Cbl is a ubiquitin-protein ligase

Ubiquitination of receptor protein-tyrosine kinases (RPTKs) terminates signaling by marking active receptors for degradation. c-Cbl, an adapter protein for RPTKs, positively regulates RPTK ubiquitination in a manner dependent on its variant SRC homology 2 (SH2) and RING finger domains. Ubiquitin-protein ligases (or E3s) are the components of ubiquitination pathways that recognize target substrates and promote their ligation to ubiquitin. The c-Cbl protein acts as an E3 that can recognize tyrosine-phosphorylated substrates, such as the activated platelet-derived growth factor receptor, through its SH2 domain and that recruits and allosterically activates an E2 ubiquitin-conjugating enzyme through its RING domain. These results reveal an SH2-containing protein that functions as a ubiquitin-protein ligase and thus provide a distinct mechanism for substrate targeting in the ubiquitin system (Joazeiro, 1999).

Receptor desensitization is accomplished by accelerated endocytosis and degradation of ligand-receptor complexes. An in vitro reconstituted system indicates that 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).

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).

Colony-stimulating factor-1 (CSF-1) activation of the CSF-1 receptor (CSF-1R) causes Cbl protooncoprotein tyrosine phosphorylation, Cbl-CSF-1R association and their simultaneous multiubiquitination at the plasma membrane. The CSF-1R is then rapidly internalized and degraded, whereas Cbl is deubiquitinated in the cytoplasm without being degraded. Primary macrophages from gene-targeted mice have been used to study the role of Cbl. Cbl-/- macrophages form denser colonies and, at limiting CSF-1 concentrations, proliferate faster than Cbl+/+ macrophages. Their CSF-1Rs fail to exhibit multiubiquitination and a second wave of tyrosine phosphorylation, as previously suggested, is thought to be involved in preparation of the CSF-1-CSF-1R complex for endocytosis. Consistent with this result, Cbl-/- macrophage cell surface CSF-1-CSF-1R complexes are internalized more slowly, yet are still lysosomally degraded, and the CSF-1 utilization by Cbl-/- macrophages is reduced approximately 2-fold. Thus, attenuation of proliferation by Cbl is associated with its positive regulation of the coordinated multiubiquitination and endocytosis of the activated CSF-1R, and a reduction in the time that the CSF-1R signals from the cell surface. The results provide a paradigm for studies of the mechanisms underlying Cbl attenuation of proliferative responses induced by ligation of receptor tyrosine kinases (Lee, 1999).

Ubiquitin-protein ligases (E3s) regulate diverse cellular processes by mediating protein ubiquitination. The c-Cbl proto-oncogene is a RING family E3 that recognizes activated receptor tyrosine kinases, promotes their ubiquitination by a ubiquitin-conjugating enzyme (E2) and terminates signaling. The crystal structure of c-Cbl bound to a cognate E2 (UbcH7) and a kinase peptide shows how the RING domain recruits the E2. A comparison with a HECT family E3-E2 complex indicates that a common E2 motif is recognized by the two E3 families. The structure reveals a rigid coupling between the peptide binding and the E2 binding domains and a conserved surface channel leading from the peptide to the E2 active site, suggesting that RING E3s may function as scaffolds that position the substrate and the E2 optimally for ubiquitin transfer (Zheng, 2000).

Cbl and FGF receptor signaling

Attenuation of growth factor signaling is essential for the regulation of developmental processes and tissue homeostasis in most organisms. The product of Cbl protooncogene is one such regulator, which functions as an ubiquitin ligase that ubiquitinates and promotes the degradation of a variety of cell signaling proteins. Grb2 bound to tyrosine-phosphorylated FRS2 alpha forms a ternary complex with Cbl by means of its Src homology 3 domains, resulting in the ubiquitination of fibroblast growth factor (FGF) receptor and FRS2 alpha in response to FGF stimulation. These observations highlight the importance of FRS2 alpha in the assembly of both positive (i.e., Sos, phosphatidylinositol 3-kinase) and negative (i.e., Cbl) signaling proteins to mediate a balanced FGF signal transduction. However, the partial inhibition of FGF receptor down-regulation in FRS2 alpha-/- cells indicates that the attenuation of signaling by FGF receptor is regulated by redundant or multiple mechanisms (Wong, 2004).

Interaction of Cbl with Sprouty

Drosophila Sprouty (dSpry) was genetically identified as a novel antagonist of fibroblast growth factor receptor (FGFR), epidermal growth factor receptor (EGFR) and Sevenless signaling, ostensibly by eliciting its response on the Ras/MAPK pathway. Four mammalian sprouty genes have been cloned, which appear to play an inhibitory role mainly in FGF-mediated lung and limb morphogenesis. Evidence is presented that describes the functional implications of the direct association between human Sprouty2 (hSpry2) and c-Cbl, and its impact on the cellular localization and signaling capacity of EGFR. Contrary to the consensus view that Spry2 is a general inhibitor of receptor tyrosine kinase signaling, hSpry2 was shown to abrogate EGFR ubiquitylation and endocytosis, and sustain EGF-induced ERK signaling that culminates in differentiation of PC12 cells. Correlative evidence showed the failure of hSpryDelta2N11 and mSpry4, both deficient in c-Cbl binding, to instigate these effects. hSpry2 interacts specifically with the c-Cbl RING finger domain and displaces UbcH7 from its binding site on the E3 ligase. It is concluded that hSpry2 potentiates EGFR signaling by specifically intercepting c-Cbl-mediated effects on receptor down-regulation (Wong, 2002).

