be expected to result in increased signaling output, if receptor quantity is a limiting factor. In addition, even if overall receptor quantity is not limiting, alterations in subcellular localization or the dynamics of receptor trafficking may have dramatic effects on receptor function. To ask if loss of Merlin and Expanded result in increased output from signaling pathways that regulate eye development and cell proliferation, markers specific for downstream activation of the EGFR, Wingless, and Notch signaling pathways were used. First, double mutant clones were stained with an antibody that recognizes the phosphorylated, activated form of MAP kinase (anti-dpERK), a downstream effector of the EGFR pathway. In addition to the normal anti-dpERK pattern in the wing imaginal disc, increased staining was observed in Mer; ex clones relative to their wild-type neighbors, suggesting upregulation of EGFR pathway activity. Similarly, output from the Wingless pathway was monitored by looking at expression of Distalless, a target of Wingless signaling and it was found to be dramatically higher in the double mutant wing clones. In contrast, similar experiments with the mAb323 antibody to E(spl) bHLH proteins, a marker for Notch pathway activity, did not show upregulation of Notch signaling. This result is consistent with the observation that overexpression of Notch in a wild-type genetic background has little or no phenotype. To examine this further, a genetic context was analyzed in which Notch receptor quantities are known to be limiting, that is, in animals that are heterozygous for a null Notch mutation. Such animals display a dominant, haploinsufficient phenotype characterized by notching along the wing margin. To ask if reduction in Merlin and Expanded in this context can cause upregulation of Notch pathway output, animals triply heterozygous for Notch, Merlin, and expanded were generated and it was found that the characteristic Notch wing phenotype was strongly suppressed (Maitra, 2006).

Taken together, these results are consistent with the observation that the steady-state level of multiple receptors is elevated in Mer; ex cells and indicate that, depending on the precise developmental or genetic context, loss of Merlin and Expanded can result in increased output from the corresponding signaling pathways. In Mer; ex eyes, upregulation of Wingless signaling may be a primary contributor to the observed defect in ommatidial development. Previous studies have shown that ectopic Wingless signaling produces remarkably similar eye phenotypes, and preliminary data suggest that inhibiting Wingless signaling partially suppresses the Mer; ex eye phenotype. In the wing, the dramatic overproliferation of Mer; ex cells may be the combined result of upregulation of several pathways, including EGFR and Wingless (Maitra, 2006).

Merlin and Expanded are associated with the apical junctional region in imaginal epithelia and with endocytic vesicles in cultured cells. Results shown in this study indicate that loss of these proteins affects abundance, cell-surface localization, and endocytic trafficking of Notch, EGFR, and other signaling and adhesion receptors in epithelial cells. Recent studies of endocytic trafficking in receptor/ligand regulation suggest aspects of endocytosis that could relate to Merlin and Expanded function. For example, it is possible that Merlin and Expanded function at the plasma membrane to recruit or anchor transmembrane proteins at sites on the membrane from which they are endocytosed or in the sorting between recycling endosomes and lysosomal degradation by promoting receptor degradation. Both possibilities are consistent with observations of increased receptor levels at the plasma membrane in Mer; ex mutant cells and colocalization of Merlin and Expanded with Notch in punctate structures at the plasma membrane. In addition, a partial colocalization was observed of Merlin and Expanded with Rab 11, a marker for recycling endosomes, and with EEA-1, which labels early endosomes. Intriguingly, it has been suggested that the closely related ERM protein Ezrin functions to promote recycling rather than degradation of the β2-adrenergic receptor via its interactions with filamentous actin. Understanding the exact relationship of Merlin and Expanded to endocytosis and recycling of receptors, as well as their possible relationship to ERM proteins in this process, will require further analysis (Maitra, 2006).

A recent study has proposed that Merlin and Expanded function upstream of Hippo in the Warts signaling pathway, which regulates proliferation. Merlin and expanded mutants display similar phenotypes to those seen in hippo mutants. However, there are significant phenotypic differences between Mer; ex and hippo mutations, most notable of which is that hippo mutations have not been reported to block induction of eye morphogenesis. In addition, there is no evidence to suggest that the Hippo pathway regulates output of the EGFR, Wingless, or Notch signaling pathways. Thus, the relationship of Merlin and Expanded to the Hippo pathway may be more complicated than the linear pathway proposed. One possibility is that Hippo activation is a downstream consequence of Merlin and Expanded's effects on output of multiple signaling pathways (Maitra, 2006).

More than a decade after its molecular characterization, the precise cellular functions of Merlin in regulating cell proliferation remain unclear. Based on the current studies, it is proposed that Merlin's tumor suppressor phenotype results from defects in endocytic trafficking of signaling receptors and accompanying hyperactivation of associated signaling pathways. Recent studies highlight the importance of endocytosis in regulation of signaling pathways. Based on the results presented in this study, it is suggested that proper regulation of membrane trafficking also may have important implications for understanding the cellular basis of tumor suppression in flies and mammals (Maitra, 2006).

Abi induces ectopic sensory organ formation by stimulating EGFR signaling

One of the central regulators coupling tyrosine phosphorylation with cytoskeletal dynamics is the Abelson interactor (Abi). Its activity regulates WASP-/WAVE mediated F-actin formation and in addition modulates the activity of the Abelson tyrosine kinase (Abl). Drosophila Abi is capable of promoting bristle development in a wasp dependent fashion. This study reports that Drosophila Abi induces sensory organ development by modulating EGFR signaling. Expression of a membrane-tethered activated Abi protein (AbiMyr) leads to an increase in MAPK activity. Additionally, suppression of EGFR activity inhibits the induction of extra-sensory organs by AbiMyr, whereas co-expression of activated AbiMyr and EGFR dramatically enhances the neurogenic phenotype. In agreement with this observation Abi is able to associate with the EGFR in a common complex. Furthermore, Abi binds the Abl tyrosine kinase. A block of Abl kinase-activity reduces Abi protein stability and strongly abrogates ectopic sensory organ formation induced by AbiMyr. Concomitantly, changes were noted in tyrosine phosphorylation supporting previous reports that Abi protein stability is linked to tyrosine phosphorylation mediated by Abl (Stephan, 2008).

A variety of morphogenetic events during metazoan development such as cell fate determination, proliferation and differentiation require a highly regulated network of signaling pathways resulting in distinct cellular responses. Cell-surface receptors and tyrosine kinases often play an important role in the control of these processes. Among the growth factor receptors, the best understood example is the epidermal growth factor receptor (EGF receptor), which plays important roles in physiological and pathological processes. Unlike the vertebrate homologues the Drosophila genome contains only a single gene encoding a member of the EGF receptor family, called DER/EGFR. In Drosophila, the EGFR pathway is required at multiple times during fly development such as oogenesis, axis determination and sensory organ formation during embryonic and imaginal disc development (Stephan, 2008).

The emergence of Drosophila sensory organs depends on a selection process called lateral inhibition where only one cell is singled out of a group of equivalent proneural cells to become a sensory organ precursor (SOP). The Drosophila notum bears many mechano-sensory bristles, 22 large macrochaetes and about 200 smaller microchaetes. In both cases, bristle formation and spacing requires cell–cell communication mediated by lateral inhibition. The microchaetes on the notum are arranged in rows and are variable in number and position within the rows. Unlike the microchaetes, the large macrochaetes are not arranged in rows, their number is constant and they are formed from proneural clusters at fixed stereotypic positions (Stephan, 2008 and references therein).

Once determined, the SOP undergoes a series of stereotyped asymmetric cell divisions giving rise to the different cells comprising an individual sensory organ. In Drosophila, two major antagonistic signaling pathways regulate the formation of SOPs, the Notch and the EGFR signaling pathways. Notch is pivotal for the process of lateral inhibition which restricts the formation of neural cells. Alternatively, activation of the EGF receptor (EGFR) positively regulates SOP formation. Loss of EGFR function leads to a loss of sensory macrochaetes whereas gain of EGFR-function evokes the formation of additional macrochaetes by stimulating proneural gene autoregulation (Stephan, 2008).

Upon EGFR activation in Drosophila a membrane-localized protein complex is formed acting through the Ras-signaling. The activation of Ras results in the translocation of di-phosphorylated MAPK from the cell surface to the nucleus, where it leads to the expression of the proneural gene scute, which in turn promotes sensory organ development (Stephan, 2008).

This study shows that Abi regulates sensory organ development by modulating EGFR signaling. Following expression of an activated Abi (AbiMyr) in proneural clusters, an increase was noted in MAPK activity. Inhibition of the EGFR pathway suppresses the formation of ectopic sensory organs evoked by AbiMyr. Furthermore, co-expression of activated AbiMyr and EGFR dramatically enhances the induction of extra-sensory organs. This effect also depends on the presence of active Abl tyrosine kinase, which stabilizes and further stimulates Abi by tyrosine phosphorylation (Juang, 1999). The physical interaction of Abi and EGFR depends on the membrane recruitment whereas the ability of the complex to induce neural development depends on both the membrane localization and the C-terminal SH3 domain of Abi, which can bind Wasp as well as the Abl kinase. These data suggest that once Abi is recruited to the membrane, EGFR signaling becomes activated to promote SOP development (Stephan, 2008).

Thus, overexpression of the Abelson kinase and its activator Abi is sufficient to promote the formation of ectopic sensory organs. Previous work has shown a partial loss of sensory organs in adult flies expressing a double-stranded abi RNA (Bogdan, 2005). However, expression of double-stranded RNA can lead to off target effects and confirmation of the mutant phenotypes by genetic analysis is needed. To address this question abi null alleles were generated. However, animals lacking zygotic abi function die relatively late during development as early pupae and no defects can be noted in the SOP pattern. This suggests that either abi is not required for SOP development or that its function is obscured by maternal contributions. In support for this notion, residual Abi protein can be detected in homozygous abi mutant larvae. In addition as previously reported for the abl kinase gene, abi might be integrated into a complex genetic network that masks direct requirements. The role of Abl is only exposed by the simultaneous loss of interaction partners such as disabled or failed axon connections (Stephan, 2008).

