Cbl

Protein Interactions

Drosophila Cbl, when expressed in human cultured cells becomes associated with the EGF receptor. Expression of human c-Cbl is accompanied by an EGF-dependent increase in a tyrosine phosphorylated EGF-receptor bound to c-Cbl immunoprecipitates. Transfection with Drosophila Cbl cDNA reveals a p60 polypeptide corresponding to Drosophila Cbl. In nonstimulated cells, Drosophila Cbl is tyrosine phosphorylated, and activation with EGF leads to increased phosphorylation and association of Drosophila Cbl with the EGF-receptor. A Glycine315-to-Glu point mutation mutation in C. elegans Cbl suppresses the ability of this protein to act as a negative regulator of the C. elegans EGF-receptor signaling. The association of Drosophila Cbl and the EGF-receptor is prevented by an analogous mutation in Drosophila Cbl. The analgous mutation totally prevents tyrosine phosphorylation of the expressed protein by the EGF receptor (Meisner, 1997).

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).

Sprouty (Spry) was first identified in a genetic screen in Drosophila as an antagonist of fibroblast and epidermal growth factor receptors and Sevenless signaling, seemingly by inhibiting the receptor tyrosine kinase (RTK)/Ras/MAPK pathway. To date, four mammalian Sprouty genes have been identified; the primary sequences of the gene products share a well conserved cysteine-rich C-terminal domain with their Drosophila counterpart. The N-terminal regions do not, however, exhibit a large degree of homology. This study was aimed at identifying proteins with which human SPRY2 (hSPRY2) interacts in an attempt to understand the mechanism by which Sprouty proteins exert their down-regulatory effects. hSPRY2 associates directly with c-Cbl, a known down-regulator of RTK signaling. A short sequence in the N terminus of hSPRY2 was found to bind directly to the Ring finger domain of c-Cbl. Parallel binding was apparent between the Drosophila homologs of Sprouty and Cbl, with cross-species associations occurring at least in vitro. Coexpression of hSPRY2 abrogates an increase in the rate of epidermal growth factor receptor internalization induced by c-Cbl, whereas a mutant hSPRY2 protein unable to bind c-Cbl showed no such effect. These results suggest that one function of hSPRY2 in signaling processes downstream of RTKs may be to modulate c-Cbl physiological function such as that seen with receptor-mediated endocytosis (Wong, 2001).

In examining the sequence of Drosophila Sprouty, a region was found between amino acids 179 and 199 that shows a low homology to residues 36-53 in hSPRY2. Residues 36-53 lie within region 11-53, which is important for c-Cbl binding. It was therefore investigated if the binding domain could be further refined to a smaller region. FLAG-tagged hSPRY2 truncation and deletion constructs were expressed in 293T cells, and cell lysates were immunoprecipitated with anti-FLAG antibody and probed with anti-c-Cbl antibody. No binding is seen between c-Cbl and the 53C (without residues 1-53) and DeltaN36 (lacking amino acids 36-53) mutants, whereas binding is apparent between c-Cbl and the 30C (without residues 1-30) and full-length constructs. A reciprocal precipitation experiment was performed in which cell lysates from the same transfections were subjected to immunoprecipitation with anti-c-Cbl antibody and immunoblotted with anti-FLAG antibody. The result is in agreement with the above data. Thus, the c-Cbl-binding region of hSPRY2 is contained within sequence 36-53 (Wong, 2001).

To extend the binding investigation to Drosophila Spry and Drosophila Cbl, various constructs were made. (1) To investigate whether the Ring finger domain of Cbl is involved in binding to Spry, 293T cells were transiently transfected with Cbl, dCblDeltaRF (Drosophila Cbl lacking the ring finger domain), or vector alone. Cell lysates were subjected to pull-down assays with GST-Spry or GST alone. Whereas full-length Cbl binds to Spry, dCblDeltaRF does not. This result is indicative of the Ring finger domain of Cbl being involved in its binding to Spry. (2) Binding domain analyses were performed to ascertain the site in Spry that binds to the Ring finger domain of Cbl. The possible involvement of residues 179-199 in Spry was directly addressed. FLAG-tagged full-length Spry and the dSPRYN210 (amino acids 1-210), dSPRY202C (amino acids 202-592), and dSPRYDeltaN179 (mutant with a deletion of amino acids 179-199) constructs were transiently expressed in 293T cells, and lysates were incubated with either GST alone or GST-Cbl. Spry-derived proteins that contain residues 179-199 bind to Cbl, whereas those that lack the sequence do not. The region comprising residues 179-199 of Spry is therefore responsible for its interaction with Cbl; deletion of this region of Spry can similarly abolish its binding to c-Cbl (Wong, 2001).