The means by which receptor ubiquitylation influences protein trafficking remain obscure, including the role of mono- and poly-ubiquitylation. The consensus view is that monoubiquitin or short ubiquitin chains are sufficient to direct internalization of cell surface proteins, whereas the proteasomal machinery recognizes polyubiquitylated proteins in Saccharomyces cerevisiae. In mammalian cells however, the situation is not as clear because a number of plasma membrane proteins that are ubiquitylated appear to be degraded through both the proteasomal and lysosomal pathways. Other classical endocytic signals include reversible modification such as phosphorylation, damage to the protein, genetically encoded sequence motifs (e.g. YXXPhi, where Phi is a bulky hydrophobic amino acid; MPXY or di-leucine), as well as sorting events that are coupled to clathrin-dependent or -independent routes. There are currently disparate views on how and where c-Cbl ubiquitylates its target RTKs; evidence derived from studies with yeast, growth hormone receptor and inhibition of ErbB-1/EGFR (and other diverse receptors) uptake into internalized vesicles using dynamin mutants suggest that ubiquitylation may be associated with sorting at the plasma membrane. c-Cbl-mediated ubiquitylation of EGFRs has been shown to occur at the plasma membrane, which then facilitates recruitment of activated EGFRs into clathrin-coated pits and the complex remains associated throughout the endocytic route. The results of this study further support the notion that c-Cbl is likely to act on EGFR at the cell surface, and inhibition of this interaction by hSpry2 attenuates early stages of receptor internalization. The data concur with previous evidence pertaining to the endocytic events governing mCSF-1R, where internalization of the macrophage receptor is retarded in c-Cbl-defective cells and yeast membrane receptor regulation, but is seemingly at odds with reports on EGFR endocytosis where c-Cbl has been arguably implicated as an endosomal sorting protein with signaling potential (Wong, 2002 and references therein).

In recent studies involving the analysis of crystal structures, it was demonstrated that UbcH7 interacts closely with both the RING finger domain and the N-terminal 70Z linker region of c-Cbl, apparently initiated upon tyrosine phosphorylation on residue 371 on the linker sequence by activated EGFR. The present study provides additional insights into the mechanism of c-Cbl's mediatory effect on receptor ubiquitylation, in that the binding of UbcH7 can be successfully competed off by hSpry2. Much remains to be elucidated regarding the specific details of c-Cbl-dependent ubiquitylation, such as: resolving the identity of the candidate lysine residue on c-Cbl that becomes ubiquitylated, elucidating the structural conformation of phosphorylated c-Cbl (on Y371) and determining whether dimerization of c-Cbl might be important in its function as a ubiquitin ligase -- all of which will advance understanding of how hSpry2 intercepts and disrupts the functional role of its E3-binding partner (Wong, 2002).

Growth factors and their receptor tyrosine kinases play pivotal roles in development, normal physiology, and pathology. Signal transduction is regulated primarily by receptor endocytosis and degradation in lysosomes ('receptor downregulation'). c-Cbl is an adaptor that modulates this process by recruiting binding partners, such as ubiquitin-conjugating enzymes. The role of another group of adaptors, Sprouty proteins, is less understood; although, studies in insects have implicated the founder protein in the negative regulation of several receptor tyrosine kinases. By utilizing transfection of living cells, as well as reconstituted in vitro systems, a dual regulatory mechanism has been identified that combines human Sprouty2 and c-Cbl. Upon activation of the receptor for the epidermal growth factor (EGFR), Sprouty2 undergoes phosphorylation at a conserved tyrosine that recruits the Src homology 2 domain of c-Cbl. Subsequently, the flanking RING finger of c-Cbl mediates poly-ubiquitination of Sprouty2, which is followed by proteasomal degradation. Because phosphorylated Sprouty2 sequesters active c-Cbl molecules, it impedes receptor ubiquitination, downregulation, and degradation in lysosomes. This competitive interplay occurs in endosomes, and it regulates the amplitude and longevity of intracellular signals. It is concluded that Sprouty2 is an inducible antagonist of c-Cbl, and together they set a time window for receptor activation. When incorporated in signaling networks, the coupling of positive (Sprouty) to negative (Cbl) feedback loops can greatly enhance output diversification (Rubin, 2003).

Genetic screens performed in invertebrates have identified several negative regulators of RTKs, including Cbl, Sprouty, Kekkon, and Argos. However, unlike the Cbl pathway, which is present in worms, the other three pathways do not exist in C. elegans, suggesting that they were added as secondary regulators that fine-tune RTK function. Consistent with this notion, biochemical studies have identified an interlinked dual feedback loop that combines c-Cbl and Spry2. A previously unidentified stimulus-dependent phosphorylation of Spry2, on an evolutionary conserved tyrosine residue (tyrosine 55), plays a key role in these interactions. Once phosphorylated, this residue acts as the core of an inducible docking site for the SH2 domain of c-Cbl, and, subsequently, it enables the flanking RING finger of c-Cbl to covalently link ubiquitin molecules to Spry2. Similar to trans-phosphorylation of Spry2, a c-Cbl docking site is established upon autophosphorylation of RTKs (e.g., tyrosine 1045 of EGFR), and it consequently enables receptor ubiquitination. These results imply that these two analogous processes occur concurrently, most likely at the plasma membrane and in endosomes, and that they are mutually competitive. As a result, Spry2 regulates ubiquitination and subsequent degradation of EGFR, and EGFR targets Spry2 to proteasomal degradation. The uncovered interplay between c-Cbl and Spry2 fine-tunes downstream signaling, which explains the role of these adaptor molecules in balancing between activation and repression of RTKs (Rubin, 2003).

An interesting outcome of the coupling between phosphorylation and ubiquitination of Spry2 and EGFR is a predicted oscillation of the Spry2 level. Studies in insects and in mammalian cells have revealed that Sprouty proteins, along with other regulators like Kekkon and Argos, are transcriptionally induced upon activation of RTKs. Hence, transcription from the spry2 gene is expected to follow the rapid, ligand-induced degradation of Spry2. Interestingly, a similar compensatory loop enables EGF to upregulate transcription from the egfr gene. Thus, the protein degradation-based feedback loops that regulate Sprouty and RTKs are mirrored at the gene transcriptional level, and this bimodal circuit ensures receptor homeostasis. Consistent with the critical role of tyrosine 55 of Spry2 in regulating EGFR signaling, a screen of Spry2 and Spry4 mutants has identified the respective Y55F and Y53F point mutants as dominant-negative proteins capable of potentiating MAPK activation by FGF. Likewise, deletion of residues 11-53 of Spry2 abolishes c-Cbl binding, probably because the Spry2 deletion construct impairs the amino-terminal part of the Cbl's docking site. In contrast to the current results, previous studies have identified the RING finger of c-Cbl, not the SH2 domain, as the site of interaction with Spry2. While the current results do not exclude weak binding with the RING finger, the strong interaction between Spry2 and an SH2-only mutant of Cbl indicates that the phosphotyrosine-dependent interactions govern the functional outcome in living cells (Rubin, 2003).