The Abi proteins are versatile molecules comprised of multiple protein-protein interaction domains allowing the formation of different protein complexes. Abi does not only function as an activator of the Abelson kinase but in turn Abl positively regulates Abi stability through phosphorylation (Juang, 1999; Huang, 2007). Moreover Abi plays an important role in regulating F-actin dynamics by activating Wave and Wasp proteins, two main activators of Arp2/3-dependent actin polymerization (Stephan, 2008 and references therein).

How can alterations in actin dynamics modulate EGFR signaling? Previous studies have shown that cytoskeletal rearrangements are likely to induce the asymmetric distribution and clustering of the EGFR in neuronal stem cells to generate diverse CNS progenitor cells. Since Abi functions as a potent activator of Wasp (Bogdan, 2005; Innocenti, 2005), the enhanced formation of F-actin may lead to clustering of the EGF receptor at the membrane to stimulate its sustained activation (Stephan, 2008).

The enhancement of actin polymerization may also affect endocytosis of the EGFR, which is F-actin dependent and as recently shown can be promoted by Abi and Wasp activity. Although it has long been assumed that endocytosis is the main pathway involved in down-regulation of the EGFR, endocytosis is also directly linked to EGFR activation. Block of endocytosis of the EGFR specifically abrogates MAPK activity. Thus Abi could be involved establishing active signaling compartments. Unfortunately, at present the molecular tools to determine the subcellular localization of the activated EGFR in the developing fly are unavailable (Stephan, 2008).

In addition, activation of the Abi complex may recruit kinases such as Abl to the EGF receptor. Abi acts as a positive regulator of the Abl kinase activity promoting the phosphorylation of several substrates including Mena/Ena, Wave, N-Wasp, the B-cell adaptor for phosphoinositide 3-kinase (BCAP) or Cdc2. Interestingly, Abi is also a substrate of Abl resulting in increased protein stability. This positive regulatory loop could hint to an important mechanism for regulating the level of Abl kinase activity during sensory organ development. Indeed, co-expression of AbiMyr with a kinase-defective Abl suppresses tyrosine hyperphosphorylation as well as reduces the level of the AbiMyr protein expression, corresponding to a strong suppression of the number of ectopic sensory organs induced by AbiMyr (Stephan, 2008).

However, the induction of ectopic sensory bristles by Abl does not depend on an intact EGFR signaling or on an activation of the Ras/Raf/MAPK pathway. Co-expression of Abl and EGFR results in an increase of sensory organs but inhibition of EGFR by co-expression of Kekkon1 or heterozygous loss of pole hole function are not sufficient to suppress the Abl-induced neurogenic phenotype suggesting that Abi and Abl act in parallel. In conclusion, it is proposed that the complex of Abi and the EGFR is joined by Abl, which stabilizes not only Abi but also stimulates Wasp function, highlighting the intricate interaction between cytoskeletal dynamics and signaling during sensory organ development (Stephan, 2008).

Myopic acts in the endocytic pathway to enhance signaling by the Drosophila EGF receptor

Endocytosis of activated receptors can control signaling levels by exposing the receptors to novel downstream molecules or by instigating their degradation. Epidermal growth factor receptor (EGFR) signaling has crucial roles in development and is misregulated in many cancers. Myopic, the Drosophila homolog of the Bro1-domain tyrosine phosphatase HD-PTP, promotes EGFR signaling in vivo and in cultured cells. myopic is not required in the presence of activated Ras or in the absence of the ubiquitin ligase Cbl, indicating that it acts on internalized EGFR, and its overexpression enhances the activity of an activated form of EGFR. Myopic is localized to intracellular vesicles adjacent to Rab5-containing early endosomes, and its absence results in the enlargement of endosomal compartments. Loss of Myopic prevents cleavage of the EGFR cytoplasmic domain, a process controlled by the endocytic regulators Cbl and Sprouty. It is suggested that Myopic promotes EGFR signaling by mediating its progression through the endocytic pathway (Miura, 2008).

The Epidermal growth factor receptor (EGFR) is required for cell differentiation and proliferation in numerous developmental systems, and activation of the human EGFR homologs, ERBB1-4, is implicated in many cancers. EGFR signaling events are terminated following removal of the receptor from the cell membrane by endocytosis. Ubiquitylation of EGFR by Cbl, an E3 ubiquitin ligase, initiates its internalization into clathrin-coated vesicles and its transit through early and late endosomes, which differ by the exchange of Rab7 for Rab5. EGFR can either return to the cell surface in Rab11-containing recycling endosomes, or reach the lysosome for degradation. Delivery of receptors to the lysosome requires sorting from the limiting membrane of late endosomes into the internal vesicles of multivesicular bodies (MVBs), a process mediated by four endosomal sorting complexes required for transport (ESCRT-0, I, II and III). Ubiquitylated receptors are bound by Hepatocyte growth factor regulated tyrosine kinase substrate (Hrs) in ESCRT-0, Tumor susceptibility gene 101 (Tsg101; also known as Erupted) in ESCRT-I and Vacuolar protein sorting 36 (Vps36) in ESCRT-II, and their deubiquitylation and internalization are coordinated by ESCRT-III (Miura, 2008).

Genetic or pharmacological blocks of endocytosis prevent degradation of EGFR and other receptors. In Drosophila, Hrs mutations block MVB invagination, trapping receptor tyrosine kinases (RTKs) and other receptors on the outer membrane of the MVB, and sometimes leading to enhanced signaling. Mutations in the ESCRT complex subunits Tsg101 (ESCRT-I) and Vps25 (ESCRT-II) cause overproliferation owing to the accumulation of mitogenic receptors such as Notch and Thickveins. In mammalian cells, loss of Hrs (also known as Hgs) or Tsg101 results in increased EGFR signaling. However, other studies have demonstrated a positive role for endocytosis in receptor signaling. Mutations affecting the Drosophila trafficking protein Lethal giant discs dramatically increase Notch signaling only in the presence of Hrs, indicating that signaling is maximized at a specific point in the endocytic process. Wingless (Wg) signaling is enhanced by internalization into endosomes, where it colocalizes with downstream signaling molecules. In mammalian cells, EGFR encounters the scaffolding proteins Mek1 partner (Mp1) and p14, which are required for maximal phosphorylation of the downstream component mitogen-activated protein kinase (MAPK), only on endosomes (Miura, 2008).

The characterization of the novel Drosophila gene myopic (mop) is described here. Loss of mop affects EGFR-dependent processes in eye and embryonic development, and reduces MAPK phosphorylation by activated EGFR in cultured cells. Mop acts upstream of Ras activation to promote the function of activated, internalized EGFR. Mop is homologous to human HD-PTP (PTPN23 - Human Gene Nomenclature Database) (Toyooka, 2000), which contains a Bro1 domain that is able to bind the ESCRT-III complex component SNF7 (CHMP4B - Human Gene Nomenclature Database) (Ichioka, 2007; Kim, 2005) and a tyrosine phosphatase domain. Mop is present on intracellular vesicles, and cells lacking mop have enlarged endosomes and reduced cleavage of the EGFR cytoplasmic domain. It is proposed that Mop potentiates EGFR signaling by enhancing its progression through endocytosis. Consistent with this hypothesis, it was found that components of the ESCRT-0 and ESCRT-I complexes are also required for EGFR signaling in Drosophila cells (Miura, 2008).

This study shows that Mop is necessary for EGFR signaling in vivo and in cultured cells. Mop is located on endosomes and affects endosome size, promotes cleavage of EGFR and lysosomal entry of its ligand, and is not required in the absence of the Cbl or Sty proteins that regulate endocytic trafficking of EGFR. These data suggest that Mop enhances EGFR signaling by facilitating its progression through the endocytic pathway. Consistent with this model, Hrs and ESCRT-I subunits also have a positive effect on EGFR signaling (Miura, 2008).

The Bro1 domain of yeast Bro1 is sufficient for localization to late endosomes through its binding to the ESCRT-III subunit Snf7 (Kim, 2005), and this domain is present in many proteins involved in endocytosis. Bro1 itself is required for transmembrane proteins to reach the vacuole for degradation; it promotes protein deubiquitylation by recruiting and activating Doa4, a ubiquitin thiolesterase. Since mutations in the E3 ubiquitin ligase gene Cbl can rescue mop mutant clones, recruiting deubiquitylating enzymes might be one of the functions of Mop. The vertebrate Bro1-domain protein Alix (also known as AIP1 and Pdcd6ip) inhibits EGFR endocytosis by blocking the ubiquitylation of EGFR by Cbl, and by preventing the binding of Ruk (Sh3kbp1), which recruits endophilins, to the EGFR-Cbl complex. However, CG12876, not Mop, is the Drosophila ortholog of Alix (Miura, 2008).

A closer vertebrate homolog of Mop, which has both Bro1 and tyrosine phosphatase domains, has been named HD-PTP in human (Toyooka, 2000) and PTP-TD14 in rat. HD-PTP shares with Alix the ability to bind Snf7 and Tsg101, but does not bind to Ruk (Ichioka, 2007). PTP-TD14 was found to suppress cell transformation by Ha-Ras, and required phosphatase activity for this function (Cao, 1998). The activity of Mop described in this study appears distinct in that Mop acts upstream of Ras activation, and no requirement was demonstrated for the catalytic cysteine in its predicted phosphatase domain. If Mop does act as a phosphatase, Hrs would be a candidate substrate because tyrosine phosphorylation of Hrs by internalized receptors promotes its degradation, and Hrs levels appear reduced in mop mutant clones (Miura, 2008).

Endocytosis has been proposed to play several different roles in receptor signaling. Most commonly, endocytosis followed by receptor degradation terminates signaling. However, endocytosis can also prolong the duration of signaling or influence its subcellular location. Receptors may also signal through different downstream pathways localized to specialized endosomal compartments (Miura, 2008).

Genetic studies in Drosophila have emphasized the importance of endocytic trafficking for receptor silencing. Mutations in Hrs, Vps25 or Tsg101 result in the accumulation of multiple receptors on the perimeter membrane of the MVB, leading to enhanced signaling. Depletion of Hrs or Tsg101 in mammalian cells also results in increased EGFR signaling, although the two molecules have distinct effects on MVB morphology. By contrast, this study found that mop and Hrs mutants exhibit diminished EGFR signaling in vivo, and depletion of mop, Tsg101 or Vps28 reduces EGFR signaling in S2 cells. Progression through the endocytic pathway may thus be required for maximal EGFR signaling, at least in some contexts (Miura, 2008).

