Interactive Fly, Drosophila

Signaling downstream of EGFR

A paradigm has been established whereby mutant tyrosine kinase receptors such as the v-erbB and v-fms gene products function as oncoproteins in the absence of ligand. A spontaneously occurring deletional mutant of the human epidermal growth factor receptor (EGFR-vIII) has been isolated from astrocytic neoplasms and transforms NIH3T3 cells in the absence of ligand. The EGFRvIII is constitutively complexed with the majority of cellular GRB2, suggesting a link to the Ras-Mitogen-activated protein (MAP) kinase pathway. The presence of EGFRvIII suppresses activation of p42 and p44 MAP kinases by phorbol and serum; however, MEK activation by PMA is not suppressed by EGFRvIII. Basal and PMA-stimulated MAP kinase activity in EGFRvIII-transfected cells is augmented by the tyrosine phosphatase inhibitor sodium vanadate. EGFR-vIII is capable of transducing downstream signals through MAP kinase (See the Drosophila homolog rolled) as evidenced by activation of cytoplasmic phospholipase A2 at levels similar to that induced by intact EGFR. These results suggest that EGFR-vIII constitutively activates downstream signal transduction through MAP kinase. This chronic stimulation of the MAP kinase pathway may represent one means by which mutant EGFR transduces an oncogenic signal (Montgomery, 1995).

The expression of an oncogenic rasHa gene in epidermal keratinocytes stimulates the tyrosine phosphorylation of protein kinase C delta (See Drosophila PKC) and inhibits its enzymatic activity. Keratinocytes expressing an activated rasHa gene secrete transforming growth factor alpha (TGFalpha) and have an altered response to differentiation signals involving protein kinase C. The neoplastic phenotype of v-rasHa expressing keratinocytes can be partially mimicked in vitro by chronic treatment with TGF alpha. Treatment of primary keratinocyte cultures for 4 days with TGFalpha induces tyrosine phosphorylation of PKCdelta. The PKCdelta that is tyrosine-phosphorylated in response to TGFalpha has reduced activity compared with the nontyrosine-phosphorylated PKCdelta. Treatment of keratinocytes expressing a normal epidermal growth factor receptor (EGFR) with TGFalpha or epidermal growth factor for 5 min induces PKCdelta tyrosine phosphorylation. This acute epidermal growth factor treatment does not induce tyrosine phosphorylation of PKCdelta in keratinocytes isolated from waved-2 mice that have a defective epidermal growth factor receptor. Recombinant PKC delta in vitro is not phosphorylated by purified EGFR, however it is phosphorylated by members of the Src family (c-Src, c-Fyn: see Drosophila Src oncogene 1) and membrane preparations from keratinocytes. Furthermore, clearing c-Src or c-Fyn from keratinocyte membrane lysates decreases PKCdelta tyrosine phosphorylation; c-Src and c-Fyn isolated from keratinocytes treated with TGFalpha have increased kinase activity. Acute or chronic treatment with TGFalpha does not induce significant PKCdelta translocation in contrast to the phorbol ester, which induces both translocation and tyrosine phosphorylation of PKCdelta. This suggests that TGFalpha-induced tyrosine phosphorylation of PKC delta results from the activation of a tyrosine kinase rather than physical association of PKCdelta with a membrane-anchored tyrosine kinase. Taken together, these results indicate that PKCdelta activity is inhibited by tyrosine phosphorylation in response to EGFR-mediated signaling. Activation of a member of the Src kinase family may be the proximal tyrosine kinase acting on PKCdelta in keratinocytes (Denning, 1996).