Along with the prospect that Sprouty proteins interfere with signaling events at a point upstream to that predicted by genetic and biochemical analyses, these results imply that Sprouty acts as a positive rather than a negative regulator of EGF signaling. It is reasonable to assume that autophosphorylation of EGFR and subsequent recruitment of c-Cbl precede the indirect transphosphorylation of Spry2. Thus, delayed activation of the Spry-mediated loop is expected to occur after c-Cbl initiates receptor ubiquitination and endocytosis. In the next step, Spry2 itself is inactivated, by means of ubiquitination and degradation, which enables a new cycle of receptor activation/inactivation. At the gene expression level, EGF-induced synthesis of Spry2 is expected to replenish the cellular pool of active Spry2 molecules. This highly regulated sequence of events predicts staggering waves of active RTKs and Sprouty proteins in growth factor-stimulated tissues. In the context of a signaling network, such as ErbB, temporal regulation of the levels of RTKs and Sprouty proteins would enable repeated stimulation by growth factors, resist perturbations, and diversify the output by balancing receptor desensitization and resensitization (Rubin, 2003).

Sprouty was originally identified in a genetic screen in Drosophila as an antagonist of fibroblast (FGF) and epidermal growth factor (EGF) signaling. Subsequently, four vertebrate homologs were discovered; among these, the human homolog Sprouty 2 (hSpry2) contains the highest degree of sequence homology to the Drosophila protein. It has been shown that hSpry2 interacts directly with c-Cbl, an E3-ubiquitin ligase, which promotes the downregulation of receptor tyrosine kinases (RTKs). In this study, the functional consequences of the association between hSpry2 and c-Cbl has been investigated. hSpry2 is found to be ubiquitinated by c-Cbl in an EGF-dependent manner. EGF stimulation induces the tyrosine phosphorylation of hSpry2, which in turn enhances the interaction of hSpry2 with c-Cbl. The c-Cbl-mediated ubiquitination of hSpry2 targets the protein for degradation by the 26S proteasome. An enhanced proteolytic degradation of hSpry2 is also observed in response to FGF stimulation. The FGF-induced degradation of hSpry2 limits the duration of the inhibitory effect of hSpry2 on extracellular signal-regulated kinase (ERK) activation and enables the cells to recover their sensitivity to FGF stimulation. These results indicate that the interaction of hSpry2 with c-Cbl might serve as a mechanism for the downregulation of hSpry2 during receptor tyrosine kinase signaling (Hall, 2003).

Sprouty proteins are recently identified receptor tyrosine kinase (RTK) inhibitors potentially involved in many developmental processes. Sprouty proteins become tyrosine phosphorylated after growth factor treatment. Tyr55 was identified as a key residue for Sprouty2 phosphorylation; phosphorylation is required for Sprouty2 to inhibit RTK signaling, because a mutant Sprouty2 lacking Tyr55 augments signaling. Tyrosine phosphorylation of Sprouty2 affects neither its subcellular localization nor its interaction with Grb2, FRS2/SNT, or other Sprouty proteins. In contrast, Sprouty2 tyrosine phosphorylation is necessary for its binding to the Src homology 2-like domain of c-Cbl after fibroblast growth factor (FGF) stimulation. To determine whether c-Cbl is required for Sprouty2-dependent cellular events, Sprouty2 was introduced into c-Cbl-wild-type and -null fibroblasts. Sprouty2 efficiently inhibited FGF-induced phosphorylation of extracellular signal-regulated kinase 1/2 in c-Cbl-null fibroblasts, thus indicating that the FGF-dependent binding of c-Cbl to Sprouty2 is dispensable for its inhibitory activity. However, c-Cbl mediates polyubiquitylation/proteasomal degradation of Sprouty2 in response to FGF. Last, using Src-family pharmacological inhibitors and dominant-negative Src, it was shown that a Src-like kinase was required for tyrosine phosphorylation of Sprouty2 by growth factors. Thus, these data highlight a novel negative and positive regulatory loop that allows for the controlled, homeostatic inhibition of RTK signaling (Mason, 2004).

The ubiquitin ligase Cbl mediates ubiquitination of activated receptor tyrosine kinases (RTKs) and interacts with endocytic scaffold complexes, including CIN85/endophilins, to facilitate RTK endocytosis and degradation. Several mechanisms regulate the functions of Cbl to ensure the fine-tuning of RTK signalling and cellular homeostasis. One regulatory mechanism involves the binding of Cbl to Sprouty2, which sequesters Cbl away from activated epidermal growth factor receptors (EGFRs). Sprouty2 associates with CIN85 and acts at the interface between Cbl and CIN85 to inhibit EGFR downregulation. The CIN85 SH3 domains A and C bind specifically to proline-arginine motifs present in Sprouty2. Intact association between Sprouty2, Cbl and CIN85 is required for inhibition of EGFR endocytosis as well as EGF-induced differentiation of PC12 cells. Moreover, Sprouty4, which lacks CIN85-binding sites, does not inhibit EGFR downregulation, providing a molecular explanation for functional differences between Sprouty isoforms. Sprouty2 therefore acts as an inducible inhibitor of EGFR downregulation by targeting both the Cbl and CIN85 pathways (Haglund, 2005).

