Src-catalyzed phosphorylation of c-Cbl leads to the interdependent ubiquitination of both proteins.

The protooncogene c-Cbl has recently emerged as an E3 ubiquitin ligase for activated receptor tyrosine kinases. We report here that c-Cbl also mediates the ubiquitination of another protooncogene, the non-receptor tyrosine kinase c-Src, as well as of itself. The c-Cbl-dependent ubiquitination of Src and c-Cbl requires c-Cbl's RING finger, Src kinase activity, and c-Cbl's tyrosine phosphorylation, probably on Tyr-371. In vitro, c-Cbl forms a stable complex with the ubiquitin-conjugating enzyme UbcH7, but active Src destabilizes this interaction. In contrast, Src inhibition stabilizes the c-Cbl. UbcH7.Src complex. Finally, c-Cbl reduces v-Src protein levels and suppresses v-Src-induced STAT3 activation. Thus, in addition to mediating the ubiquitination of activated receptor tyrosine kinases, c-Cbl also acts as a ubiquitin ligase for the non-receptor tyrosine kinase Src, thereby down-regulating Src.     

The RING finger of c-Cbl mediates desensitization of the epidermal growth factor receptor.

Ligand-induced activation of surface receptors, including the epidermal growth factor receptor (EGFR), is followed by a desensitization process involving endocytosis and receptor degradation. c-Cbl, a tyrosine phosphorylation substrate shared by several signaling pathways, accelerates desensitization by recruiting EGFR and increasing receptor polyubiquitination. Here we demonstrate that the RING type zinc finger of c-Cbl is essential for ubiquitination and subsequent desensitization of EGFR. Mutagenesis of a single cysteine residue impaired the ability of c-Cbl to enhance both down-regulation and ubiquitination of EGFR in living cells, although the mutant retained binding to the activated receptor. Consequently, the mutant form of c-Cbl acquired a dominant inhibitory function and lost the ability to inhibit signaling downstream to EGFR. In vitro reconstitution of EGFR ubiquitination implies that the RING finger plays an essential direct role in ubiquitin ligation. Our results attribute to the RING finger of c-Cbl a causative role in endocytic sorting of EGFR and desensitization of signal transduction.     

Negative regulation of EphA2 receptor by Cbl

The c-Cbl proto-oncogene product Cbl has emerged as a negative regulator of receptor and non-receptor tyrosine kinases, a function dependent on its recently identified ubiquitin ligase activity. Here, we report that EphA2, a member of Eph receptor tyrosine kinases is negatively regulated by Cbl. The negative regulation of EphA2 mediated by Cbl is dependent on the activity of EphA2, as the kinase inactive mutant of EphA2 cannot be regulated by Cbl. Moreover, a point mutation (G306E-Cbl) in TKB region of Cbl that has been reported to abolish Cbl binding to RTKs and non-receptor tyrosine kinases impaired the binding to active EphA2. The dominant negative mutant 70Z-Cbl, which has a 17-amino acids deletion in the N-boundary of the RING finger domain, defuncted negative regulatory function of Cbl to EphA2. These results demonstrate that the TKB domain and RING finger domain of Cbl are essential for this negative regulation.     

RING finger mutations that abolish c-Cbl-directed polyubiquitination and downregulation of the EGF receptor are insufficient for cell transformation

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, we 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.     

Sprouty2 attenuates epidermal growth factor receptor ubiquitylation and endocytosis,and consequently enhances Ras/ERK signalling

Drosophila Sprouty (dSpry) was genetically identified as a novel antagonist of fibroblast growth factor receptor (FGFR), epidermal growth factor receptor (EGFR) and Sevenless signalling, 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 herein that describes the functional implications of the direct association between human Sprouty2 (hSpry2) and c-Cbl, and its impact on the cellular localization and signalling capacity of EGFR. Contrary to the consensus view that Spry2 is a general inhibitor of receptor tyrosine kinase signalling, hSpry2 was shown to abrogate EGFR ubiquitylation and endocytosis, and sustain EGF-induced ERK signalling that culminates in differentiation of PC12 cells. Correlative evidence showed the failure of hSpry2DeltaN11 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. We conclude that hSpry2 potentiates EGFR signalling by specifically intercepting c-Cbl-mediated effects on receptor down-regulation.     

c-Cbl is involved in Met signaling in B cells and mediates hepatocyte growth factor-induced receptor ubiquitination