Phospholipase C-gamma (PLC gamma) is required for EGF-induced motility; however, the molecular basis for how PLC gamma modulates the actin filament network underlying cell motility remains undetermined. It has been proposed that one connection to the actin cytoskeleton is direct hydrolysis of PIP2 with subsequent mobilization of membrane-associated actin modifying proteins. Gelsolin anti-sense oligonucleotide treatment of NR6 cells expressing the motogenic full-length (WT) and truncated c'1000 EGFR decreases endogenous gelsolin by 30-60%; this results in preferential reduction of EGF-induced cell movement by > 50% with little effect on the basal motility. EGF treatment decreases the gelsolin mass associated with the membrane fraction in motogenic WT and c'1000 EGFR NR6 cells but not in cells expressing the fully mitogenic (but nonmotogenic c'973) EGFR. Blocking PLC activity diminishes both this mobilization of gelsolin and EGF-induced motility, suggesting that gelsolin mobilization is downstream of PLC. Concomitantly, there is a reorganization of submembranous actin filaments correlating directly with PLC activation and gelsolin mobilization. In vivo expression of a peptide that is reported to compete in vitro with gelsolin in binding to PIP2 dramatically increases basal cell motility in NR6 cells expressing either motogenic or nonmotogenic EGFR; EGF does not further augment cell motility and gelsolin mobilization. Cells expressing this peptide demonstrate actin reorganization similar to that observed in EGF-treated control cells. These data suggest that much of the EGF-induced motility and cytoskeletal alterations can be reproduced by displacement of select actin-modifying proteins from a PIP2-bound state. This provides a signaling mechanism for translating cell surface receptor-mediated biochemical reactions to the cell movement machinery (Chen, 1996).

Human squamous cell carcinomas (SCC) frequently express elevated levels of epidermal growth factor receptor (EGFR). EGFR overexpression in SCC-derived cell lines correlates with the cellular ability to invade in an in vitro invasion assay in response to EGF, whereas benign epidermal cells, which express low levels of EGFR, do not invade. EGF-induced invasion of SCC-derived A431 cells is inhibited by sustained expression of the dominant negative mutant of c-Jun, TAM67, suggesting a role for the transcription factor AP-1 (activator protein-1) in regulating invasion. Significantly, sustained TAM67 expression inhibits growth factor-induced cell motility and the reorganization of the cytoskeleton and cell-shape changes essential for this process: TAM67 expression inhibits EGF-induced membrane ruffling, lamellipodia formation, cortical actin polymerization and cell rounding. Introduction of a dominant negative mutant of Rac and of the Rho inhibitor C3 transferase into A431 cells indicates that EGF-induced membrane ruffling and lamellipodia formation are regulated by Rac, whereas EGF-induced cortical actin polymerization and cell rounding are controlled by Rho. Constitutively activated mutants of Rac or Rho introduced into A431 or A431 cells expressing TAM67 (TA cells) induce equivalent actin cytoskeletal rearrangements, suggesting that the effector pathways downstream of Rac and Rho required for these responses are unimpaired by sustained TAM67 expression. However, EGF-induced translocation of Rac to the cell membrane, which is associated with its activation, is defective in TA cells. These data establish a novel link between AP-1 activity and EGFR activation of Rac and Rho, which in turn mediate the actin cytoskeletal rearrangements required for cell motility and invasion (Malliri, 1998).

A current model of growth factor-induced cell motility invokes integration of diverse biophysical processes required for cell motility, including dynamic formation and disruption of cell/substratum attachments along with extension of membrane protrusions. To define how these events are actuated by biochemical signaling pathways, an investigation was carried out as to whether epidermal growth factor (EGF) induces disruption of focal adhesions in fibroblasts. EGF treatment of NR6 fibroblasts presenting full-length WT EGF receptors (EGFR) reduces the fraction of cells presenting focal adhesions from approximately 60% to approximately 30% within 10 minutes. The dose dependency of focal adhesion disassembly mirrors that for EGF-enhanced cell motility, being noted at 0.1 nM EGF. EGFR kinase activity is required as cells expressing two kinase-defective EGFR constructs retain their focal adhesions in the presence of EGF. The short-term (30 minutes) disassembly of focal adhesions is reflected in decreased adhesiveness of EGF-treated cells to substratum. Known motility-associated pathways were examined to determine whether these contribute to EGF-induced effects. Phospholipase C(gamma) (PLC[gamma]) activation and mobilization of gelsolin from a plasma membrane-bound state have been shown to be required for EGFR-mediated cell motility. In contrast, this study found that short-term focal adhesion disassembly is induced by a signaling-restricted truncated EGFR (c'973) that fails to activate PLC(gamma) or mobilize gelsolin. The PLC inhibitor U73122 has no effect on this process, nor is the actin severing capacity of gelsolin required as EGF treatment reduces focal adhesions in gelsolin-devoid fibroblasts, further supporting the contention that focal adhesion disassembly is signaled by a pathway distinct from that involving PLC(gamma). Because both WT and c'973 EGFR activate the erk MAP kinase pathway, additional exploration of this signaling pathway has been carried out. The pathway has not previously been associated with growth factor-induced cell motility. Levels of the MEK inhibitor PD98059 that block EGF-induced mitogenesis and MAP kinase phosphorylation also abrogate EGF-induced focal adhesion disassembly and cell motility. In summary, EGFR kinase activity to directly stimulates focal adhesion disassembly and cell/substratum detachment, in relation to its ability to stimulate migration. A model of EGF-induced motogenic cell responses is proposed in which the PLC(gamma) pathway stimulating cell motility is distinct from the MAP kinase-dependent signaling pathway leading to disassembly and reorganization of cell-substratum adhesion (Xie, 1998).