Interaction of Cbl with Crk and Phosphatidylinositol-3 kinase

The cellular homologs of the v-Crk oncogene product are composed exclusively of Src homology region 2 (SH2) and SH3 domains. v-Crk overexpression in fibroblasts causes cell transformation and elevated tyrosine phosphorylation of specific cellular proteins. Among these proteins is a 130-kDa protein, identified as p130cas (see CAS/CSE1 segregation protein), that forms a stable complex in vivo with v-Crk. The role of endogenous Crk proteins has been explored in Bcr-Abl-transformed cells (see Drosophila Abl oncogene). In the K562 human chronic myelogenous leukemia cell line, p130cas is not tyrosine phosphorylated or bound to Crk. Instead, Crk proteins predominantly associate with the tyrosine-phosphorylated proto-oncogene product of Cbl. In vitro analysis shows that this interaction is mediated by the SH2 domain of Crk and can be inhibited with a phosphopeptide containing the Crk-SH2 binding motif. In NIH 3T3 cells transformed by Bcr-Abl, c-Cbl becomes strongly tyrosine phosphorylated and associates with c-Crk. The complex between c-Crk and c-Cbl is also seen upon T-cell receptor cross-linking or with the transforming, tyrosine-phosphorylated c-Cbl. These results indicate that Crk binds to c-Cbl in a tyrosine phosphorylation-dependent manner, suggesting a physiological role for the Crk-c-Cbl complex in Bcr-Abl tyrosine phosphorylation-mediated transformation (Ribon, 1996).

CRKL is an SH2-SH3-SH3 adapter protein that is a major substrate of the BCR/ABL oncogene. The function of CRKL in normal cells is unknown. In cells transformed by BCR/ABL, CRKL is associated with two focal adhesion proteins, tensin and paxillin, suggesting that CRKL could be involved in integrin signaling. CRKL rapidly associates with tyrosine-phosphorylated proteins after cross-linking of beta1 integrins (See Drosophila Myospheroid) with fibronectin or anti-beta1 integrin monoclonal antibodies. The major tyrosine-phosphorylated CRKL-binding protein in megakaryocytic MO7e cells was identified as p120(CBL), the cellular homolog of the v-Cbl oncoprotein. However, in the lymphoid H9 cell line, the major tyrosine-phosphorylated CRKL-binding protein is p110(HEF1). In both cases, this binding is mediated by the CRKL SH2 domain. Interestingly, although both MO7e and H9 cells express p120(CBL) and p110(HEF1), beta1 integrin cross-linking induces tyrosine phosphorylation of p120(CBL) (but not p110[HEF1]) in MO7e cells and of p110(HEF1) (but not p120[CBL]) in H9 cells. In both cell types, CRKL is constitutively complexed to C3G, SOS, and c-ABL through its SH3 domains; the stoichiometry of these complexes does not change upon integrin ligation. Thus, in different cell types CRKL and its SH3-associated proteins may form different multimeric complexes depending on whether p120(CBL) or p110(HEF1) is tyrosine-phosphorylated after integrin ligation. The shift in association of CRKL and its SH3-associated proteins from p120(CBL) to p110(HEF1) could contribute to different functional outcomes of "outside-in" integrin signaling in different cells (Sattler, 1997).

The Philadelphia chromosome, detected in virtually all cases of chronic myelogenous leukemia (CML), is formed by a reciprocal translocation between chromosomes 9 and 22 that fuses BCR-encoded sequences upstream of exon 2 of c-ABL. The BCR-ABL fusion creates a gene whose protein product, p210BCR-ABL, has been implicated as the cause of the disease. Although ABL kinase activity has been shown to be required for the transforming abilities of BCR-ABL and numerous substrates of the BCR-ABL tyrosine kinase have been identified, the requirement of most of these substrates for the transforming function of BCR-ABL is unknown. There is a direct binding site of the c-CBL proto-oncogene to the SH2 domain of BCR-ABL. This interaction only occurs under conditions where c-CBL is tyrosine-phosphorylated. Despite the direct interaction of c-CBL with the SH2 domain of BCR-ABL, the deletion of the SH2 domain of BCR-ABL does not result in an alteration in the complex formation of BCR-ABL and c-CBL. This suggests that another site of direct interaction between c-CBL and BCR-ABL exists or that another protein mediates an indirect interaction of c-CBL and BCR-ABL. Since CRKL, an SH2, SH3 domain-containing adapter protein is known to bind directly to BCR-ABL and also binds to tyrosine-phosphorylated c-CBL, the ability of CRKL to mediate a complex between c-CBL and BCR-ABL was examined. Such a three way complex is confirmed (Bhat, 1997).

Phosphatidylinositol-3 kinase (PI-3 kinase) has been implicated in cellular events such as mitogenic signaling, actin organization, and receptor sorting. The p85 subunit of PI-3 kinase contains multiple domains capable of protein-protein interactions that may contribute to mediate the multiple physiological functions of this enzyme. Antibodies raised against the p85 subunit of PI-3 kinase immunoprecipitate a single tyrosine-phosphorylated protein of 120 kDa (pp120) from lysates of activated Jurkat T cells and A20 B cells. This protein is the only significant phosphotyrosine-containing protein in p85 immunoprecipitates from these cells, and it cannot be detected in immunoprecipitates of other signaling proteins such as PLC gamma. Furthermore, antibodies specific for the beta isoform of p85 but not antibodies specific for the alpha isoform immunoprecipitate this tyrosine-phosphorylated protein. pp120 completely comigrates with the proto-oncogene c-Cbl, which is a 120 kDa protein product abundant in lymphoid cells. Furthermore, immunoblots of p85 immunoprecipitates using antibodies raised against c-Cbl detect a band at exactly the position of pp120. In addition, p85 can be detected in immunoblots of c-Cbl immunoprecipitates. Thus, pp120 appears to correspond to c-Cbl. A direct association between c-Cbl and p85 can be observed in vitro using a fusion protein comprising the Src homology 2 (SH2) domains of p85; this binding is abolished by phenyl phosphate, suggesting that the interaction is mediated through phosphotyrosine-SH2 domain interactions. Thus important functional differences exist between the alpha and beta isoforms of p85 in vivo, pointing to c-Cbl as a potentially important mediator of some of the functions of PI-3 kinase in intact cells (Hartley, 1995).