Several possible mechanisms could explain such a requirement for endocytic progression. MAPK phosphorylation may be enhanced in the presence of signaling components present on late endosomes. Cleavage of the EGFR cytoplasmic domain, which requires Mop activity, might enhance EGFR signaling. The cleaved intracellular domain of ErbB4 has been shown to enter the nucleus and regulate gene expression (Sardi, 2006), suggesting the possibility that Mop affects a nuclear function of EGFR in addition to promoting MAPK phosphorylation. Alternatively, the reduction in EGFR signaling in mop mutants could be due to a failure to recycle the receptor to the cell surface. Mutations in the yeast Vps class C genes, which are required for trafficking to late endosomes, also prevent the recycling of cargo proteins. Recycling is essential for EGFR-induced proliferation of mammalian cells, and may promote the localized RTK signaling that drives directional cell migration (Miura, 2008).

Despite the reduction in EGFR signaling in mop mutants, signaling by other receptors such as Notch, Smoothened and Torso is unaffected. This phenotypic specificity could be due to a dedicated function of Mop in the EGFR pathway, or to high sensitivity of EGFR signaling to a general process that requires Mop. Although the Mop-related protein Alix has been found in a complex with EGFR (Schmidt, 2004), no physical interaction of Mop with EGFR could be detected. The function of mop is not limited to promoting EGFR signaling; it also promotes trafficking of Wg and expression of the Wg target gene sens. In addition, mop is required for normal cellularization of the embryo, and its cellularization phenotype is not rescued by removal of Cbl(Miura, 2008).

Additional studies will be required to determine whether all endosomes, or only a specific subclass, are affected by mop. Interestingly, EGF treatment of mammalian cells induces EGFR trafficking through a specialized class of MVBs. Although significant colocalization of activated EGFR with Mop was not seen, EGFR may transiently pass through Mop-containing endosomes before accumulating in another compartment. The wing disc appears less sensitive than the eye disc to the effect of mop on EGFR signaling. This might be due to differences in the endogenous levels of Cbl or other mediators of EGFR internalization, or in the strength or duration of signaling necessary to activate target genes, or to the use of a different ligand with distinct effects on receptor trafficking (Miura, 2008).

Taken together, these results identify a positive role for progress through the endocytic pathway and for the novel molecule Mop in EGFR signaling in Drosophila. The importance of upregulation of the trafficking proteins Rab11a, Rab5a and Tsg101 for EGFR signaling in hepatomas and breast cancers highlights the potential value of specific effectors of EGFR endocytosis as targets for anti-cancer therapies (Miura, 2008).

Receptor tyrosine phosphatases control tracheal tube geometries through negative regulation of Egfr signaling

The formation of epithelial tubes with defined shapes and sizes is essential for organ development. This study describes a unique tracheal tubulogenesis phenotype caused by loss of both Drosophila type III receptor tyrosine phosphatases (RPTPs), Ptp4E and Ptp10D. Ptp4E is the only widely expressed Drosophila RPTP, and is the last of the six fly RPTPs to be genetically characterized. Mutations have been isolated in in Ptp4E; although Ptp4E null mutants have no detectable phenotypes, double mutants lacking both Ptp4E and Ptp10D display synthetic lethality at hatching owing to respiratory failure. In these double mutants, unicellular and terminal tracheal branches develop large bubble-like cysts that selectively incorporate apical cell surface markers. Cysts in unicellular branches are enlargements of the lumen that are sealed by adherens junctions, whereas cysts in terminal branches are cytoplasmic vacuoles. Cyst size and number are increased by tracheal expression of activated Egfr tyrosine kinase, and decreased by reducing Egfr levels. Ptp10D forms a complex with Egfr in transfected cells. Downregulation of Egfr signaling by the RPTPs is required for the construction of tubular lumens, whether extracellular or intracellular, by cells that undergo remodeling during branch morphogenesis. The Ptp4E Ptp10D phenotype represents the first evidence of an essential role for RPTPs in epithelial organ development. These findings might be relevant to organ development and disease in mammals, because PTPRJ (DEP-1), an ortholog of Ptp4E/Ptp10D, interacts with the hepatocyte growth factor receptor tyrosine kinase. PTPRJ corresponds to the murine Scc1 (suppressor of colon cancer) gene (Jeon, 2009).

This paper analyzes the phenotypes of Drosophila embryos lacking both type III RPTPs, Ptp10D and Ptp4E. Ptp10D regulates axon guidance in embryos and larvae. It is selectively expressed on CNS axons and tracheal branches, and is apically localized in tracheal cells (Jeon, 2009).

Ptp4E is broadly expressed, and Ptp4E single mutants have no detectable phenotypes. The Ptp10D/Ptp4E gene pair was generated by a relatively recent duplication event, and the two proteins are closely related to each other. Although Ptp10D and Ptp4E single mutants are viable and fertile, double mutants die at the end of embryogenesis. These data show that Ptp10D and Ptp4E have partially redundant functions during development. The lethality of the double mutants is due to respiratory failure that results from the malformation of tracheal branches (Jeon, 2009).

The Drosophila tracheal system consists of a network of tubes with different sizes and architectures. The larger tubes are multicellular, and have lumens surrounded by several cells that are separated by intercellular junctions. Smaller tracheal branches are composed of unicellular tubes with autocellular AJs. The lumen of a unicellular tube is surrounded by the apical surface of a single cell, and is sealed by a longitudinal AJ. The smallest tubes, called tracheoles, are intracellular, seamless structures that form within the cytoplasm of terminal tracheal cells (Jeon, 2009).

Loss of both type III RPTPs alters the shape of tracheal lumens, so that unicellular and seamless tubes develop large bubble-like cysts. Multicellular tubes, however, are unaffected. This is a unique phenotype that has never been seen in any single mutant. The cysts are modified apical compartments, because they selectively incorporate apical but not basolateral cell surface markers (Jeon, 2009).

Cysts in seamless tubes are necessarily intracellular compartments. Cysts in unicellular tubes, however, are expanded segments of lumen, because they are associated with single AJs. Accumulation of luminal matrix markers in unicellular tubes is reduced in double mutants, suggesting that they have a selective defect in lumen formation in certain tube types (Jeon, 2009).

RPTPs are regulators of tyrosine kinase signaling pathways. By examining the effects of manipulating tyrosine kinase activity on the cyst phenotype, this study showed that the Egfr receptor tyrosine kinase is central to cyst formation. Increasing Egfr activity enhances the cyst phenotype, while reducing Egfr suppresses it. The Btl (FGF receptor) tyrosine kinase also contributes to the phenotype. Egfr physically associates with Ptp10D in cultured Drosophila cells (Jeon, 2009).

The data suggest that the phosphatases are negative regulators of Egfr signaling, and that the loss of this negative regulation is partially responsible for the cystic phenotype of Ptp4E Ptp10D double mutants. Cysts are not observed when activated Egfr (EgfrElp) is expressed in tracheae in a wild-type background, but it is not known if Egfr is activated to the same extent by the Elp mutation as it is by loss of the RPTPs. If it is, then excess tyrosine phosphorylation of RPTP substrates in other signaling pathways, perhaps including the FGF receptor pathway, might be required together with Egfr upregulation in order to produce the cyst phenotype (Jeon, 2009).

Ptp10D and Ptp4E, which have catalytic domain sequences that are 89% identical, are likely to dephosphorylate the same targets, explaining why the phenotype is observed only when both RPTPs are absent. Ptp10D forms a complex with Egfr, and it is likely that the closely related Ptp4E would do so as well. Studies in mammalian cell culture have shown that the growth factor receptor tyrosine kinase Met can be dephosphorylated by the type III RPTP DEP-1, which is 50% identical to Ptp10D and Ptp4E within the catalytic domain (Palka, 2003). These findings, together with genetic and biochemical analysis, suggest that autophosphorylated Drosophila Egfr might be a direct target for the enzymatic activities of Ptp10D and Ptp4E (Jeon, 2009).

The fact that apical and luminal cell surface markers accumulate in cysts in unicellular and terminal branches, but not in multicellular branches, suggests that lumen formation in unicellular and terminal branches involves a common mechanism that is not required in multicellular tubes. This is odd, because the apical/luminal surface appears to be generated in a very different manner in unicellular and terminal branches. Lumen in unicellular branches is an extracellular compartment created by remodeling of the cell surface, whereas lumen in terminal branches is an intracellular compartment created by vesicular trafficking (Jeon, 2009).

A possible solution to this conundrum is suggested by the observation that cells in multicellular branches increase in size during development but do not radically change their shapes. Cells in unicellular and terminal branches, however, undergo dramatic shape changes during branch outgrowth. These changes involve reorganization of the apical cell surface in unicellular branches, and the de novo generation of an apical compartment in terminal branches. In unicellular branches with autocellular AJs, the apical/luminal surface coordinates its growth with the remodeling of the cell as a whole, wrapping around the luminal matrix and forming a junction with itself. During elongation of terminal branches, apical surface and lumen markers are localized to growing lines within cells, indicating that lumen and its enclosing apical membrane form in synchrony with the extension of the rest of the cell (Jeon, 2009).

It is speculated that negative regulation of Egfr signaling by Ptp10D and Ptp4E is important for synchronizing apical membrane expansion with the remodeling of the rest of the cell. This could be mediated by phosphotyrosine-regulated interactions between the cytoskeleton and the apical membrane. In this context, it is interesting that loss of the C. elegans ERM (ezrin-radixin-moesin) homolog, which links the apical membrane to the underlying cortical actin network, causes cystic phenotypes in the intestine and the excretory canal (Jeon, 2009).