Acetylcholine receptor (AChR) genes are transcribed selectively in synaptic nuclei of skeletal muscle fibers, leading to accumulation of the mRNAs encoding AChR subunits at synaptic sites. The signals that regulate synapse-specific transcription remain elusive, though Neuregulin-1 is considered a favored candidate. Motor neurons and terminal Schwann cells express neuregulin-2, a neuregulin-1-related gene. In skeletal muscle, Neuregulin-2 protein is concentrated at synaptic sites, where it accumulates adjacent to terminal Schwann cells. Neuregulin-2 stimulates AChR transcription in cultured myotubes expressing ErbB4, as well as ErbB3 and ErbB2, but not in myotubes expressing only ErbB3 and ErbB2. Thus, Neuregulin-2 is a candidate for a signal that regulates synaptic differentiation (Rimer, 2004).

Proteolytic processing and ectodomain shedding have been described for a broad spectrum of transmembrane proteins under both normal and pathophysiological conditions and has been suggested as one mechanism to regulate a protein's function. It has also been documented for the receptor-like protein tyrosine phosphatase PTP-LAR, induced by treating cells with the tumor promoter TPA or the calcium ionophor A23187. The epidermal growth factor receptor (EGFR) has been identified as both an association partner of PTP-LAR, that mediates phosphorylation of the latter, as well as an inducer of LAR-cleavage. Both overexpression of this kinase and stimulation of endogenous EGFR in various tumor cell lines have been shown to induce proteolytic processing of the catalytic LAR-P-subunit. In contrast to TPA-induced shedding of PTP-LAR, EGFR-mediated cleavage does not require PKC-activity. For both stimuli, however, processing of the P-subunit turns out to be dependent on the activation of the MAP kinases ERK1 and ERK2, and is completely abrogated upon pre-treating cells with Batimastat, indicating the involvement of a metalloproteinase in this pathway. Being strongly impaired in fibroblasts derived from ADAM-17/TACE-knockout-mice or tumor cells that express a dominant negative mutant of ADAM-17/TACE, cleavage of PTP-LAR is suggested to be mediated by this metalloproteinase. Paralleled by rapid reduction of cell surface-localized LAR-E-subunit, EGFR-induced cleavage could be shown to lead to degradation of the catalytic LAR-P-subunit, thereby resulting in a significantly reduced overall cellular phosphatase activity of PTP-LAR. These results for the first time identify a protein tyrosine phosphatase as a potential substrate of TACE and describe proteolytic processing of PTP-LAR as a means of regulating phosphatase activity downstream and thus under the control of EGFR-mediated signaling pathways (Ruhe, 2006).