Hepatocyte growth factor/scatter factor (HGF) and its receptor tyrosine kinase Met are key regulators of epithelial motility and morphogenesis. Recent studies indicate that the HGF/Met pathway also plays a role in B cell differentiation, whereas uncontrolled Met signaling may lead to B cell neoplasia. These observations prompted us to explore HGF/Met signaling in B cells. In this study, we demonstrate that HGF induces strong tyrosine phosphorylation of the proto-oncogene product c-Cbl in B cells and increases Cbl association with the Src family tyrosine kinases Fyn and Lyn, as well as with phosphatidylinositol-3 kinase and CrkL. In addition, we demonstrate that c-Cbl mediates HGF-induced ubiquitination of Met. This requires the juxtamembrane tyrosine Y1001 (Y2) of Met, but not the multifunctional docking site (Y14/15) or any additional C-terminal tyrosine residues (Y13-16). In contrast to wild-type c-Cbl, the transforming mutants v-Cbl and 70Z/3 Cbl, which lack the ubiquitin ligase RING finger domain, suppress Met ubiquitination. Our findings identify c-Cbl as a negative regulator of HGF/Met signaling in B cells, mediating ubiquitination and, consequently, proteosomal degradation of Met, and suggest a role for Cbl in Met-mediated tumorigenesis.     

The herpes simplex virus type 1 (HSV-1) regulatory protein ICP0 interacts with and Ubiquitinates p53

Herpes simplex virus type 1 regulatory protein ICP0 contains a zinc-binding RING finger and has been shown to induce the proteasome-dependent degradation of a number of cellular proteins in a RING finger-dependent manner during infection. This domain of ICP0 is also required to induce the formation of unanchored polyubiquitin chains in vitro in the presence of ubiquitin-conjugating enzymes UbcH5a and UbcH6. These data indicate that ICP0 has the potential to act as a RING finger ubiquitin ubiquitin-protein isopeptide ligase (E3) and to induce the degradation of certain cellular proteins through ubiquitination and proteasome-mediated degradation. Here we demonstrate that ICP0 is a genuine RING finger ubiquitin E3 ligase that can interact with and mediate the ubiquitination of the major oncoprotein p53 both in vitro and in vivo. Ubiquitination of p53 requires ICP0 to have an intact RING finger domain and occurs independently of its ability to bind to the ubiquitin-specific protease USP7.     

Ubiquitin ligase activity and tyrosine phosphorylation underlie suppression of growth factor signaling by c-Cbl/Sli-1

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. We infer a sequential process 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. Our study identifies Cbl proteins as components of the ubiquitin ligation machinery and implies that they similarly suppress many other signaling pathways.     

E2-c-Cbl recognition is necessary but not sufficient for ubiquitination activity

The E2 ubiquitin-conjugating enzymes UbcH7 and UbcH5B both show specific binding to the RING (really interesting new gene) domain of the E3 ubiquitin-protein ligase c-Cbl, but UbcH7 hardly supports ubiquitination of c-Cbl and substrate in a reconstituted system. Here, we found that neither structural changes nor subtle differences in the E2-E3 interaction surface are possible explanations for the functional specificity of UbcH5B and UbcH7 in their interaction with c-Cbl. The quick transfer of ubiquitin from the UbcH5B∼Ub thioester to c-Cbl or other ubiquitin acceptors suggests that UbcH5B might functionally be a relatively pliable E2 enzyme. In contrast, the UbcH7∼Ub thioester is too stable to transfer ubiquitin under our assay conditions, indicating that UbcH7 might be a more specific E2 enzyme. Our results imply that the interaction specificity between c-Cbl and E2 is required but not sufficient for transfer of ubiquitin to potential targets.     

Requirements of multiple domains of SLI-1, a Caenorhabditis elegans homologue of c-Cbl, and an inhibitory tyrosine in LET-23 in regulating vulval differentiation

