EGF receptor
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).
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).
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).
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 (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).
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).
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 proteinñprotein 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).
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).
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
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