Cbl and Sprint regulate early steps of RTK endocytosis

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Differential effects of Cbl isoforms on Egfr signaling in Drosophila

The Cbl family of proteins downregulate epidermal growth factor receptor (Egfr) signaling via receptor internalization and destruction. These proteins contain two functional domains, a RING finger domain with E3 ligase activity, and a proline rich domain mediating the formation of protein complexes. The Drosophila cbl gene encodes two isoforms, D-CblS and D-CblL. While both contain a RING finger domain, the proline rich domain is absent from D-CblS. Expression of either isoform is sufficient to rescue both the lethality of a D-cbl null mutant and the adult phenotypes characteristic of Egfr hyperactivation, suggesting that both isoforms downregulate Egfr signaling. Interestingly, targeted overexpression of D-CblL, but not D-CblS, results in phenotypes characteristic of reduced Egfr signaling and suppresses the effect of constitutive Egfr activation. The level of D-CblL was significantly correlated with the phenotypic severity of reduced Egfr signaling, suggesting that D-CblL controls the efficiency of downregulation of Egfr signaling. Furthermore, reduced dynamin function suppresses the effects of D-CblL overexpression in follicle cells, suggesting that D-CblL promotes internalization of activated receptors. D-CblL is detected in a punctate cytoplasmic pattern, whereas D-CblS is mainly localized at the follicle cell cortex. Therefore, D-CblS and D-CblL may downregulate Egfr through distinct mechanisms (Pai, 2006).

D-CblL has domains that allow it to act both as an ubiquitin ligase and as an adaptor mediating protein–protein interaction through its proline rich regions, while D-CblS lacks the proline rich region in the C terminus that may promote receptor internalization. Both isoforms rescue lethality in a D-cbl mutant background and can control Egfr signaling roughly correctly in the wings, eyes, and follicle cells. A low level of D-CblS partially rescues lethality and is accompanied by a mild wing phenotype characteristic of hyperactivated Egfr. However, when the expression levels are sufficient for better survival, no patterning defects are observed. This full rescue suggests interactions mediated through the C-terminus of D-CblL are not essential for viability, and D-CblS can downregulate activated Egfr properly by itself. Indeed, a mutant encoding a similar protein in C. elegans, sli-1ΔC, also retains some ability to negatively regulate Egfr signaling during vulval development of C. elegans. Furthermore, Cbl-3, which does not bind to CIN85, promotes the ubiquitination and degradation of activated Egfr in mammals (Pai, 2006).

Although the levels of D-CblS are critical for animal and cell survival as indicated by the lethal effect produced by high ubiquitous expression of transgenes driven by e22cGal4 or heat shock, no or only a slight patterning defect is observed when D-CblS is overexpressed using tissue specific Gal4 lines in the wings, eyes and egg shells. These data have demonstrated that D-CblS has the ability to control Egfr signaling in a manner similar to the wild-type D-cbl gene and high expression levels of D-CblS reduce survival but exert very little or no effects on patterning in specific tissues. Therefore, D-CblS appears to have a higher dynamic range than D-CblL in controlling proper Egfr signaling, which may reflect its higher level of expression in vivo compared to D-CblL (Pai, 2006).

Internalization and degradation of receptors is an efficient way to terminate signaling, and protein complexes interacting with the C-terminus of Cbl are involved in ligand-induced downregulation of Egfr through internalization. For example, the C terminal proline rich region of Cbl interacts with the multiple SH3 domain-containing protein, Grb2, which is required for Egfr endocytosis. Grb2 has been demonstrated to regulate the rate of Egfr internalization through clathrin-coated pits by recruiting c-Cbl. The Drosophila homolog of Grb2, Drk, binds to several kinds of proline rich motif through its SH3 domains. Interestingly, one classic SH3 binding motif PXXPXR was found in the C terminus of D-CblL. Moreover, two such motifs exist in Sli-1, which showed genetic interactions with SEM5, the C. elegans homolog of Grb2. Therefore, it is possible that D-CblL more efficiently promotes Egfr degradation in part through association with Drk, which does not bind to D-CblS (Pai, 2006).