SLI-1, a Caenorhabditis elegans homologue of the proto-oncogene product c-Cbl, is a negative regulator of LET-23-mediated vulval differentiation. Lack of SLI-1 activity can compensate for decreased function of the LET-23 epidermal growth factor receptor, the SEM-5 adaptor, but not the LET-60 RAS, suggesting that SLI-1 acts before RAS activation. SLI-1 and c-Cbl comprise an N-terminal region (termed SLI-1:N/Cbl-N, containing a four-helix bundle, an EF hand calcium-binding domain, and a divergent SH2 domain) followed by a RING finger domain and a proline-rich C-terminus. In a transgenic functional assay, the proline-rich C-terminal domain is not essential for sli-1(+) function. A protein lacking the SH2 and RING finger domains has no activity, but a chimeric protein with the SH2 and RING finger domains of SLI-1 replaced by the equivalent domains of c-Cbl has activity. The RING finger domain of c-Cbl has been shown recently to enhance ubiquitination of active RTKs by acting as an E3 ubiquitin-protein ligase. We find that the RING finger domain of SLI-1 is partially dispensable. Further, we identify an inhibitory tyrosine of LET-23 requiring sli-1(+) for its effects: removal of this tyrosine closely mimics the loss of sli-1 but not of another negative regulator, ark-1. Thus, we suggest that this inhibitory tyrosine mediates its effects through SLI-1, which in turn inhibits signaling upstream of LET-60 RAS in a manner not wholly dependent on the ubiquitin-ligase domain.     

Mutation of the c-Cbl TKB domain binding site on the Met receptor tyrosine kinase converts it into a transforming protein.

The c-Cbl protooncogene is a negative regulator for several receptor tyrosine kinases (RTKs) through its ability to promote their polyubiquitination. Hence, uncoupling c-Cbl from RTKs may lead to their deregulation. In testing this, we show that c-Cbl promotes ubiquitination of the Met RTK. This requires the c-Cbl tyrosine kinase binding (TKB) domain and a juxtamembrane tyrosine residue on Met. This tyrosine provides a direct binding site for the c-Cbl TKB domain, and is absent in the rearranged oncogenic Tpr-Met variant. A Met receptor, where the juxtamembrane tyrosine is replaced by phenylalanine, is not ubiquitinated and has transforming activity in fibroblast and epithelial cells. We propose the uncoupling of c-Cbl from RTKs as a mechanism contributing to their oncogenic activation.     

Structural model of the UbcH5B/CNOT4 complex revealed by combining NMR, mutagenesis, and docking approaches

The protein CNOT4 possesses an N-terminal RING finger domain that acts as an E3 ubiquitin ligase and specifically interacts with UbcH5B, a ubiquitin-conjugating enzyme. The structure of the CNOT4 RING domain has been solved and the amino acids important for the binding to UbcH5B have been mapped. Here, the residues of UbcH5B important for the binding to CNOT4 RING domain were identified by NMR chemical shift perturbation experiments, and these data were used to generate structural models of the complex with the program HADDOCK. Together with the NMR data, additional biochemical data were included in a second docking, and comparisons of the resulting model with the structure of the c-Cbl/UbcH7 complex reveal some significant differences, notably at specific residues, and give structural insights into the E2/E3 specificity.     

Herpes simplex virus type 1 immediate-early protein ICP0 and is isolated RING finger domain act as ubiquitin E3 ligases in vitro.

Proteasome-dependent degradation of ubiquitinated proteins plays a key role in many important cellular processes. Ubiquitination requires the E1 ubiquitin activating enzyme, an E2 ubiquitin conjugating enzyme, and frequently a substrate-specific ubiquitin protein ligase (E3). One class of E3 ubiquitin ligases has been shown to contain a common zinc-binding RING finger motif. We have previously shown that herpes simplex virus type 1 ICP0, itself a RING finger protein, induces the proteasome-dependent degradation of several cellular proteins and induces the accumulation of colocalizing conjugated ubiquitin in vivo. We now report that both full-length ICP0 and its isolated RING finger domain induce the accumulation of polyubiquitin chains in vitro in the presence of E1 and the E2 enzymes UbcH5a and UbcH6. Mutations within the RING finger region that abolish the in vitro ubiquitination activity also cause severe reductions in ICP0 activity in other assays. We conclude that ICP0 has the potential to act as an E3 ubiquitin ligase during viral infection and to target specific cellular proteins for destruction by the 26S proteasome.     

Cbl promotes ubiquitination of the T cell receptor zeta through an adaptor function of Zap-70

Triggering of the T cell antigen receptor (TCR).CD3 complex induces its ubiquitination. However, the molecular events that lead to ubiquitin conjugation to these cell surface molecules have not been defined. Here we report that Cbl, a RING-type E3 ubiquitin-protein ligase, promotes ubiquitination of TCR zeta chain, which requires its functional variant Src homology 2 domain and an intact RING finger. The tyrosine kinase Zap-70, which binds to both TCR zeta and Cbl, plays an adaptor role in these events. Mutations in TCR zeta, Zap-70, or Cbl that disrupt the interaction between TCR zeta and Zap-70 or between Zap-70 and Cbl reduce ubiquitination of TCR zeta. Our results suggest a novel mechanism by which Cbl negatively regulates T cell development and activation by inducing ubiquitination of the TCR.CD3 components.     