Interactions between Type III receptor tyrosine phosphatases and growth factor receptor tyrosine kinases regulate tracheal tube formation in Drosophila

The respiratory (tracheal) system of the Drosophila melanogaster larva is an intricate branched network of air-filled tubes. Its developmental logic is similar in some ways to that of the vertebrate vascular system. A unique embryonic tracheal tubulogenesis phenotype has been described caused by loss of both of the Type III receptor tyrosine phosphatases (RPTPs), Ptp4E and Ptp10D. In Ptp4E Ptp10D double mutants, the linear tubes in unicellular and terminal tracheal branches are converted into bubble-like cysts that incorporate apical cell surface markers. This tube geometry phenotype is modulated by changes in the activity or expression of the epidermal growth factor receptor (Egfr) tyrosine kinase (TK). Ptp10D physically interacts with Egfr. This study demonstrates that the Ptp4E Ptp10D phenotype is the consequence of the loss of negative regulation by the RPTPs of three growth factor receptor TKs: Egfr, Breathless and Pvr. Reducing the activity of any of the three kinases by tracheal expression of dominant-negative mutants suppresses cyst formation. By competing dominant-negative and constitutively active kinase mutants against each other, it was shown that the three RTKs have partially interchangeable activities, so that increasing the activity of one kinase can compensate for the effects of reducing the activity of another. This implies that SH2-domain downstream effectors that are required for the phenotype are likely to be able to interact with phosphotyrosine sites on all three receptor TKs. It was also shown that the phenotype involves increases in signaling through the MAP kinase and Rho GTPase pathways (Jeon, 2012).

The Drosophila tracheal system is an intricate branched network of air-filled tubes that delivers oxygen to tissues. Tube formation in the tracheal system involves complex morphogenetic events that differ between tube types. Multicellular tubes have lumens that are surrounded by the apical surfaces of several cells. Unicellular tubes are formed by rolling up of single cells to form junctions with themselves. Seamless tubes are intracellular structures within terminal cells. Many genes have been identified that affect the formation and morphology of tracheal tubes (Jeon, 2012).

The absence of the two Type III RPTPs, Ptp4E and Ptp10D, changes the geometries of the tubes in unicellular and terminal branches, so that they form spherical cysts in place of continuous tubular lumen. The phenotype involves a loss of negative regulation of the Egfr RTK, and Ptp10D physically associates with Egfr (Jeon, 2009; Jeon, 2012).

One of the mammalian Ptp4E/Ptp10D orthologs, PTPRJ (DEP-1), is a direct regulator of multiple growth factor receptor TKs (Arora, 2011; Berset, 2005; Chabot, 2009; Lampugnani, 2003; Kappert, 2007; Palka, 2003; Tarcic, 2009). This led to a test of the hypothesis that Btl and Pvr, the other two Drosophila growth factor receptor TK orthologs that are expressed in embryonic tracheae, are also required for the Ptp4E Ptp10D phenotype (Jeon, 2012).

A quantitative analysis of cyst size at the TC/LT junction showed that tracheally expressed DN mutants of Egfr, Btl, and Pvr all suppress the Ptp4E Ptp10D phenotype almost to wild-type, suggesting that dysregulation of all three RTKs is required for the replacement of linear tubes by spherical cysts. Also, CA mutants of each RTK enhance the phenotype, producing enlarged cysts. These effects cannot be produced by all RTKs, since expression of a CA mutant of InR did not enhance the phenotype (Jeon, 2012).

If the Ptp4E Ptp10D cyst phenotype is the consequence of simultaneous deregulation of all three RTKs, it might be possible to generate the phenotype in a wild-type background by expressing multiple RTK CA mutants. This did not work, even when all three CA mutants were expressed at once. Possible explanations include: 1) because PTP activity normally dominates over RTK activity, the effect on PTyr levels of removing negative regulation by the RPTPs is much greater than that produced by expressing CA RTK mutants in the presence of the RPTPs; 2) other TKs are important for the phenotype, and their activities must also be increased; 3) the RPTPs have PTP-independent activities as adhesion molecules, and generation of the phenotype requires both elevation of RTK activity and the absence of the PTP-independent functions of the RPTPs (Jeon, 2012).

Whether Ptp4E and Ptp10D both regulate all three RTKs was investigated. If they have specificity for particular RTKs, one might be able to generate the cyst phenotype by removing only one RPTP in the presence of a CA RTK mutant. Such combinations were made for Egfr (Ptp4E, Btl>Egfr-CA and Ptp10D, Btl>Egfr-CA), but neither of them had cysts. Since the PTP domains of Ptp4E and Ptp10D are 89% identical, it is likely that they have the same enzymatic targets. The idea that the RPTPs have redundant functions is also consistent with the observation that Ptp4E and Ptp10D single mutants have no detectable phenotypes, while the double mutant is lethal and has both tracheal (Jeon, 2009) and nervous system defects (Jeon, 2008: Jeon, 2012).

When each of the DN RTK mutants was expressed together with a CA mutant of one of the other RTKs in the Ptp4E Ptp10D background, the DN mutant was now unable to suppress the phenotype back to wild-type. Instead the phenotype returned to the strength of unmodified Ptp4E Ptp10D mutants. This shows that if the activity of one RTK is sufficiently elevated, it can replace the requirement for another RTK. Thus, none of the three RTKs is uniquely required to generate the phenotype. Rather, the formation of tubes vs. cysts is controlled by the total amount of activity of certain RTKs in tracheal cells. This implies that the downstream pathways whose increased activity causes cyst formation use SH2-domain effectors that can bind to PTyr sites on any of the three RTKs (Jeon, 2012).

It is interesting that the three RTKs can substitute for each other in regulating the tube vs. cyst decision when they are deregulated by loss of the RPTPs, since the RTKs do not seem to have interchangeable activities in wild-type tracheal cells (or in other tissues). Loss or gain of Btl function produces defects in primary, secondary, and terminal tracheal branching. Loss of Egfr function produces a much more subtle tracheal phenotype affecting tissue integrity. Maintenance of tissue integrity requires signaling through the MAP kinase pathway downstream of Egfr, but is unaffected by reduction of MAP kinase signaling downstream of Btl (Jeon, 2012).

These findings can be explained by the fact that growth factor receptor TKs are usually in an inactive state, due to insufficient levels of ligands and to negative regulation by PTPs. They become active only when they come into contact with elevated levels of their ligands at specific times and places. The activities of Type III RPTPs that dephosphorylate the RTKs might also be transiently reduced at some of these times and places, possibly through interactions of their XC domains with as yet unidentified ligands. As a consequence of the tight control of RTK activity, only those downstream signaling pathways that are most responsive to a particular RTK are likely to be activated by that RTK at any time in wild-type embryos, and the outcomes of signaling through these pathways may also be controlled by the subcellular distributions of ligands, RTKs, RPTPs, and downstream effectors. By contrast, in the absence of Ptp4E and Ptp10D, basal levels of RTK ligands may be able to drive all of the growth factor RTKs to a high level of activity, resulting in strong signaling through all of the downstream pathways they can control. Loss of negative regulation might also cause delocalization of signaling, so that effectors whose activity is normally restricted to particular parts of the cell become activated in a cell-wide manner. Under these conditions, a reduction in the activity of any one of the RTKs by a DN mutant will decrease signaling through multiple downstream pathways. Adding a CA mutant of another RTK can then turn signaling through all of these pathways back up, compensating for the effects of the DN mutant (Jeon, 2012).

The ability of RTKs to substitute for each other in control of cyst formation is conceptually similar to cell transformation through elevation of RTK signaling. RTK activity in cultured cells is tightly controlled, and only a few endogenous RTKs are normally involved in cellular responses to the mitogenic growth factors in their culture medium. Many RTKs can signal through the Ras/MAP kinase pathway, however, and elevated Ras/MAP kinase transduction is sufficient to cause transformation of established cell lines. Thus, oncogenic (CA) mutants of a variety of RTKs are able to transform fibroblastic cell lines when expressed at high levels, regardless of whether the endogenous versions of the RTKs are normally used to regulate proliferation in those cell lines (or are even expressed there) (Jeon, 2012).

The observations on the interchangeability of the RTKs suggests that the decision to form cysts rather than tubes in Ptp4E Ptp10D mutants is very sensitive to the levels of PTyr on the effector binding sites on autophosphorylated RTKs, and that cysts appear when total PTyr rises above a critical threshold. This conclusion is based on the complete suppression of the Ptp4E Ptp10D cyst phenotype that is produced by expression of any of the three DN mutants, even though each DN would eliminate only about 1/3 of total PTyr on the RTKs, if they have roughly equal activities. In wild-type embryos, negative regulation by the RPTPs keeps RTK signaling well below this threshold, so the system is insulated against random fluctuations in phosphorylation or downstream signaling (Jeon, 2012).

The Ptp4E Ptp10D GB cyst number phenotype is completely suppressed by Btl-DN, but not suppressed at all by Egfr-DN and only slightly by Pvr-DN. This might be taken as evidence that elevation of Btl activity is uniquely required to replace GB tubes with cysts. However, when Btl-DN is competed against Pvr-CA, it is only able to suppress the Ptp4E Ptp10D, Btl>Pvr-CA phenotype back to that of unmodified Ptp4E Ptp10D, indicating that Btl can be replaced by Pvr if its activity is sufficiently elevated. These findings can be explained if Btl activity is much higher than Egfr or Pvr activity in GB cells, so that a DN mutant that knocks down endogenous Btl activity by a DN mutant has a greater effect on phosphorylation of effector binding sites than mutants that reduce Egfr or Pvr activities. The activity of CA RTK mutants is independent of the endogenous levels of RTKs, so the Pvr CA mutants could still reverse the effect of Btl-DN even if Pvr-DN has no effect on its own (Jeon, 2012).

To evaluate whether the MAP kinase signaling pathway is involved in the determination of tube geometry, a CA mutant of Phl, the Drosophila Raf kinase, was expressed in the Ptp4E Ptp10D background. Phl-CA enhances the TC/LT phenotype almost as strongly as Egfr-CA. When Phl-CA is combined with Egfr or Btl DN mutants, the phenotype is suppressed back to that of unmodified Ptp4E Ptp10D. Since these RTKs are upstream of Raf, the fact that suppression occurs suggests that pathways other than the MAP kinase pathway are required to generate the phenotype. However, these other pathways may be stimulated by elevation of MAP kinase signaling, since one would expect suppression back to a near-wild-type phenotype if they were completely independent of the MAP kinase pathway (Jeon, 2012).