The adaptor molecule Crkl is a major in vivo substrate for the Bcr/Abl tyrosine kinase (see below and Drosophila Abl), and it is thought to connect Bcr/Abl with downstream effectors. In the current study, a tyrosine-phosphorylated protein with a molecular mass of approximately 120 kDa was identified which binds only to the Crkl Src homology 2 (SH2) domain in cells, including Philadelphia chromosome-positive patient material, containing an active Bcr/Abl protein. The 120 kDa protein is Cbl, originally discovered as an oncogene that induces B-cell and myeloid leukemias in mice. The Crkl SH2 domain binds specifically to Cbl. The Src homology 3 (SH3) domains of Crkl do not bind to Cbl, but do bind Bcr/Abl. These findings suggest the existence of a trimolecular complex involving Bcr/Abl, Crkl, and Cbl and are consistent with a model in which Crkl mediates the oncogenic signal of Bcr/Abl to Cbl (de Jong, 1995).

In primary leukemic neutrophils from patients with chronic myelogenous leukemia (CML), the major tyrosine phosphorylated protein is CRKL, an SH2-SH3-SH3 adapter protein that has an overall homology of 60% to CRK, the human homolog of the v-crk oncogene. In cell lines transformed by BCR/ABL, CRKL is tyrosine phosphorylated, while CRK is not. There was a striking qualitative difference in the proteins coprecipitating with CRKL and CRK II. In untransformed cells, three major proteins coprecipitate with CRKL: C3G, SOS and c-ABL. Each of these proteins is found to interact with the CRKL-SH3 domains, but not the SH2 domain. After BCR/ABL transformation, the CRKL SH3-domain binding proteins do not change, with the exception that BCR/ABL now coprecipitate with CRKL. Compared to CRKL, very few proteins coprecipitated with CRK II in untransformed, quiescent cells. After BCR/ABL transformation, both the CRKL- and CRK-SH2 domains bind to a new complex of proteins of approximate molecular weight 105-120 kDa. The major protein in this complex is p120CBL. Thus, in these hematopoietic cell lines, CRKL is involved to a greater extent than CRK II in normal signaling pathways that involve c-ABL, C3G and SOS. In BCR/ABL-transformed cells, CRKL but not CRK II, appears to form complexes that potentially link BCR/ABL, c-ABL, C3G, and SOS to the protooncoprotein, p120CBL (Uemura, 1997).

Crkl is an adapter protein and phosphotyrosine-containing substrate implicated in transformation by the bcr-abl oncogene and in signaling by cytokines. When phosphorylated, Crkl binds through its Src homology 2 (SH2) domain to other tyrosine phosphoproteins such as paxillin and Cbl. Overexpression of Crkl in fibroblasts induces transformation. The role of Crkl in hematopoietic cells was examined. Overexpression of Crkl confers a signal leading to increased adhesion to fibronectin. In both fibroblasts and hematopoietic cells, individual mutations or deletions of each SH2 and SH3 domain abrogate transformation and adhesion, respectively, indicating that interactions with other proteins such as Cbl and paxillin (SH2 domain) and Abl, Sos, and C3G (N-terminal SH3 domain) are essential for biological activity. In vivo and in vitro tryptic phosphopeptide mapping studies have shown that Crkl is phosphorylated on multiple tyrosine residues when overexpressed or when activated by Bcr-Abl. Mutation at tyrosine 207, a residue conserved in c-Crk, abrogates all in vivo tyrosine phosphorylation of Crkl. Despite this loss of phosphotyrosine, mutation at this site enhances Crkl function as measured by complex formation with SH2 binding proteins, signal transduction to Jun Kinase, and fibroblast transformation. These observations implicate Crkl in cellular adhesion and demonstrate that Y207 functions as a negative regulatory site (Senechal, 1998).

Mutant macrophages that are deficient in expression of Src-family kinases have been used to define an integrin signaling pathway that is required for macrophage adhesion and migration. Following the ligation of surface integrins by fibronectin, the p120c-cbl (Cbl) protein rapidly becomes tyrosine phosphorylated and associates with the Src-family kinases Fgr and Lyn. In hck-/-fgr-/-lyn-/- triple mutant cells, which are defective in spreading on fibronectin-coated surfaces in vitro and show impaired migration in vivo, Cbl tyrosine phosphorylation is blocked; Cbl protein levels are low; adhesion-dependent translocation of Cbl to the membrane is impaired, and Cbl-associated, membrane-localized, phosphatidylinositol 3 (PI-3)-kinase activity is dramatically reduced. In contrast, adhesion dependent activation of total cellular PI-3 kinase activity is normal in mutant cells, demonstrating that it is the membrane-associated fraction of PI-3 kinase that is most critical in regulating the actin cytoskeletal rearrangements that lead to cell spreading. Treatment of wild-type cells with the Src-family-specific inhibitor PP1, or with Cbl antisense oligonucleotides or pharmacological inhibitors of PI-3 kinase, blocks cell spreading on fibronectin surfaces. These data provide a molecular description for the role of Src-family kinases Hck, Fgr and Lyn in beta1-integrin signal transduction in macrophages (Meng, 1998).

The central role of PI-3 kinase in initiating actin cytoskeletal rearrangements, membrane ruffling and cell migration following integrin clustering has been demonstrated in several cell systems, including neutrophils, fibroblasts, platelets and, most recently, carcinoma cells. In all these systems, different integrin subunits are involved; however, in each case, integrin-mediated adhesion leads to activation of PI-3 kinase and accumulation of D3 phosphoinositides. Treatment of cells with PI-3 kinase-specific inhibitors leads to the blockade of cell spreading. In carcinoma cells, activation of PI-3 kinase has been associated with a specific integrin-dependent function: promotion of invasion in vitro. However, none of the previous studies recognized the recognition that association with Cbl (or other adaptor proteins expressed in non-hematopoietic cells) and cytoskeletal translocation of PI-3 kinase are both critical events leading to actin cytoskeletal rearrangement. In this regard, it is the fortuitous observation in the Src-family kinase knockout macrophages that total cellular activation of PI-3 kinase is normal, yet the association of PI-3 kinase with Cbl and translocation of PI-3 kinase activity are impaired. This allows for the inference that the cytoskeletal localization of the PI-3 kinase-Cbl complex (not just overall activation of lipid kinase activity) is the critical event in regulating integrin-induced cytoskeletal rearrangements. It is believed that the abnormal subcellular localization of PI-3 kinase in adherent hck-/-fgr-/-lyn-/- macrophages is a cause of the abnormal cytoskeletal actin in these cells, rather than an effect of it. This is based on the rationale that treatment of wild-type cells with Cbl antisense oligonucleotides or PI-3 kinase inhibitors produces the same cellular phenotype of impaired Fn-induced cell spreading observed in the mutant macrophages. In macrophages the downstream signaling pathways such as MAP kinase or NF-kappaB activation that follow integrin clustering do not require the major Src-family kinases expressed in these cells. FAK has also been shown not to be required for integrin-induced MAP kinase activation in fibroblasts. Hence the possibility that dual and separable integrin signaling pathways lead to actin cytoskeletal rearrangements, versus activation of the MAP kinase cascade, may be a generalizable conclusion (Meng, 1998 and references).