Intracellular feedback on EGFR activity

The mammalian proto-oncoprotein Cbl and its homologs in C. elegans and Drosophila (see Drosophila Cbl) are evolutionarily conserved negative regulators of the epidermal growth factor receptor (EGF-R). In a mammalian cell culture system, overexpression of wild-type Cbl enhances down-regulation of activated EGF-R from the cell surface. The Cbl tyrosine kinase-binding (TKB) domain is essential for this activity. Whereas wild-type Cbl enhances ligand-dependent EGF-R ubiquitination, down-regulation from the cell surface, accumulation in intracellular vesicles, and degradation, a Cbl TKB domain-inactivated mutant (G306E) does not have these effects. Furthermore, the transforming truncation mutant Cbl-N (residues 1-357), comprising only the Cbl TKB domain, functions as a dominant negative protein. It colocalizes with EGF-R in intracellular vesicular structures, yet it suppresses down-regulation of EGF-R from the surface of cells expressing endogenous wild-type Cbl. Therefore, Cbl-mediated down-regulation of EGF-R requires the integrity of both the N-terminal TKB domain and additional C-terminal sequences. A Cbl truncation mutant comprising amino acids 1-440 functions like wild-type Cbl in down-regulation assays. This mutant includes the evolutionarily conserved TKB and RING finger domains but lacks the less conserved C-terminal sequences. It is concluded that the evolutionarily conserved N terminus of Cbl is sufficient to effect enhancement of EGF-R ubiquitination and down-regulation from the cell surface (Lill, 2000).

Clues to a potential mechanism for Cbl-mediated down-regulation of EGF-R activity were obtained by studying other tyrosine kinases that are negatively regulated by Cbl. Suppression by Cbl of the Syk non-receptor tyrosine kinase and the PDGF-R-alpha tyrosine kinase correlates with Cbl-induced degradation of these proteins. In the case of PDGF-R-alpha, overexpression of Cbl enhances ligand-dependent receptor ubiquitination. Thus, the unifying theme in Cbl-mediated negative regulation of tyrosine kinases appears to be the facilitation of activated tyrosine kinase degradation. The molecular basis for this facilitation has not been defined. However, several lines of evidence suggest that Cbl might act at the level of endocytosis of activated EGF-R. (1) Whereas individual null mutations in the C. elegans SLI-1 (Cbl) and UNC-101 proteins induce no phenotype on a wild-type LET-23 (EGF-R) background, their combination greatly enhances LET-23 signaling, resulting in a multi-vulva phenotype. The UNC-101 protein is a homolog of mammalian AP-47, an adaptin protein involved in clathrin-mediated endocytosis. Therefore, UNC-101 and SLI-1 may have linked but distinct functions in the process of clathrin-mediated endocytosis of EGF-R, or their functions may be in part redundant. (2) Both mammalian Cbl and Drosophila Cbl associate with EGF-R and colocalize with it to intracellular vesicles upon cell stimulation. Although the nature of these vesicles has not been defined, their appearance soon after Egf stimulation is consistent with that of endosomal compartments, which have been demonstrated to function in EGF-R down-regulation. (3) Whereas Cbl undergoes tyrosine phosphorylation and EGF-R association upon Egf stimulation of cells, detectable Cbl phosphorylation is not induced upon ligation of other ErbB family members by their ligands. Notably, the EGF-R is efficiently endocytosed, whereas the other ErbB family members have been reported to be endocytosis-impaired. The correlation of Cbl tyrosine phosphorylation and EGF-R binding with the ability of the EGF-R to be endocytosed in response to activating ligand indicates that Cbl may be a regulator involved in the receptor endocytosis process (Lill, 2000 and references therein).