Identification of the domain in ErbB2 that restricts ligand-induced degradation

Ligand-induced receptor degradation is an important process for down-regulation of plasma membrane receptors. While epidermal growth factor receptor (EGFR) is rapidly internalised and degraded upon ligand stimulation, ErbB2, the closest member to EGFR in ErbB receptor family, is resistant in ligand-induced degradation. To understand the molecular mechanisms underlying the impairment in ligand-induced degradation of ErbB2, we attempted to determine structural factor in ErbB2 that restricts the degradation. By analysis of ligand-induced degradation of EGFR/ErbB2 chimeras, we have identified a region between amino acid residues F1030 and L1075 in ErbB2 as the domain that restricts the ligand-induced degradation. We designated this domain as the Blocking ErbB2 Degradation or the BED domain. Replacement of the BED domain in an EGFR/ErbB2 chimera with the corresponding region of EGFR changed this chimera from a non-degradable to a degradable receptor, indicating that the BED domain is the factor restricting the ligand-induced degradation of ErbB2. In addition, we found that a non-degradable EGFR/ErbB2 chimera was not defective in tyrosine phosphorylation, ubiquitination and interaction with c-Cbl, rather, was defective in ligand-induced internalisation, suggesting that the endocytosis defect is the cause restricting the degradation of ErbB2, and that c-Cbl-catalysed mono-ubiquitination is not involved in the impairment in ligand-induced degradation of ErbB2.     

A conserved DpYR motif in the juxtamembrane domain of the Met receptor family forms an atypical c-Cbl/Cbl-b tyrosine kinase binding domain binding site required for suppression of oncogenic activation

The activation and phosphorylation of Met, the receptor tyrosine kinase (RTK) for hepatocyte growth factor, initiates the recruitment of multiple signaling proteins, one of which is c-Cbl, a ubiquitin-protein ligase. c-Cbl promotes ubiquitination and enhances the down-modulation of the Met receptor and other RTKs, targeting them for lysosomal sorting and subsequent degradation. The ubiquitination of Met by c-Cbl requires the direct interaction of the c-Cbl tyrosine kinase binding (TKB) domain with tyrosine 1003 in the Met juxtamembrane domain. Although a consensus for c-Cbl TKB domain binding has been established ((D/N)XpYXX(D/E0phi), this motif is not present in Met, suggesting that other c-Cbl TKB domain binding motifs may exist. By alanine-scanning mutagenesis, we have identified a DpYR motif including Tyr(1003) as being important for the direct recruitment of the c-Cbl TKB domain and for ubiquitination of the Met receptor. The substitution of Tyr(1003) with phenylalanine or substitution of either aspartate or arginine residues with alanine impairs c-Cbl-recruitment and ubiquitination of Met and results in the oncogenic activation of the Met receptor. We demonstrate that the TKB domain of Cbl-b, but not Cbl-3, binds to the Met receptor and requires an intact DpYR motif. Modeling studies suggest the presence of a salt bridge between the aspartate and arginine residues that would position pTyr(1003) for binding to the c-Cbl TKB domain. The DpYR motif is conserved in other members of the Met RTK family but is not present in previously identified c-Cbl-binding proteins, identifying DpYR as a new binding motif for c-Cbl and Cbl-b.     

NK lytic-associated molecule, involved in NK cytotoxic function, is an E3 ligase

NK lytic-associated molecule (NKLAM) is a protein involved in the cytolytic function of NK cells and CTLs. It has been localized to the cytolytic granules in NK cells and is up-regulated when cells are exposed to cytokines IL-2 or IFN-beta. We report in this study that NKLAM contains a cysteine-rich really interesting new gene (RING) in between RING-RING domain, and that this domain possesses strong homology to the RING domain of the known E3 ubiquitin ligase, Dorfin. To determine whether NKLAM functions as an E3 ligase, we performed coimmunoprecipitation binding assays with ubiquitin conjugates (Ubcs) UbcH7, UbcH8, and UbcH10. We demonstrated that both UbcH7 and UbcH8 bind to full-length NKLAM. We then performed a similar binding assay using endogenous NKLAM and UbcH8 expressed by human NK clone NK3.3 to show that the protein interaction occurs in vivo. Using the yeast two-hybrid system, we identified uridine kinase like-1 (URKL-1) protein as a substrate for NKLAM. We confirmed that NKLAM and URKL-1 interact in mammalian cells by using both immunoprecipitation and confocal microscopy. We demonstrated decreased protein expression and enhanced ubiquitination of URKL-1 in the presence of NKLAM. These data indicate that NKLAM is a RING finger protein that binds Ubcs and has as one of its substrates, URKL-1, thus defining this cytolytic protein as an E3 ubiquitin ligase.     