Interaction of Cbl with transmembrane receptors

Vulval induction during C. elegans development is mediated by LET-23, a homolog of the mammalian epidermal growth factor receptor tyrosine kinase. The sli-1 gene is a negative regulator of LET-23 and encodes a protein similar to c-Cbl, a mammalian proto-oncoprotein. SLI-1 and c-Cbl share approximately 55 percent amino acid identity over a stretch of 390 residues, which includes a C3HC4 zinc-binding motif (a ring finger), and multiple consensus binding sites for Src homology 3 (SH3) domains. SLI-1 and c-Cbl may define a new class of proteins that modify receptor tyrosine kinase-mediated signal transduction (Yoon, 1995).

Upon stimulation of human epidermal growth factor (EGF) receptor, p120cbl becomes strongly tyrosine-phosphorylated and associates with activated EGF receptor in vivo. A GST fusion protein containing amino acids 1-486 of p120cbl, including a region that is highly conserved in nematodes, binds directly to the autophosphorylated carboxyl-terminal tail of the EGF receptor. Platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), or nerve growth factor (NGF) stimulation also results in tyrosine phosphorylation of p120cbl. p120cbl is involved in an early step in the EGF signaling pathway that is conserved from nematodes to mammals (Galisteo, 1995).

The mammalian proto-oncoprotein Cbl and its homologs in C. elegans and Drosophila 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).

The proto-oncogene product, Cbl, is a 120-kDa protein present in lymphocytes that contains numerous PXXP motifs in its COOH-terminal region and constitutively binds the SH3-containing adaptor protein Grb2. Cross-linking of CD3 and CD4 receptors in Jurkat T cells causes tyrosine phosphorylation of Cbl and its association with phosphatidylinositol 3'-kinase. Cbl is also present in nonlymphoid cells; epidermal growth factor (EGF) elicits its rapid tyrosine phosphorylation in a human embryonic cell line. Immunoprecipitates of Cbl from lysates of these cells contain Grb2 in the basal state, while EGF stimulation causes co-precipitation of tyrosine-phosphorylated EGF receptors. Similarly, EGF receptor immunoprecipitates from EGF-treated cells contain Cbl and Grb2. Both Grb2 and EGF receptors are released from Cbl in the presence of a proline-rich peptide that binds the NH2-terminal SH3 domain of Grb2. These results indicate that autophosphorylated EGF receptors associate with the SH2 domain of Grb2, which is complexed through its SH3 domain with proline-rich regions of Cbl. Such recruitment of Cbl to EGF receptors may reflect an important mechanism for its tyrosine phosphorylation and for assembling signaling components that mediate or modulate EGF actions (Meisner, 1995b).

The human cell surface antigen CD38 is a 46-kDa type II transmembrane glycoprotein with a short N-terminal cytoplasmic domain and a long Cys-rich C-terminal extracellular domain. The extracellular domain of CD38 has NAD+ glycohydrolase (NADase) activity and the ecto-form NADase activity induced in HL-60 cells during cell differentiation by retinoic acid is due to CD38. The addition of anti-CD38 monoclonal antibody to the cells induces rapid tyrosine phosphorylation of the cellular proteins with molecular weights of 120,000, 87,000, and 77,000. An increase in tyrosine kinase activity in the anti-phosphotyrosine immunoprecipitates of the cells is also observed after the addition of anti-CD38 monoclonal antibody. Moreover, one of the prominent tyrosine-phosphorylated proteins stimulated by the anti-CD38 monoclonal antibody was identified as the c-cbl proto-oncogene product, p120c-Cbl. These results indicate that tyrosine phosphorylation of cellular proteins, including p120c-Cbl, is possibly involved in transmembrane signaling mediated by CD38 (Kontani, 1996).

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).

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).

Fibroblast growth factor receptor signaling is an important mechanism regulating osteoblast function. To gain an insight into the regulatory role of FGF receptor-2 (FGFR2) signaling in osteoblasts, integrin-mediated attachment and cell survival were investigated in human calvarial osteoblasts expressing activated FGFR2. FGFR2 activation reduces osteoblast attachment on fibronectin. This is associated with reduced expression of the alpha5 integrin subunit normally expressed in human calvarial osteoblasts in vivo. Treatment with lactacystin, a potent inhibitor of proteasome, restores alpha5 integrin levels in FGFR2 mutant osteoblasts. Immunoprecipitation analysis shows that alpha5 integrin interacts with both the E3 ubiquitin ligase Cbl and ubiquitin. Immunocytochemistry revealed that alpha5 integrin colocalizes with FGFR2 and Cbl at the leading edge in membrane ruffle regions. Transfection with the 70Z-Cbl mutant lacking the RING domain required for Cbl-ubiquitin interaction, or with the G306E Cbl mutant that abolishes the binding ability of Cbl phosphotyrosine-binding domain restores alpha5 integrin levels. This suggests that Cbl-mediated ubiquitination plays an essential role in alpha5 integrin proteasome degradation induced by FGFR2 activation. Reduced alpha5 integrin expression is associated with an increased Bax/Bcl-2 ratio and increased caspase-9 and -3 activities in FGFR2 mutant osteoblasts. Forced expression of alpha5 integrin rescues cell attachment and corrects both the Bax/Bcl-2 ratio and caspase-3 and caspase-9 activities in FGFR2 mutant osteoblasts. Cbl recruitment induced by FGFR2 activation triggers alpha5 integrin degradation by the proteasome, which results in reduced osteoblast attachment on fibronectin and caspase-dependent apoptosis. This identifies a functional role of the alpha5 integrin subunit in the induction of apoptosis triggered by FGFR2 activation in osteoblasts, and reveals that a Cbl-dependent mechanism is involved in the coordinated regulation of cell apoptosis induced by alpha5 integrin degradation (Kaabeche, 2005).