These results indicate that at least two Cbl domains are required for enhanced degradation of activated EGF-R and that they may differentially impact the EGF-R intracellular trafficking pathway. For efficient colocalization of Cbl and EGF-R in endocytic vesicles, a functional Cbl TKB domain appears to be necessary (the Cbl-G306E mutant shows poor colocalization with EGF-R). However, the TKB domain is not sufficient to effect down-regulation of internalized EGF-R (Cbl-N colocalizes with EGF-R in endocytic vesicular structures, yet fails to enhance receptor degradation). Apparently, Cbl-mediated enhancement of EGF-R transit through the endocytic pathway involves a commitment step downstream of Cbl·EGF-R recruitment to endosomes, and it is at this point that the fates of Cbl·EGF-R and Cbl-N·EGF-R complexes diverge. A second Cbl domain is clearly required at this point to route Cbl·EGF-R complexes toward the lysosomal degradation pathway. In conjunction with the TKB domain, Cbl amino acids 358-440 are sufficient to effect enhanced degradation of internalized EGF-R. The latter region of Cbl encompasses the evolutionarily conserved RING finger domain that is present in all Cbl homologs that suppress EGF-R signaling in vivo, and that plays a role in EGF-R down-regulation. Ligand-activated, internalized EGF-R is either targeted to the lysosome or recycled back to the cell surface, and the point of divergence for these routing pathways lies within the multivesicular bodies. Further studies will determine whether the Cbl TKB and 358-440 domains effect enhanced receptor degradation at this stage of the EGF-R trafficking pathway (Lill, 2000).

One remarkable aspect of data presented here is that overexpressed wild-type Cbl enhances ligand-dependent EGF-R down-regulation beyond the level observed for cells expressing only the endogenous Cbl. These results raise two possibilities: either Cbl is a limiting factor required to route EGF-R·wild-type Cbl complexes into the endosomal/lysosomal pathway for protein degradation, or overexpression of Cbl inactivates a repressor that normally limits entry of EGF-R into the endosomal/lysosomal degradation pathway. Both interpretations invoke the existence of an EGF-R endocytosis pathway that is saturable. Although the current results do not favor one interpretation over the other, they implicate Cbl as a limiting factor that could set the saturation level of the EGF-R down-regulation pathway (Lill, 2000).

c-Cbl plays a negative regulatory role in tyrosine kinase signaling by an as yet undefined mechanism. Using the yeast two-hybrid system and an in vitro binding assay, it has been demonstrated that the c-Cbl RING finger domain interacts with UbcH7, a ubiquitin-conjugating enzyme (E2). UbcH7 interacts with the wild-type c-Cbl RING finger domain but not with a RING finger domain that lacks the amino acids that are deleted in 70Z-Cbl, an oncogenic mutant of c-Cbl. The in vitro interaction is enhanced by sequences on both the N- and C-terminal sides of the RING finger. In vivo and in vitro experiments reveal that c-Cbl and UbcH7 synergistically promote the ligand-induced ubiquitination of the epidermal growth factor receptor (EGFR). In contrast, 70Z-Cbl markedly reduces the ligand-induced, UbcH7-mediated ubiquitination of the EGFR. MG132, a proteasome inhibitor, significantly prolongs the ligand-induced phosphorylation of both the EGFR and c-Cbl. Thus, c-Cbl plays an essential role in the ligand-induced ubiquitination of the EGFR by a mechanism that involves an interaction of the RING finger domain with UbcH7. This mechanism participates in the down-regulation of tyrosine kinase receptors and loss of this function, as occurs in the naturally occurring 70Z-Cbl isoform, probably contributes to oncogenic transformation (Yokouchi, 1999).

The epidermal growth factor receptor (EGFR) is regulated by at least two mechanisms involving protein kinase C (PKC): inhibition of EGF binding and inhibition of EGF-stimulated tyrosine kinase activity. Does mitogen-activated protein kinase (MAPK) mediate the inhibitory effects of PKC on either EGFR binding or kinase activity? The results indicate that (1) PKC down-regulates EGFR tyrosine kinase activity by an MEK-dependent mechanism presumably involving MAPK; (2) the inhibition by PKC is not a direct result of phosphorylation of the EGFR by PKC or MAPK; (3) activation of MAPK is not sufficient to regulate EGFR kinase activity; and (4) PKC-mediated down-regulation of EGF binding and EGFR kinase activity occurs by different mechanisms. These data are consistent with a model for regulation of the EGFR by other receptors whereby their activation of PKC, in conjunction with MAPK, results in the phosphorylation of a protein(s) that modulates EGFR kinase activity (Morrision, 1996).