The involvement of Rho GTPases was tested by expressing DN mutants of Rho1, Rac1, and Cdc42 in the Ptp4E Ptp10D background. Rho1-DN and Rac1-DN completely suppress the TC/LT phenotype, and Cdc42-DN and a tracheally expressed Rho1 RNAi construct produces partial suppression. The DN mutant data do not necessarily show that Rho and Rac are both required for generation of the phenotype. DN mutants may occlude binding of wild-type Rho family GTPases to their GEFs, some of which can act on both Rho and Rac. Therefore high-level expression of Rac-DN might inhibit Rho activation, and vice versa. However, the suppression of the TC/LT phenotype by Rho1 RNAi, which is a specific inhibitor, implicates Rho1 itself in the phenotype. Rho1-CA enhances the Ptp4E Ptp10D phenotype, producing cysts on most DBs (Jeon, 2012).

The differences in the abilities of the three RTK DN mutants to suppress the effects of Phl-CA and Rho-CA may provide clues to the pathways that act downstream of these kinases. The MAP kinase and Rho pathways are not necessarily independent, since Ras/MAP kinase pathway activation can increase Rho-GTP in some cell lines. Also, each of the three RTKs is likely to activate both pathways to some extent, as well as many other downstream pathways. Defining the specific outputs of the MAP kinase and Rho pathways that control tracheal tube geometry and identifying other RTK pathways that regulate tube formation is likely to require genome-wide screens for suppressors and enhancers of the Ptp4E Ptp10D phenotype (Jeon, 2012).

Spatial restriction of receptor tyrosine kinase activity through a polarized endocytic cycle controls border cell migration

Border cell migration is a stereotyped migration occurring during the development of the Drosophila egg chamber. During this process, a cluster composed of six to eight follicle cells migrates between nurse cells toward the oocyte. Receptor tyrosine kinases (RTKs) are enriched at the leading edge of the follicle cells and establish the directionality of their migration. Endocytosis has been shown to play a role in the maintenance of this polarization; however, the mechanisms involved are largely unknown. This study shows that border cell migration requires the function of the small GTPases Rab5 and Rab11 that regulate trafficking through the early and the recycling endosome, respectively. Expression of a dominant negative form of rab11 induces a loss of the polarization of RTK activity, which correlates with a severe migration phenotype. In addition, it was demonstrated that the exocyst component Sec15 is distributed in structures that are polarized during the migration process in a Rab11-dependent manner and that the down-regulation of different subunits of the exocyst also affects migration. Together, these data demonstrate a fundamental role for a plasma membrane-endosome trafficking cycle in the maintenance of active RTK at the leading edge of border cells during their migration (Assaker, 2010).

During migration, the cell needs to rearrange its cytoskeleton, its plasma membrane content, and its interaction with other cells. Many of these features can be controlled by vesicular trafficking. For example, Integrins, Cadherins, and other cell-cell or cell-matrix attachment proteins are transmembrane proteins tightly regulated by trafficking. Furthermore, the distribution of proteins and lipids at the plasma membrane is directly controlled by vesicular trafficking, as well as the localization of some actin remodeling proteins. During the process of border cell migration, endocytosis has been shown to regulate the polarity of RTK activity. This paper shows that the endocytic process plays a role in regulating the spatial localization of RTK activity by trafficking through the recycling endosome and by the polarized redelivery of endocytosed material to the plasma membrane (Assaker, 2010).

The key endocytic proteins previously involved in border cell migration - Sprint, Cbl, and Shibire - regulate the polarization of RTK activity during border cell migration. Different possible mechanisms are proposed to explain their action and have not been addressed in this landmark article (Jékely, 2005). Recently, it was shown that both the degradative pathway and the recycling pathway might be involved in this process. Thus, at least two models, which are not mutually exclusive, could explain the role of endocytosis in establishing this polarity. First, active RTKs could be endocytosed and degraded when diffusing away from the leading edge. Second, polarized recycling of endocytosed active RTKs could concentrate these active receptors at the leading edge. From the current experiments it can be concluded that the recycling of active RTKs or of a cofactor at the plasma membrane is necessary for border cell migration. Furthermore, it was demonstrated that the slow recycling route, through the recycling endosome, is used and that polarized redelivery at the plasma membrane is mediated by the exocyst subunit sec15 (Assaker, 2010).

It seems logical to think that RTKs, or active RTKs, are the cargo transported through this endocytic cycle. However, the identity of the protein being recycled remains to be determined. Indeed, there is no indication that RTKs are recycled. Immunofluorescence staining of the EGFR was performed. The signal obtained was diffuse and inconclusive in both control and rab11SN-expressing border cells. In addition, if active RTKs were trafficking through the recycling endosome, the pTyr in endocytic vesicles would be expected to be marked by Rab11. However, such a colocalization has never been observed. These data do not rule out a potential recycling of RTKs, because they could be present in the recycling endosome in quantities below detection levels or in an inactive form. However, the data suggest that the main cargo of this trafficking cycle is of another nature. This cargo could be a plasma membrane diffusion barrier, because polarized cells, such as epithelial cells and neurons, maintain different membrane domains, which rely on such barriers: the tight junctions and the axon hillock, respectively. Moreover, diffusion barriers have been proposed to define plasma membrane domains in migrating cells. These diffusion barriers appear to be linked to the actin cytoskeleton, but their exact nature is unknown. Because E-cadherin is involved in cell migration, it would have been an ideal candidate to play such a role, but it is unaffected by rab11SN expression (Assaker, 2010).

Another possibility is that endocytosis acts indirectly. For example, it might regulate key components of the plasma membrane or of the cytoskeleton. Recent evidence has shown that endocytosis and recycling can play a critical role in creating a positive feedback loop during polarity establishment in the budding yeast. In this particular case, endocytosis is critical for the localization of regulators of small GTPases of the Rho family. Furthermore, mammalian Tiam1, a GDP-to-GTP exchange factor (GEF) for Rac, has been shown to localize to endosomes, leading to the loading of active Rac at the plasma membrane through an endocytic-recycling cycle. Interestingly, a robust genetic interaction has been found between Rab11 and Rac1, which directs border cell migration (Wang, 2010). Until now, two Rac-GEFs, Myoblast city and Elmo, have been involved in border cell migration, but not the Drosophila Tiam1 homolog still life. Further studies will be necessary to determine if Rac1 is the main cargo of the endocytic-recycling cycle that regulates border cell migration (Assaker, 2010).

In the past few years, trafficking via the recycling endosome has been involved in the establishment or rearrangement of cell polarity in various events. In particular, a role for the recycling endosome has been observed when a rapid and dramatic rearrangement of the cell organization is required, including cellularization, cell-cell boundary rearrangement, asymmetric cell division, and cell migration. Trafficking through the recycling endosome is an ideal mechanism to polarize a cell rapidly, because it hijacks material already available in the cell at a new location. Furthermore, it is a very efficient mechanism to reinforce polarity by feedback loops. Similarly the exocyst plays a key role in the majority of these cell polarizations. In the case of cell migration, the recycling endosome may transform the diffuse extracellular gradient of RTK ligand into a robust intracellular polarization of RTK activity that is crucial for directed migration (Assaker, 2010).

There is much evidence that the function of the recycling endosome in the regulation of directed migration is conserved in mammals. The mammalian homologs of Drosophila Rab11 are Rab11A and -B and Rab25. They have been directly implicated in the migration of cancerous cells and in the formation of metastasis, a cell migration event resembling border cell migration. Rab11 effectors are also involved in mammalian cell migration. More specifically, PDGF receptor-dependent cell migration has been shown to be regulated by endocytosis in a mammalian cell culture assay and the recycling endosome has been indirectly implicated in the regulation of migration guided by the EGFR. Given the involvement of the recycling endosome in so many processes, targeting its function to reduce metastasis is unlikely to be efficient. However, identifying the main cargo of this recycling cycle could help identify more specific targets for drugs blocking the formation of metastasis (Assaker, 2010).

Rhomboid family pseudoproteases use the ER quality control machinery to regulate intercellular signaling

Intramembrane proteolysis governs many cellular control processes, but little is known about how intramembrane proteases are regulated. iRhoms are a conserved subfamily of proteins related to rhomboid intramembrane serine proteases that lack key catalytic residues. This study has used a combination of genetics and cell biology to determine that these 'pseudoproteases' inhibit rhomboid-dependent signaling by the epidermal growth factor receptor pathway in Drosophila, thereby regulating sleep. iRhoms prevent the cleavage of potential rhomboid substrates by promoting their destabilization by endoplasmic reticulum (ER)-associated degradation; this mechanism has been conserved in mammalian cells. The exploitation of the intrinsic quality control machinery of the ER represents a new mode of regulation of intercellular signaling. Inactive cognates of enzymes are common, but their functions are mostly unclear; these data indicate that pseudoenzymes can readily evolve into regulatory proteins, suggesting that this may be a significant evolutionary mechanism (Zettl, 2011).

Phylogenetic analysis of rhomboids identified a subgroup of rhomboid-like proteins that lack essential catalytic residues (Lemberg, 2007). These 'iRhoms' are mysterious because, despite their predicted lack of protease activity, they are present in all sequenced metazoans, and their high degree of sequence identity implies selective pressure. Little is known about their function, but human iRhom1/Rhbdf1 has been reported to be necessary for the survival of some epithelial cancer cells (Yan, 2008) and may be linked to GPCR-mediated EGFR transactivation (Zou, 2009; Zettl, 2011).

iRhoms exemplify a more general phenomenon: the existence of conserved but catalytically inactive cognates of enzymes. The widespread occurrence of this type of predicted protein has only been apparent since genome sequences have been available, and their function is largely unknown. They are not generally encoded by pseudogenes and are therefore presumed to be expressed. Bioinformatic analysis has led to the suggestion that inactive enzyme cognates are disproportionately involved in regulatory processes (Zettl, 2011).

In Drosophila, active rhomboids are cardinal regulators of epidermal growth factor receptor (EGFR) signaling, and although it is unclear whether this activity is conserved in mammals, recent evidence supports some evolutionary conservation (Adrain, 2011). Until now, nothing has been reported about iRhom function in Drosophila. This study used a combination of genetics and cell biology to discover that Drosophila iRhom regulates EGFR signaling. The EGFR pathway in Drosophila has become a model for understanding how signaling pathways are regulated with the precision that is necessary to control their multiple developmental and physiological roles. Drosophila iRhom is expressed predominantly in neuronal cells, and its loss causes a 'sleep'-like phenotype that is indistinguishable from gain-of-EGFR signaling in the central nervous system. Consistent with this, genetic interactions show that Drosophila iRhom counteracts the function of active rhomboids, specifically acting to inhibit EGFR signaling. As well as revealing the biological role of Drosophila iRhom, this study investigated the cellular mechanism of iRhom function. Both fly and mammalian iRhoms, which are localized in the ER, inhibit secretion and intracellular levels of specific client proteins by promoting proteasomal degradation. Overall, these data imply that iRhoms regulate secretion of specific client proteins, which include the EGF family of growth factors. They can target clients for proteasomal removal by ER-associated degradation (ERAD). In this way, fundamental cellular quality control machinery is exploited as a mechanism for regulating growth factor signaling (Zettl, 2011).