Knockdown of growth factor receptor binding protein 2 (Grb2) by RNA interference strongly inhibits clathrin-mediated endocytosis of the epidermal growth factor receptor (EGFR). To gain insights into the function of Grb2 in EGFR endocytosis, cell lines were generated in which endogenous Grb2 was replaced by yellow fluorescent protein (YFP)-tagged Grb2 expressed at the physiological level. In these cells, Grb2-YFP fully reversed the inhibitory effect of Grb2 knockdown on EGFR endocytosis and, moreover, trafficked together with EGFR during endocytosis. Overexpression of Grb2-binding protein c-Cbl does not restore endocytosis in Grb2-depleted cells. However, EGFR endocytosis is rescued in Grb2-depleted cells by chimeric proteins consisting of the Src homology (SH) 2 domain of Grb2 fused to c-Cbl. The 'knockdown and rescue' analysis revealed that the expression of Cbl-Grb2/SH2 fusions containing RING finger domain of Cbl restores normal ubiquitylation and internalization of the EGFR in the absence of Grb2, consistent with the important role of the RING domain in EGFR endocytosis. In contrast, the carboxy-terminal domain of Cbl, when attached to Grb2 SH2 domain, had 4 times smaller endocytosis-rescue effect compared with the RING-containing chimeras. Together, the data suggest that the interaction of Cbl carboxy terminus with CIN85 has a minor and a redundant role in EGFR internalization. It is concluded that Grb2-mediated recruitment of the functional RING domain of Cbl to the EGFR is essential and sufficient to support receptor endocytosis (Huang, 2005).

Interaction of Cbl with Abl and other non-receptor tyrosine kinases

The 120-kD protein product of the c-cbl oncogene is tyrosine phosphorylated in tumor cells generated by BCR-ABL or v-Abl and p120cbl associates with these proteins in vivo. An oncogenic form of Cbl protein in the 70Z/3 pre-B cell lymphoma exhibits deregulated tyrosine phosphorylation. Dbl is the major 120-kD tyrosine phosphorylated protein in cells which express activated forms of the abl oncogene. Tyrosine phosphorylation of pl20cbl in BCR-ABL transformed cells does not alter its subcellular localization. The oncogenic 7OZ/3 form of Cbl exhibits enhanced tyrosine phosphorylation in v-Abl infected cells and Cbl is heavily tyrosine phosphorylated in hemopoietic cells transformed by v-Src. Two sites are essential for the tyrosine phosphorylation of Cbl in Abl-transformed cells. These sites conform to the preferred Abl kinase substrate sequence of YXXP; following phosphorylation they mediate an association with the CrkL SH2 domain (Andoniou, 1996).

Chronic myelogenous leukemia (CML) and some acute lymphoblastic leukemias (ALL) are caused by the t(9;22) chromosome translocation, which produces the constitutively activated BCR/ABL tyrosine kinase. When introduced into factor dependent hematopoietic cell lines, BCR/ABL induces the tyrosine phosphorylation of many cellular proteins. One prominent BCR/ABL substrate is p120CBL, the cellular homolog of the v-Cbl oncoprotein. In addition to p210BCR/ABL and c-ABL, p120CBL coprecipitates with an 85 kDa phosphoprotein, which is identified as the p85 subunit of PI3K. Anti-p120CBL immunoprecipitates from BCR/ABL-transformed, but not from untransformed, cell lines contained PI3K lipid kinase activity. Interestingly, the adaptor proteins CRKL and c-CRK are also found in these complexes. In vitro binding studies indicated that the SH2 domains of CRKL and c-CRK bind directly to p120CBL, while the SH3 domains of c-CRK and CRKL bind to BCR/ABL and c-ABL. The N-terminal and the C-terminal SH2 and the SH3 domain of p85PI3K bind directly in vitro to p120CBL. The ABL-SH2, but not ABL-SH3, can also bind to p120CBL. These data suggest that BCR/ABL may induce the formation of multimeric complexes of signaling proteins that include p120CBL, PI3K, c-CRK or CRKL, c-ABL and BCR/ABL itself (Sattler, 1996).

Engagement of antigen and immunoglobulin receptors on hematopoietic cells is directly coupled to activation of nonreceptor protein tyrosine kinases (PTKs), which then phosphorylate critical intracellular substrates. In mast cells, stimulated through the FcepsilonRI receptor, activation of several PTKs including Syk leads to degranulation and release of such mediators of the allergic response as histamine and serotonin. Regulation of Syk function occurs through interaction with the Cbl protein, itself a PTK substrate in this system. Overexpression of Cbl leads to inhibition of Syk and suppression of serotonin release from mast cells, demonstrating Cbl's ability to inhibit a nonreceptor tyrosine kinase. Complex adaptor proteins such as Cbl can directly regulate the functions of the proteins they bind (Ota, 1997).

The proto-oncogene product Cbl has emerged as a negative regulator of a number of protein-tyrosine kinases, including the ZAP-70/Syk tyrosine kinases that are critical for signaling in hematopoietic cells. The evolutionarily conserved N-terminal tyrosine kinase-binding domain is required for Cbl to associate with ZAP-70/Syk and for their subsequent negative regulation. However, the role of the remaining C-terminal regions of Cbl remains unclear. A COS-7 cell reconstitution system has been used to address this question. Analysis of a series of C-terminally truncated Cbl mutants reveals that the N-terminal half of the protein, including the TKB and RING finger domains, is sufficient to mediate negative regulation of Syk. Further truncations, which delete the RING finger domain, abrogates the negative regulatory effects of Cbl on Syk. Point mutations of conserved cysteine residues or a histidine in the RING finger domain, which are required for zinc binding, abrogate the ability of Cbl to negatively regulate Syk in COS-7 cells and Ramos B lymphocytic cells. In addition, Syk-dependent transactivation of a serum response element-luciferase reporter in transfected 293T cells is reduced by wild type Cbl; mutations of the RING finger domain or its deletion abrogate this effect. These results establish the RING finger domain as an essential element in Cbl-mediated negative regulation of a tyrosine kinase and reveal that the evolutionarily conserved N-terminal half of the protein is sufficient for this function (Ota, 2000).