In an alternative mechanism leading to Egfr downregulation, the Cbl-CIN85-endophilin complex mediates ligand-induced internalization of Egfr as well. The CIN85-Endophilin complex is thought to play a critical role at invagination during endocytosis by inducing negative membrane curvature. The PXXXPR motif in Cbl, responsible for interaction with CIN85, is also present in D-CblL. Through an intensive data base search a putative Drosophila homolog of CIN85 (CG31012), was identified which has three SH3 domains in the N-terminus and a proline rich region prior to the coiled-coil domain at the C-terminus. This information suggests that a protein–protein interaction network for Cbl-CIN85-Endophilin exists in Drosophila (Pai, 2006).

The phenotype characteristic of Egfr hypoactivation generated in the eyes, wings and egg shells by overexpression of D-CblL demonstrates that D-CblL is critical for control of Egfr signaling and high levels of D-CblL elevate the downregulation of Egfr signaling. Indeed it was also found that the severity of the ventralized egg shell phenotype is dependent on the levels of D-CblL. In addition, a dominant negative shibire mutant suppressed the D-Cbl effects on egg shell. These data indicate that activated Egfr units can be internalized rapidly through the activity of the endocytic protein complexes when D-CblL, but not D-CblS, is present at high levels. In addition to patterning defects, overexpression of D-CblL is usually accompanied by a lethal effect. The rapid internalization may lead to hypoactivation of Egfr and thus result in lethality, since Egfr signaling is required for cell survival (Pai, 2006).

It is crucial to control both the strength and the duration of signaling during development, and internalization of receptors is a key process for this control. To date, many reports have shown that there are several redundant mechanisms of internalization of activated receptors, such as clathrin-dependent and -independent, dynamin-dependent and -independent processes, as well as caveolar endocytosis. The contribution of these pathways in Egfr downregulation in vivo is not completely understood (Pai, 2006).

The data have shown that D-CblS, which lacks the C terminal proline rich domain, regulates Egfr signaling properly, suggesting that the interactions between Cbl and adaptor proteins are dispensable. D-CblS presumably promotes internalization of activated Egfr solely through its E3 ligase activity, and ubiquitination of Egfr subsequently recruits ubiquitin binding proteins, such as Epsin family members, which then serve as clathrin or caveolin adaptors to form clathrin-coated vesicle or caveolae in endocytic pathways. In contrast, D-CblL may have additional endocytic pathways other than those that are ubiquitination dependent, such as the interaction with the CIN85-endophilin complex through its C terminus. In addition, the Drk binding site in the C terminus of D-CblL may also increase the association of D-CblL and activated Egfr. Therefore, increase of D-CblL levels may promote Egfr downregulation by the lysosomal pathway mediated through D-CblL-CIN85-endophilin complex. In addition, D-CblL may promote polyubiquitination of Egfr through its E3 ligase activity while the constitutive binding between its SH3 domain and the proline rich motif of Drk provides a stable association between activated receptor and D-CblL, thus polyubiquitinated Egfr can be degraded by the 26S proteasome. In contrast, D-CblS only allows monoubiquitination on activated Egfr, which allows lysosomal degradation (Pai, 2006).

It is known that post-translational modifications of both receptor and components of the endocytic machinery control the rate of receptor degradation. The EGFR complex is sorted into multivesicular bodies where it either fuses with the lysosome for complete degradation and termination of signaling, or is recycled which prolongs the signaling. Since, the punctate pattern of D-CblL staining indicates a possible subcellular vesicle localization, D-CblL may also promote sorting activated Egfr to the lysosome by ubiquitination modification on components of the endocytic machinery. Taken together, these data strongly suggest that CblS and CblL mediate internalization of activated Egfr through distinct mechanisms, which are characterized by different genetic behaviors. Given the different intracellular localization patterns of D-CblS and D-CblL it is very likely that these differences reflect distinct endocytic pathways, which regulate Egfr signaling with different efficiencies and in a partially redundant manner (Pai, 2006).

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date revised: 15 December 2006 

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