Intramembrane proteases are potentially dangerous enzymes: they catalyze the irreversible cleavage of proteins that trigger important cellular processes. Their regulation is therefore paramount, but little is known about how this is achieved under physiological conditions. Nevertheless, several lines of evidence suggest that intramembrane proteases rely heavily on regulated segregation of substrate and enzyme. For example, in Drosophila, the type II membrane protein Star regulates access of substrates to rhomboids. Similarly, the trafficking of the site-2 protease substrates SREBP or ATF6 to the Golgi apparatus, the location of S2P, is highly regulated. This study reports a new mechanism, the specific destabilization of substrates in the ER, ultimately preventing access to an active rhomboid. The point is not that these diverse control strategies share common mechanisms, but that they all comply with the regulatory logic of segregating substrate and enzyme. This contrasts with the regulation of soluble proteases, in which there is greater emphasis on regulating enzyme activity. This distinction reflects the greater ability to restrict and control the cellular location of membrane proteins than soluble proteins (Zettl, 2011).

ER-associated degradation (ERAD) was first discovered as a mechanism for removing misfolded components of the T cell receptor complex. It is now clear that it contributes more generally to ER quality control and homeostasis. The current data extend the biological consequences of ERAD by demonstrating that it has been recruited in metazoans as a way of regulating intercellular signaling. Although many ERAD components have been identified, the molecular details of how proteins are targeted for recognition and retrotranslocation remain unclear. iRhoms are polytopic ER membrane proteins that can bind to at least two rhomboid substrates (this work and Nakagawa, 2005). They could enhance ERAD actively by introducing clients into the retrotranslocation machinery or passively by prolonging ER retention, thereby increasing the probability of exposure to ERAD. It is not known how many proteins iRhoms affect; genetic and cell biological data show significant specificity, but the fact that mammalian iRhoms target EGF ligands that are not rhomboid substrates implies that they have evolved additional clients (Zettl, 2011).

The data also demonstrate the physiological significance of iRhoms in Drosophila: they are specific regulators of EGFR signaling. In mammals, the data also show that iRhoms can inhibit secretion of EGF family ligands, but in contrast to flies, the physiological significance is not yet clear. The Drosophila EGF receptor is probably the most genetically well-characterized growth factor receptor in any system, and it is striking how many distinct proteins contribute to its control. The current experiments demonstrate that iRhom is a new type of regulator of EGFR activity - the most upstream control element in the pathway yet discovered. A theme that has emerged is the importance of negative feedback as a way of limiting the extent and/or amplitude of signaling. The fact that iRhom is transcriptionally activated in the developing eye imaginal disc in cells with active EGFR signaling implies that, at least in that context, iRhom could also participate in a feedback loop. Intriguingly, its expression pattern shows that, in the eye at least, its expression peaks at a time when EGFR signaling needs to be switched off (Zettl, 2011).

Degradation of EGFR ligands could therefore represent a robust way of preventing inappropriate EGFR signaling. Although the role of iRhom can be detected in the development of the wing and eye under genetically sensitized conditions, the only prominent phenotype caused by loss of Drosophila iRhom is behavioral. This is consistent with its expression pattern, which is largely restricted to the nervous system. An important open question is whether the activity phenotype of iRhom mutant flies is a consequence of a developmental or physiological defect (Zettl, 2011).

Loss of iRhom causes flies to undergo excessive periods of inactivity during the daytime, when wild-type flies are active. In fact, iRhom mutants appear to be the first loss-of-function mutations to have an excess sleep-like phenotype. Drosophila has become a genetic model to investigate the neuronal and molecular mechanisms of sleep, and the data support the recent report that abnormally high EGFR activity in the CNS leads to inactivity in flies (Foltenyi, 2007; Zettl, 2011 and references therein).

The iRhoms have evolved from rhomboid proteases that lost their catalytic activity but retained their location in the secretory pathway and the ability to bind their substrates. This allowed them to acquire new functions as specific regulators of secreted proteins, without retaining a direct mechanistic interaction with the proteases. Because many mutations will lead to loss of catalytic activity, the evolution of regulatory proteins from 'dead' enzymes might be quite common. The expression pattern, cellular location, and substrate binding capacity of such proteins provide an ideal platform for the subsequent acquisition of specific regulatory properties. Consistent with this idea is the striking existence of inactive cognates of most proteases -- indeed of many enzymes of all families. Little or nothing is known about most of these pseudoenzymes, the widespread existence of which has only become apparent as genomes have been extensively sequenced. But it is an attractive idea that many will have regulatory functions related to the enzymes from which they evolved (Zettl, 2011).

Drosophila activated Cdc42 kinase has an anti-apoptotic function: Drosophila Ack possesses potent anti-apoptotic activity that is dependent on Ack kinase activity and is further activated by EGF receptor/Ras signaling

Activated Cdc42 kinases (Acks) are evolutionarily conserved non-receptor tyrosine kinases. Activating somatic mutations and increased ACK1 protein levels have been found in many types of human cancers and correlate with a poor prognosis. ACK1 is activated by epidermal growth factor (EGF) receptor signaling and functions to regulate EGF receptor turnover. ACK1 has additionally been found to propagate downstream signals through the phosphorylation of cancer relevant substrates. Using Drosophila as a model organism, it has been determined that Drosophila Ack possesses potent anti-apoptotic activity that is dependent on Ack kinase activity and is further activated by EGF receptor/Ras signaling. Ack anti-apoptotic signaling does not function through enhancement of EGF stimulated MAP kinase signaling, suggesting that it must function through phosphorylation of some unknown effector. Several putative Drosophila Ack interacting proteins were isolated, many being orthologs of previously identified human ACK1 interacting proteins. Two of these interacting proteins, Drk and Yorkie, were found to influence Ack signaling. Drk is the Drosophila homolog of GRB2, which is required to couple ACK1 binding to receptor tyrosine kinases. Drk knockdown blocks Ack survival activity, suggesting that Ack localization is important for its pro-survival activity. Yorkie is a transcriptional co-activator that is downstream of the Salvador-Hippo-Warts pathway and promotes transcription of proliferative and anti-apoptotic genes. yorkie and Ack were found to synergistically interact to produce tissue overgrowth, and yorkie loss of function interferes with Ack anti-apoptotic signaling. These results demonstrate how increased Ack signaling could contribute to cancer when coupled to proliferative signals (Schoenherr, 2012).

The founding member of tha ACK family is human ACK1, which was identified as a protein that binds to CDC42 in its active GTP bound form. Since this discovery Ack homologs have been found in chordates, arthropods and nematodes. Ack family members can be divided into three structural categories based on the presence or absence of four conserved domain motifs. All Ack family members contain an amino-terminal tyrosine kinase domain that is flanked by a sterile alpha motif (SAM) and a Src homology 3 (SH3) domain. The carboxy-terminal half of these kinases contains short proline rich sequences, but lacks any identifiable domains, with the exception of two tandemly repeated ubiquitin-associated (UBA) domains located at the extreme carboxy-terminus. ACK1 UBA domains have been shown to interact with both mono and poly-ubiquitinated proteins and are thought to play a role in ACK1 protein turnover. The Caenorhabditis elegans Ack homolog, Ark-1, contains no UBA domains, placing it in a class by itself. The other two Ack structural classes can be distinguished by the presence or absence of a Cdc42/Rac interactive binding (CRIB) domain. Human ACK1 and Drosophila PR2 are representative members of the CRIB domain containing structural class, while human TNK1 and Drosophila Ack are members of the structural class lacking a conserved CRIB domain. Variants containing a CRIB domain bind GTP liganded CDC42, but this interaction does not appear to directly influence Ack activity in vitro (Schoenherr, 2012).

Human ACK1 is the most well characterized member of the Ack family. Early studies uncovered a role for ACK1 in the promotion of internalization and down-regulation of activated epidermal growth factor (EGF) receptor. ACK1 tyrosine phosphorylation is enhanced and ACK1 is co-localized with EGF receptor after EGF stimulation. Knockdown of ACK1 reduces the rate of EGF receptor degradation following EGF stimulation. While on the surface these data suggest that ACK1 merely serves as a negative regulator of growth factor signaling, ACK1 activation may additionally propagate downstream signaling. Recent studies support this latter alternative by uncovering a role for ACK1 as a positive transducer of cell surface receptor signaling that promotes growth and survival by ACK1 mediated phosphorylation and activation of downstream components, including AKT (Mahajan, 2010) and the androgen receptor (Schoenherr, 2012 and references therein).

A pro-survival role for Ack function is consistent with reported links between activation of Ack family members and cancer genesis and metastasis. Several somatic missense mutations have been identified in ACK1 from cancer tissue samples that increase ACK1 autophosphorylation and promote cellular proliferation and migration. Amplification of the ACK1 gene in tumors correlates with a poor prognosis, and ACK1 overexpression in cancer cell lines increases invasiveness in a mouse metastasis model, while knockdown of ACK1 reduces the migration of human breast cancer cells (Schoenherr, 2012).

Activated ACK1 has been detected in advanced human prostate cancers where it has been shown to phosphorylate three cancer relevant substrates in prostate cancer cell lines: WWOX, AKT, and androgen receptor. WWOX spans the FRA16D chromosomal fragile site that is frequently disrupted in human cancers. While the molecular function of WWOX is not known, it has been shown that the growth of tumor cells lacking WWOX is strongly inhibited by restoring WWOX expression. ACK1 phosphorylation of WWOX leads to the polyubiquitination and degradation of WWOX, which correlates with a tumorigenic role. AKT is a serine/threonine kinase whose activity promotes cell survival and proliferation, while deregulation of the AKT signaling pathway is commonly associated with cancer. ACK1 activation results in tyrosine phosphorylation and apparent activation of AKT in a PI3K independent mechanism. Finally, the activity of the androgen receptor is required for growth of prostate cells. In advanced stages of prostate cancer, these cells lose their dependence on androgens for activation of this receptor to become androgen independent prostate cancer. ACK1 has been found to phosphorylate the androgen receptor, promote androgen independent growth of prostate cells, and activate transcription of androgen inducible genes in the absence of androgen (Schoenherr, 2012).