Activation of voltage-sensitive calcium channels (VSCCs) can stimulate the Ras/mitogen-activated protein kinase (MAPK) signaling pathway. Calcium influx through L-type VSCCs leads to tyrosine phosphorylation of the adaptor protein Shc and its association with the adaptor protein Grb2, which is bound to the guanine nucleotide exchange factor Sos1. In response to calcium influx, Shc, Grb2, and Sos1 inducibly associate with the epidermal growth factor receptor (EGFR), a 180-kDa tyrosine-phosphorylated protein. Calcium influx induces tyrosine phosphorylation of the EGFR to levels that can activate the MAPK signaling pathway. Thus, ion channel activation stimulates growth factor receptor signal transduction (Rosen, 1996).

Nerve growth factor (NGF) treatment causes a profound down-regulation of epidermal growth factor receptors during the differentiation of PC12 cells. This process is characterized by a progressive decrease in epidermal growth factor (EGF) receptor level. Treatment of the cells with NGF for 5 days produces a 95% reduction in the amount of EGF receptors. This down-regulation does not occur in PC12nnr5 cells, which lack the p140(trk) NGF receptor. However, in PC12nnr5 cells stably transfected with p140(trk), the NGF-induced heterologous down-regulation of EGF receptors is reconstituted in part. NGF-induced heterologous down-regulation, but not EGF-induced homologous down-regulation of EGF receptors, is blocked in Ras- and Src-dominant-negative PC12 cells. Treatment with either pituitary adenylate cyclase-activating peptide (PACAP) or staurosporine stimulates neurite outgrowth in PC12 cell variants, but neither induces down-regulation of EGF receptors. NGF treatment of PC12 cells in suspension induces down-regulation of EGF receptors in the absence of neurite outgrowth. These results strongly suggest a p140(trk)-, Ras- and Src-dependent mechanism for NGF-induced down-regulation of EGF receptors and separate this process from NGF-induced neurite outgrowth in PC12 cells (Lazarovici, 1997).

Receptor desensitization is accomplished by accelerated endocytosis and degradation of ligand-receptor complexes. An in vitro reconstituted system indicates that Cbl (see Drosophila Cbl) adaptor proteins directly control downregulation of the receptor for the epidermal growth factor (EGFR) by recruiting ubiquitin-activating and -conjugating enzymes. A sequential process is inferred initiated by autophosphorylation of EGFR at a previously identified lysosome-targeting motif that subsequently recruits Cbl. This is followed by tyrosine phosphorylation of c-Cbl at a site flanking its RING finger, which enables receptor ubiquitination and degradation. Whereas all three members of the Cbl family can enhance ubiquitination, two oncogenic Cbl variants, whose RING fingers are defective and phosphorylation sites are missing, are unable to desensitize EGFR. This study identifies Cbl proteins as components of the ubiquitin ligation machinery and implies that they similarly suppress many other signaling pathways (Levkowitz, 1999).

The 'metastasis suppressor' CD82/KAI-1, a member of the tetraspanin superfamily of transmembrane proteins, is widely distributed in normal tissues, and has been shown to be suppressed in the advanced stages of various epithelial malignancies. Although the physiological relevance of this change is unknown, in vitro data show that ectopically expressed CD82/KAI-1 can suppress tumor cell migration, a process underlying the dissemination of tumor cells in vivo. The function of CD82/KAI-1 is not known and it has been proposed that association of CD82/KAI-1 with other cell-surface proteins may be pivotal in directing its biological activities. The CD82/KAI-1 tetraspanin is directly associated with the EGF receptor (EGFR), and ectopic expression of CD82/KAI-1 in epithelial cells specifically suppresses EGF-induced lamellipodial extensions and cell migration. In cells expressing CD82/KAI-1, the initial activation of EGFR is not affected, but subsequent desensitization of EGF-induced signaling occurs more rapidly. This attenuation is correlated with an increased rate of receptor endocytosis. These results identify CD82/KAI-1 as a new regulator of EGF-induced signaling and show that the association of EGFR with the tetraspanin is critical in EGFR desensitization (Odintsova, 2000).