The kinase activity of Abl is known to be regulated by a putative trans-acting inhibitor molecule interacting with the Src homology (SH) 3 domain of Abl. The kinase-deficient Src (SrcKD) directly inhibits the tyrosine phosphorylation of Cbl and other cellular proteins by Abl. Both the SH2 and SH3 domains of SrcKD are necessary for the suppressor activity toward the Abl kinase phosphorylating Cbl. To suppress the Cbl phosphorylation by Abl, the interaction between the SH3 domain of SrcKD and Cbl is required. This interaction between SrcKD and Cbl is regulated by a closed structure of Cbl. The binding of Abl to the extreme carboxyl-terminal region of Cbl unmasks the binding site of SrcKD to Cbl. This results in a ternary complex that inhibits the Abl-mediated phosphorylation of Cbl by steric hindrance. These results illustrate a mechanism by which the enzymatically inactive Src can exert a biological function in vivo (Shishido, 2000).

Cbl can form a closed structure that prevents the binding of Src to Cbl. The binding of Abl to Cbl is considered to trigger the structural changes in Cbl. In the case of other proteins that have a closed structure such as Src or Crk, the deletions of the C-terminal regions give rise to the formation of oncogenic proteins v-Src and v-Crk. Similarly, v-Cbl has a deletion of the C-terminal region of Cbl, including the proline-rich region of Cbl. These results suggest that the oncogenic activity residing in the N-terminal region of Cbl can be released because of a disruption of a closed structure of Cbl. In addition, Cbl has another oncogenic form, 70Z Cbl, which contains a 17-aa deletion between the N-terminal and the C-terminal regions. 70Z Cbl constitutively binds to Src although WT Cbl does not. This result also suggests that the constitutive conformational changes that open the structure of Cbl contribute to its oncogenic activation. The Cbl ring finger domain exhibits the ubiquitin ligase activity. The structural transition of Cbl induced by Abl might regulate the ligase activity because the ring finger domain is localized right next to the Src binding proline-rich region (Shishido, 2000).

In summary SrcKD directly inhibits Cbl phosphorylation by the Abl kinase. The analysis of mutants suggests that the complex of SrcKD and Cbl acts as an inhibitor of Abl. Although the physiological consequence of the interaction and the regulation of Abl, Cbl, and kinase-inactive form of WT Src is still unclear at the moment, it is possible that these interactions are important for the cytoskeleton reorganization. The kinase-activated Src activates Abl and the kinase-deficient Src reduces this activation in response to platelet-derived growth factor-regulating membrane ruffling. SrcKD regulates the cytoskeleton reorganization in osteoclasts, and Cbl has been found to act downstream of Src in these cells. The mechanism of Abl inhibition by SrcKD would not be limited to the inhibition of the WT Src kinase but would rather ensue from direct inhibition of the Abl kinase by SrcKD. Thus, this study suggests caution in interpreting data using a kinase-deficient molecule as a dominant negative to study the kinase activity (Shishido, 2000 and references therein).

The signaling events downstream of integrins that regulate cell attachment and motility are only partially understood. Using osteoclasts and transfected 293 cells, it has been found that a molecular complex comprising Src, Pyk2 (a calcium-dependent tyrosine kinase that is the predominant FAK family member in the adhesion structures of osteoclasts), and Cbl functions to regulate cell adhesion and motility. The activation of integrin alphavß3 induces the [Ca2+]i-dependent phosphorylation of Pyk2 Y402, its association with Src SH2, Src activation, and the Src SH3-dependent recruitment and phosphorylation of c-Cbl. Furthermore, the PTB domain of Cbl is shown to bind to phosphorylated Tyr-416 in the activation loop of Src, the autophosphorylation site of Src, inhibiting Src kinase activity and integrin-mediated adhesion. Deletion of c-Src or c-Cbl leads to a decrease in osteoclast migration. Thus, binding of alphavß3 integrin induces the formation of a Pyk2/Src/Cbl complex in which Cbl is a key regulator of Src kinase activity and of cell adhesion and migration. These findings may explain the osteopetrotic phenotype in the Src-/- mice (Sanjay, 2001).

Cyclic attachment and detachment of individual podosomes is required as cells, particularly highly motile cells such as macrophages and osteoclasts, migrate over a substratum. Pyk2, Src, and Cbl appear to play a pivotal role in these processes. Deletion of Src significantly alters the initial distribution of podosomes in osteoclasts, eventually leading to the formation of focal adhesion-like structures. This shift from podosome to focal adhesion correlates with a decrease in the formation and motility of lamellipodia and in cell migration, although it is not possible to determine which comes first. Adhesion to vitronectin or activation of the alphavß3 receptor induces an increase in intracellular calcium that does not depend on the presence of Src. The formation of a kinase-rich trimolecular complex results in the Pyk2- and Src-dependent phosphorylation of Cbl. Therefore, Pyk2, Src, and Cbl are all involved in the 'outside-in' alphavß3 integrin signaling which results in podosome assembly. In transfected 293-VnR cells, the PTB domain of Cbl binds to Tyr 416 in the activation loop of the Src kinase domain, and this interaction downregulates both Src kinase activity and integrin-mediated adhesion. Thus, Cbl might also be crucial in 'inside-out' signaling, playing a key role in podosome detachment and subsequent disassembly. In agreement with this hypothesis, deletion of the gene encoding Cbl, like the deletion of the gene encoding Src, leads to a significant, albeit smaller, decrease in osteoclast migration. This series of events could form the basis for the cyclic attachment-detachment of single adhesion sites at the leading edge of lamellipodia in motile cells, and thereby participates in the assembly-disassembly of individual podosomes, thereby ensuring cell adhesion while still allowing cell motility (Sanjay, 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).

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).

Interaction of Cbl with cytoskeletal proteins

Continued: Cbl Evolutionary homologs part 2/2

Cbl: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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