Less is known about the function of Ack family members lacking CRIB domains, and published studies on TNK1 describe conflicting functions. TNK1 overexpression in cell culture lines inhibits cell growth in a kinase dependent manner. Mutant mice having deletions in the kinase domain of TNK1 develop spontaneous tumors at a high frequency, which is thought to originate from hyperactivation of Ras signaling and suggests that TNK1 functions as a tumor suppressor. In contrast to this function, TNK1 was identified as a potentially oncogenic tyrosine kinase in a mutagenesis screen and activated TNK1 was found in Hodgkin's lymphoma. It is possible that these conflicting findings reflect tissue specific responses or complex dosage sensitivity to TNK1 loss and gain of function (Schoenherr, 2012 and references therein).

In order to better understand the physiological role of Ack family members and determine how Ack might contribute to cancer, genetic and biochemical experiments were conducted in the model organism Drosophila melanogaster. These studies focus on Drosophila Ack, which has a domain structure resembling human TNK1, but shares significantly higher sequence identity with ACK1 in all conserved domains including the kinase domain activation loop. It was found that Drosophila Ack possesses potent anti-apoptotic properties that function downstream of EGF receptor signaling through an unknown mechanism. This activity is dependent on Ack kinase function and can be further stimulated by increased Ras signaling. A protein interaction study was conducted, and it was found that many of the same proteins that associate with human ACK1 also bind to fly Ack. The influence of these proteins on Ack anti-apoptotic activity was tested, and it was determined that the adapter protein Drk (GRB2) is required for this activity, while the transcriptional co-activator protein Yki (YAP) functions synergistically with Ack to promote cell survival and massive tissue overgrowth. These findings support both anti-apoptotic and proliferative roles for Ack family members, which may contribute to cancer genesis and progression (Schoenherr, 2012).

Drosophila has two Ack family members: Ack and PR2. Ack possesses anti-apoptotic properties, while PR2 either does not possess anti-apoptotic properties or requires activators not present in the assay system. While Ack may appear to be more closely related to vertebrate TNK1 because both proteins lack a CRIB domain, Ack is most similar to vertebrate ACK1 based on sequence identity of all shared protein domains. Additionally it was found that many of the proteins that interact with ACK1 also interact with fly Ack. Therefore, the conclusions drawn in this study will likely be applicable to vertebrate ACK1 function (Schoenherr, 2012).

Ack function can suppress programmed cell death induced by Hid or Rpr expression in the developing eye and wing discs. Overexpression studies reveal that Ack kinase activity is required for suppression of apoptosis induced by hid but is unnecessary for suppression of rpr-induced apoptosis. It was further shown that Ack loss of function enhances cell death induced by expression of both of these genes, and it was determined that Ack is critically required for the survival of rpr expressing eye tissue. The molecular mechanisms underlying these differential requirements are not known. hid and rpr are known to function in a multimeric protein complex on the mitochondria outer membrane to promote apoptosis. Both hid and rpr are able to stimulate apoptosis by competing with initiator and effector caspases for DIAP binding, but rpr additionally induces DIAP auto-ubiquitination leading to DIAP degradation. While Ack kinase activity may be important for aspects of hid complex regulation, it is tempting to speculate that the UBA domains of Ack may play a critical role in the modulation of DIAP or Rpr ubiquitination and stability (Schoenherr, 2012).

EGF receptor signaling has been shown to activate ACK1 in vertebrates, and EGF signaling was found to enhance the anti-apoptotic function of Ack in Drosophila. ACK1 negatively regulates EGF receptor signaling by stimulating endocytosis of activated receptor complexes. The evidence supports the idea that Drosophila Ack, in conjunction with SH3PX1, functions in a similar manner, which will be described elsewhere. In Drosophila, EGF signaling is anti-apoptotic through the activation of MAPK, which phosphorylates and inactivates Hid. If Ack affected apoptosis exclusively through attenuation of EGF signaling, then it would be expected that Ack loss of function would be anti-apoptotic while gain of function would be pro-apoptotic, which is opposite to what was observed. By using the hidAla5 mutant, it was demonstratde that Ack does not modulate programmed cell death through activation of MAPK (Schoenherr, 2012).

The anti-apoptotic function of Ack is surprisingly robust compared to other proteins that were have tested. The studies show that activity of the kinase domain contributes to Ack anti-apoptotic function. Based on this, it is concluded that Ack propagates anti-apoptotic signals by phosphorylating downstream targets. Several ACK1 substrates have been identified that are attractive candidates for the regulation of programmed cell death: the putative tumor suppressor WWOX, the apoptosis inhibiting protein kinase AKT and the caspase-cleaved ubiquitin E3 ligase NEDD4. Akt1 loss and gain of function alleles and Nedd4 RNAi fail to significantly modify hid induced small eye phenotypes. Wwox RNAi is able to suppress hid induced apoptosis but not nearly as robustly as Ack expression. Since ACK1 mediated phosphorylation of WWOX leads to WWOX destruction, it would be predicted that Wwox RNAi would phenocopy Ack expression in the assay system, but it is unable to reproduce the magnitude of Ack anti-apoptotic function. This does not rule out Wwox as an anti-apoptotic substrate target of Ack, but it demonstrates that Wwox is not the only cell death relevant substrate of Ack. It is worth noting that hid and rpr act fairly late within the programmed cell death pathway, being just a step upstream of initiator caspase activation. Therefore, Ack must target substrates that have activities influencing hid, rpr or events at the level of caspase activation (Schoenherr, 2012).

Several Ack physically interacting proteins were identified using a tandem affinity purification strategy. Many of these have vertebrate homologs that have been previously determined to interact with ACK1. Drk and Yki have the most pronounced effect on Ack's anti-apoptotic properties, and their contribution to Ack signaling was further characterized. Drk is the fly ortholog of vertebrate GRB2, which has previously been described as an ACK1 and TNK1 interacting protein. In the case of TNK1, GRB2 is tyrosine phosphorylated by TNK1, which disrupts the ability of GRB2/SOS complexes to activate Ras. This does not appear to be the case for Drosophila Ack, because even though Drk forms a complex with tyrosine phosphorylated Ack, no evidence of tyrosine phosphorylation on Drk was found. Rather, the data support that Drk association with Ack is required for Ack anti-apoptotic properties. It is proposed that Drk SH3 domains likely interact with PXXP motifs in the C-terminal half of Ack similar to the interaction described in vertebrates. This interaction could then lead to the recruitment of Ack into protein complexes required for Ack activation (Schoenherr, 2012).

Yki is a transcriptional co-activator that regulates expression of genes with proliferative and anti-apoptotic functions. The vertebrate homolog of Yki is Yes Associated Protein (YAP), which has not previously been identified as an ACK1 or TNK1 interacting protein. Yki and YAP studies have focused primarily on the pathways that regulate their function as transcription factors. Given the role of Yki and YAP in transcriptional control of proliferative and anti-apoptotic genes, it would seem likely that Ack activity leads to enhancement of Yki function. However, this does not appear to be the case because Ack overexpression does not lead to increased Yki nuclear localization or increased expression of yki target genes. Rather, the data indicate that Yki directly interacts with Ack in the cytoplasm and functions to regulate Ack activity. In support of this, it was found that Ack colocalizes with Yki, and yki dosage reduction suppresses Ack anti-apoptotic function. Yki contains two WW domains, which may interact with conserved PPXY motifs that are present in the region flanked by the SH3 and UBA domains of Ack family members. In vertebrates, these PPXY motifs have been shown to interact with WWOX, which also contains two WW domains. Further studies are required to define how Yki and Ack interact (Schoenherr, 2012).

Yki expression in the fly eye produces an overgrowth phenotype that is indicative of its role in regulating proliferation. Ack overexpression produces a slightly larger eye due to inhibition of apoptotic events that occur during normal eye development. Simultaneous expression of yki and Ack results in a synergistic effect that produces enormous eyes. These results reveal that in addition to anti-apoptotic function, Ack can also enhance proliferation. This illustrates how increased Ack signaling could contribute to cancer when coupled to proliferative signals. Indeed, the results are consistent with recent reports of ACK1 activating somatic mutations and gene amplification being associated with human cancers. At present the key anti-apoptotic substrates of Ack and their mechanisms of action remain to be determined. With their discovery will come a better understanding of Ack signaling and potentially new targets for cancer interventions (Schoenherr, 2012).

FAK acts as a suppressor of RTK-MAP kinase signalling in Drosophila melanogaster epithelia and human cancer cells

Receptor Tyrosine Kinases (RTKs) and Focal Adhesion Kinase (FAK) regulate multiple signalling pathways, including mitogen-activated protein (MAP) kinase pathway. FAK interacts with several RTKs but little is known about how FAK regulates their downstream signalling. This study investigated how FAK regulates signalling resulting from the overexpression of the RTKs RET and EGFR. FAK was found to suppress RTKs signalling in Drosophila epithelia by impairing MAPK pathway. This regulation was also observed in MDA-MB-231 human breast cancer cells, suggesting it is a conserved phenomenon in humans. Mechanistically, FAK reduces receptor recycling into the plasma membrane, which results in lower MAPK activation. Conversely, increasing the membrane pool of the receptor increases MAPK pathway signalling. FAK is widely considered as a therapeutic target in cancer biology; however, it also has tumour suppressor properties in some contexts. Therefore, the FAK-mediated negative regulation of RTK/MAPK signalling described in this study may have potential implications in the designing of therapy strategies for RTK-driven tumours (Macagno, 2014).