The c-Cbl protooncogene can function as a negative regulator of receptor protein tyrosine kinases (RPTKs) by targeting activated receptors for polyubiquitination and downregulation. This function requires its tyrosine kinase binding (TKB) domain for targeting RPTKs and RING finger domain to recruit E2 ubiquitin-conjugating enzymes. It has therefore been proposed that oncogenic Cbl proteins act in a dominant-negative manner to block this c-Cbl activity. In testing this hypothesis, it was found that although mutations spanning the RING finger abolish c-Cbl-directed polyubiquitination and downregulation of RPTKs, they do not induce transformation. In contrast, it is mutations within a highly conserved alpha-helical structure linking the SH2 and RING finger domains that render Cbl proteins oncogenic. Thus, Cbl transformation involves effects additional to polyubiquitination of RPTKs that are independent of the RING finger and its ability to recruit E2-conjugating enzymes (Thien, 2001).

It is proposed that oncogenic mutations are those that induce structural changes to the alpha helix that are sufficiently profound to disrupt contacts with both the tyrosine kinase binding (TKB) domain and the ubiquitin-conjugating enzyme (Ubc). This would explain why all the transforming mutants of Cbl also lose the ability to promote EGF receptor polyubiquitination. The linker helix is likely to be a critical determinant of c-Cbl's E3 function by precisely regulating the position and orientation of the TKB-bound substrate relative to the RING finger-bound E2 enzyme. Thus, mutations that alter linker/TKB interactions could also prevent substrate polyubiquitination, even in the presence of an intact RING finger. Indeed, a Y368-deleted GST-Cbl construct spanning amino acids 359-447 still binds E2, and has functional ubiquitin ligase activity in vitro, yet the full-length DeltaY368-Cbl is unable to direct polyubiquitination of the EGF receptor in vivo. Since abolishing c-Cbl's ability to direct receptor polyubiquitination alone is insufficient to induce transformation, this implicates deregulation of Cbl's TKB activity as being the key event in transformation. How this leads to enhanced signaling, and ultimately transformation, is unknown, but it is an important question that requires further investigation (Thien, 2001).

Ligand-induced desensitization of the epidermal growth factor receptor (EGFR) is controlled by c-Cbl, a ubiquitin ligase that binds multiple signaling proteins, including the Grb2 adaptor. Consistent with a negative role for c-Cbl, defective Tyr1045 of EGFR, an inducible c-Cbl docking site, enhances the mitogenic response to EGF. Signaling potentiation is due to accelerated recycling of the mutant receptor and a concomitant defect in ligand-induced ubiquitylation and endocytosis of EGFR. Kinetic as well as morphological analyses of the internalization-defective mutant receptor imply that c-Cbl-mediated ubiquitylation sorts EGFR to endocytosis and to subsequent degradation in lysosomes. Unexpectedly, however, the mutant receptor displays significant residual ligand-induced ubiquitylation, especially in the presence of an overexpressed c-Cbl. The underlying mechanism seems to involve recruitment of a Grb2 c-Cbl complex to Grb2-specific docking sites of EGFR, and concurrent acceleration of receptor ubiquitylation and desensitization. Thus, in addition to its well-characterized role in mediating positive signals, Grb2 can terminate signal transduction by accelerating c-Cbl-dependent sorting of active tyrosine kinases to destruction (Waterman, 2002).

Cbl is a multi-adaptor protein involved in ligand-induced down-regulation of receptor tyrosine kinases. It is thought that Cbl-mediated ubiquitination of active receptors is essential for receptor degradation and cessation of receptor-induced signal transduction. Cbl additionally regulates epidermal growth factor (EGF) receptor endocytosis. Cbl rapidly recruits CIN85 (Cbl-interacting protein of 85K) and endophilins (regulatory components of clathrin-coated vesicles) to form a complex with activated EGF receptors, thus controlling receptor internalization. CIN85 is constitutively associated with endophilins, whereas CIN85 binding to the distal carboxy terminus of Cbl is increased on EGF stimulation. Inhibition of these interactions is sufficient to block EGF receptor internalization, delay receptor degradation and enhance EGF-induced gene transcription, without perturbing Cbl-directed receptor ubiquitination. Thus, the evolutionary divergent C terminus of Cbl uses a mechanism that is functionally separable from the ubiquitin ligase activity of Cbl to mediate ligand-dependent downregulation of receptor tyrosine kinases (Soubeyran, 2002).