Protein tyrosine phosphatase PTPN3 inhibits lung cancer cell proliferation and migration by promoting EGFR endocytic degradation

Epidermal growth factor receptor (EGFR) regulates multiple signaling cascades essential for cell proliferation, growth and differentiation. Using a genetic approach, this study found that Drosophila FERM and PDZ domain-containing protein tyrosine phosphatase, dPtpmeg, negatively regulates border cell migration and inhibits the EGFR/Ras/mitogen-activated protein kinase signaling pathway during wing morphogenesis. EGFR pathway substrate 15 (Eps15) was further identified as a target of dPtpmeg and its human homolog PTPN3. Eps15 is a scaffolding adaptor protein known to be involved in EGFR endocytosis and trafficking. Interestingly, PTPN3-mediated tyrosine dephosphorylation of Eps15 promotes EGFR for lipid raft-mediated endocytosis and lysosomal degradation. PTPN3 and the Eps15 tyrosine phosphorylation-deficient mutant suppress non-small-cell lung cancer cell growth and migration in vitro and reduce lung tumor xenograft growth in vivo. Moreover, depletion of PTPN3 impairs the degradation of EGFR and enhances proliferation and tumorigenicity of lung cancer cells. Taken together, these results indicate that PTPN3 may act as a tumor suppressor in lung cancer through its modulation of EGFR signaling (Li, 2014).

Reversible tyrosine protein phosphorylation by protein tyrosine kinases and protein tyrosine phosphatases (PTPs) acts as a molecular switch that regulates a variety of biological processes. The receptor tyrosine kinase epidermal growth factor receptor (EGFR), the best characterized member of the ErbB family receptors, acts as a critical regulator of numerous cellular processes, including growth, proliferation and differentiation. Upon activation by its growth factor ligands, EGFR undergoes dimerization and activation, leading to tyrosine phosphorylation of the intracellular region of the receptor as well as many cytoplasmic substrates. The activated EGFR is then internalized by clathrin-mediated endocytosis and sorted into the endosomal compartments, through which it is either recycled back to the plasma membrane or transported to the lysosome for degradation. Because overexpression or constitutive activation of EGFR has been implicated in the pathogenesis and progression of a variety of human malignancies, it is therefore crucial to understand how EGFR signaling is regulated. Several PTPs have been implicated in the regulation of EGFR signaling. Among them, PTPrk, DEP-1 (PTPRJ), PTP1B (PTPN1), SHP-1 (PTPN6), TCPTP (PTPN2), PTPN9 and PTPN12 have been shown to downregulate EGFR signaling by dephosphorylating EGFR. The receptor-type PTP DEP-1 dephosphorylates EGFR on the cell surface and inhibits its internalization. On the other hand, the endoplasmic reticulum-localized PTP1B has been reported to regulate EGFR signaling from endosomes. PTP1B promotes the sequestration of EGFR onto internal vesicles of multivesicular bodies. The ESCRT (endosomal sorting complex required for transport) complexes are known to play an important role in sorting EGFR to multivesicular bodies. Recently, it has been shown that the ESCRT accessory protein HD-PTP/PTPN23 coordinates with the ubiquitin-specific peptidase UBPY to drive EGFR sorting to the multivesicular bodies. A better understanding of the role of PTPs in regulating EGFR signaling will help to provide insights into the molecular mechanisms behind EGFR-mediated tumorigenesis (Li, 2014).

PTPN3 (PTPH1) and the closely-related PTPN4 (PTPMEG) are non-transmembrane PTPs that contain an N-terminal FERM (Band 4.1, Ezrin, Radixin, Moesin homology) domain followed by a single PDZ (PSD95, Dlg, ZO1) domain and the C-terminal PTP domain. They have been implicated in the regulation of cell growth and proliferation. However, their role in receptor protein tyrosine kinase signaling is not clear. The dPtpmeg is the Drosophila homolog of mammalian PTPN3 and PTPN4. Phenotypic analyses have revealed that dptpmeg mutants exhibit aberrant mushroom body axon projection patterns in the brain. Besides its role in regulating neuronal wiring, the molecular function of dPtpmeg has remained largely unknown. This study has identified EGFR pathway substrate 15 (Eps15) as a substrate of dPtpmeg and PTPN3. Eps15 is known to be an endocytic adaptor involved in the regulation of EGFR trafficking. PTPN3 dephosphorylated Eps15 and promoted EGFR for lipid raft-mediated endocytosis and lysosomal degradation. The ectopic expression of PTPN3 or Eps15-Y850F mutant in the non-small-cell lung cancer (NSCLC) cells inhibited cell proliferation, migration and tumor growth. These findings uncover a novel role for PTPN3 in the regulation of EGFR endocytic trafficking, degradation and signaling (Li, 2014).

The EGFR belongs to the ErbB family of protein tyrosine kinases and is a major regulator for both normal development and cancer progression. PTPs, which include receptor-like PTPs and nonreceptor PTPs, are a group of tightly regulated enzymes thought to regulate tyrosine phosphorylation by antagonizing the action protein tyrosine kinases. This study provides the first evidence that PTPN3 inhibits the EGFR signaling by targeting the receptor for lysosomal degradation. In Drosophila, dPtpmeg antagonizes receptor tyrosine kinase activity and plays a role in controlling border cell migration during oogenesis. Moreover, dPtpmeg negatively regulates the EGFR/Ras/MAPK pathway during wing morphogenesis. Substrate-trapping and biochemical analysis further identified Eps15 as a substrate for dPTPmeg and PTPN3. The results demonstrate that PTPN3 dephosphorylates Eps15 and promotes EGFR for degradation in lung cancer cells (Li, 2014).

Eps15 is a multidomain adaptor protein that plays an important role in regulating endocytic trafficking. In mammalian cells, Eps15 can be phosphorylated by EGFR at tyrosine residue 850 upon EGF stimulation. It has been shown that ectopic expression of Eps15-Y850F mutant impairs the internalization of EGFR. On the contrary, using immunofluorescence and flow cytometry, this study shows that ectopic expression of PTPN3 and Eps15-Y850F does not affect the internalization of EGFR. Since a dramatic reduction of EGFR levels was found in cells expressing PTPN3 and Eps15-Y850F, one explanation for the results being contradictory to previous findings might be because of the difference in detection sensitivity. In addition to its role in endocytic trafficking, Eps15 was reported to localize at the trans-Golgi network and regulate vesicle trafficking during the secretory process. However, immunofluorescence analysis of the intracellular distribution of endogenous PTPN3 or exogenously expressed HA-tagged PTPN3 indicated no significant localization at the trans-Golgi network, and this might suggest that PTPN3 is not involved in Eps15-mediated protein sorting and vesicle trafficking at the trans-Golgi network (Li, 2014).

The ligand-activated EGFR and the transforming growth factor-β receptor have been reported to be endocytosed through a clathrin-dependent as well as a clathrin-independent pathway. Segregation of these cell surface receptors through distinct endocytic pathways is known to regulate downstream signal duration and receptor trafficking, although it is unclear how it does this. Several lines of evidence indicate that PTPN3 and Eps15-Y850F accelerate downregulation of EGFR via a clathrin-independent but lipid raft-dependent pathway. First, EGF-488 trafficking assay revealed that EGF-488 was largely colocalized with lipid raft-associated protein caveolin-1 but not with clathrin in cells expressing PTPN3 or Eps15-Y850F. Second, analysis of EGFR profile by sucrose gradient fractionation showed that EGFR was concentrated in clathrin-enriched non-lipid fractions in control cells. However, overexpression of PTPN3 and Eps15-Y850F led to a redistribution of EGFR to caveolin-1-enriched lipid raft fractions. Third, disruption of lipid rafts with MβCD and filipin suppressed PTPN3- or Eps15-Y850F-induced EGFR degradation. How does PTPN3-mediated tyrosine dephosphorylation of Eps15 regulate EGFR for lipid raft-dependent endocytosis and lysosomal degradation? Accumulating evidence has shown that EGFR ubiquitination is not essential for its internalization, but appears to play an important role in endosomal sorting and lysosomal targeting of the receptor. One possibility is that tyrosine dephosphorylation of Eps15 by PTPN3 may affect the ubiquitination status of EGFR, accelerating EGFR for lysosomal degradation. Recently, an endosomally localized Eps15 isoform (Eps15b) has been identified that interacts with the Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) complex to mediate EGFR degradation. This study found that like Eps15, Eps15b is also a substrate of PTPN3, indicating a dual role for PTPN3 in the regulation of endocytic trafficking and endosomal sorting of EGFR (Li, 2014).

Accumulating evidence has indicated that PTPs can function as tumor suppressors or oncogenes depending on the substrate involved and the cellular context. It has been reported that PTPN3 expresses in gastric cancer cells and may play a role in gastric cancer progression and differentiation. PTPN3 has been found to coordinate with p38γ MAPK to promote Ras oncogenesis in colon cancer, and it has been found to stimulate breast cancer growth by inducing and stabilizing the protein expression of vitamin D receptor. Interestingly, recent studies have also indicated that PTPN3 plays a role in tumor suppression. Mutational analysis of the tyrosine phosphatome found PTPN3 along with five other PTPs (PTPRF, PTPRG, PTPRT, PTPN13 and PTPN14) are mutated in colorectal cancer. Moreover, the transcriptome of two NSCLC cell lines were analyzed, and one allele of PTPN3 was found to be mutated in the NSCLC cell line H2228. It was further shown that ectopic expression of PTPN3 inhibits the growth of NSCLC cells, although the molecular mechanisms underlying the growth inhibition remain unknown. The current data support a model in which PTPN3-mediated tyrosine dephosphorylation of Eps15 leads to EGFR degradation and tumor suppression in NSCLC cells. This study demonstrated that PTPN3 and Eps15-Y850F overexpression reduced EGFR protein levels and impeded the proliferation and migration of NSCLC cells. Moreover, PTPN3 and Eps15-Y850F significantly suppressed NSCLC tumor growth in a subcutaneous xenograft model. Conversely, depletion of PTPN3 enhanced EGFR stabilization and promoted NSCLC tumorigenicity both in vitro and in vivo. The findings are consistent with the idea that PTPN3 acts as a tumor suppressor in NSCLC (Li, 2014).

In conclusion, this study has identified Eps15 as an evolutionarily conserved dPtpmeg/PTPN3 substrate that regulates EGFR signaling. The finding that PTPN3-mediated tyrosine dephosphorylation of Eps15 modulates EGFR-dependent cancer progression may help contribute to the development of a targeting intervention in NSCLC (Li, 2014).


EGF receptor : Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

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