Phospholipase C-gamma1 (PLC-gamma1) plays pivotal roles in cellular growth and proliferation through its two Src homology (SH) 2 domains and its single SH3 domain, which interact with signaling molecules in response to various growth factors and hormones. However, the role of the SH domains in the growth factor-induced regulation of PLC-gamma1 is unclear. By peptide-mass fingerprinting analysis Cbl has been identified as a binding protein for the SH3 domain of PLC-gamma1 from rat pheochromatocyte PC12 cells. Association of Cbl with PLC-gamma1 is induced by epidermal growth factor (EGF) but not by nerve growth factor (NGF). Upon EGF stimulation, both Cbl and PLC-gamma1 are recruited to the activated EGF receptor through their SH2 domains. Mutation of the SH2 domains of either Cbl or PLC-gamma1 abrogates the EGF-induced interaction of PLC-gamma1 with Cbl, indicating that SH2-mediated translocation is essential for the association of PLC-gamma1 and Cbl. Overexpression of Cbl attenuates EGF-induced tyrosine phosphorylation and the subsequent activation of PLC-gamma1 by interfering competitively with the interaction between PLC-gamma1 and EGFR. Taken together, these results provide the first indications that Cbl may be a negative regulator of intracellular signaling following EGF-induced PLC-gamma1 activation (Choi, 2003).

Regulation of the EGF-R promoter

Exogenously introduced wild-type and mutant p53 have recently been reported to enhance the human epidermal growth factor receptor (EGF-R) gene promoter activity in p53-deficient Saos2 osteosarcoma cells. A p53 binding site residing at position -265/-239 in the EGF-R proximal promoter has also been identified. Wild-type and mutant p53Ala143 enhance the EGF-R core promotor activity in cells that are either p53-deficient or contain wild-type or mutant endogenous p53. Upon further characterization of the various deletion fragments of the EGF-R promoter, a wild-type p53 responsive 62 bp region was identified residing at position -167/-105. The -167/-105 segment is responsive only to wild-type p53 but not to mutant p53Ala143 or p53His273. The -167/-105 segment of the EGF-R promotor contains one perfect and several imperfect consensus p53-binding half sites; indeed, in gel shift experiments, the 62 bp -167/-105 segment as well as the oligonucleotides corresponding to two p53 consensus half-sites within the 62 bp fragment, exhibit binding to p53-containing protein complexes. Thus, an additional wild-type p53 responsive site has been identified in the human EGF-R promoter. This site containing consensus p53-binding sequences resides at position -167/-105 and is proximal to a recently identified p53 binding element located at position -265/-239 in the EGF-R promotor (Sheikh, 1997).

Epidermal growth factor signaling induces behavioral quiescence in Caenorhabditis elegans

The epidermal growth factor receptor (EGFR)/ErbB receptor tyrosine kinases regulate several aspects of development, including the development of the mammalian nervous system. ErbB signaling also has physiological effects on neuronal function, with influences on synaptic plasticity and daily cycles of activity. However, little is known about the effectors of EGFR activation in neurons. This study shows that EGF signaling has a nondevelopmental effect on behavior in Caenorhabditis elegans. Ectopic expression of the EGF-like ligand LIN-3 at any stage induces a reversible cessation of feeding and locomotion. These effects are mediated by neuronal EGFR (also called LET-23) and phospholipase Cγ (PLC-γ), diacylglycerol-binding proteins, and regulators of synaptic vesicle release. Activation of EGFR within a single neuron, ALA, is sufficient to induce a quiescent state. This pathway modulates the cessation of pharyngeal pumping and locomotion that normally occurs during the lethargus period that precedes larval molting. These results reveal an evolutionarily conserved role for EGF signaling in the regulation of behavioral quiescence (Van Buskirk, 2007).

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