Features of the parkin/ariadne-like ubiquitin ligase, HHARI, that regulate its interaction with the ubiquitin-conjugating enzyme, Ubch7

We recently reported the identification of a RING finger-containing protein, HHARI (human homologue of Drosophila ariadne), which binds to the human ubiquitin-conjugating enzyme UbcH7 in vitro. We now demonstrate that HHARI interacts and co-localizes with UbcH7 in mammalian cells, particularly in the perinuclear region. We have further defined a minimal interaction region of HHARI comprising residues 186-254, identified individual amino acid residues essential for the interaction, and determined that the distance between the RING1 finger and IBR (in between RING fingers) domains is critical to maintaining binding. We have also established that the RING1 finger of HHARI cannot be substituted for by the highly homologous RING finger domains of either of the ubiquitin-protein ligase components c-CBL or Parkin, despite their similarity in structure and their independent capabilities to bind UbcH7. Furthermore, mutation of the RING1 finger domain of HHARI from a RING-HC to a RING-H2 type abolishes interaction with UbcH7. These studies demonstrate that very subtle changes to the domains that regulate recognition between highly conserved components of the ubiquitin pathway can dramatically affect their ability to interact.     

Dasatinib Targets B-Lineage Cells but Does Not Provide an Effective Therapy for Myeloproliferative Disease in c-Cbl RING Finger Mutant Mice

This study aimed to determine whether the multi-kinase inhibitor dasatinib would provide an effective therapy for myeloproliferative diseases (MPDs) involving c-Cbl mutations. These mutations, which occur in the RING finger and linker domains, abolish the ability of c-Cbl to function as an E3 ubiquitin ligase and downregulate activated protein tyrosine kinases. Here we analyzed the effects of dasatinib in a c-Cbl RING finger mutant mouse that develops an MPD with a phenotype similar to the human MPDs. The mice are characterized by enhanced tyrosine kinase signaling resulting in an expansion of hematopoietic stem cells, multipotent progenitors and cells within the myeloid lineage. Since c-Cbl is a negative regulator of c-Kit and Src signaling we reasoned that dasatinib, which targets these kinases, would be an effective therapy. Furthermore, two recent studies showed dasatinib to be effective in inhibiting the in vitro growth of cells from leukemia patients with c-Cbl RING finger and linker domain mutations. Surprisingly we found that dasatinib did not provide an effective therapy for c-Cbl RING finger mutant mice since it did not suppress any of the hematopoietic lineages that promote MPD development. Thus we conclude that dasatinib may not be an appropriate therapy for leukemia patients with c-Cbl mutations. We did however find that dasatinib caused a marked reduction of pre-B cells and immature B cells which correlated with a loss of Src activity. This study is therefore the first to provide a detailed characterization of in vivo effects of dasatinib in a hematopoietic disorder that is driven by protein tyrosine kinases other than BCR-ABL.     

Cbl-c Ubiquitin Ligase Activity Is Increased via the Interaction of Its RING Finger Domain with a LIM Domain of the Paxillin Homolog,Hic 5

Cbl proteins (Cbl, Cbl-b and Cbl-c) are ubiquitin ligases that are critical regulators of tyrosine kinase signaling. In this study we identify a new Cbl-c interacting protein, Hydrogen peroxide Induced Construct 5 (Hic-5). The two proteins interact through a novel interaction mediated by the RING finger of Cbl-c and the LIM2 domain of Hic-5. Further, this interaction is mediated and dependent on specific zinc coordinating complexes within the RING finger and LIM domain. Binding of Hic-5 to Cbl-c leads to an increase in the ubiquitin ligase activity of Cbl-c once Cbl-c has been activated by Src phosphorylation or through an activating phosphomimetic mutation. In addition, co-transfection of Hic-5 with Cbl-c leads to an increase in Cbl-c mediated ubiquitination of the EGFR. These data suggest that Hic-5 enhances Cbl-c ubiquitin ligase activity once Cbl-c has been phosphorylated and activated. Interactions between heterologous RING fingers have been shown to activate E3s. This is the first demonstration of enhancement of ubiquitin ligase activity of a RING finger ubiquitin ligase by the direct interaction of a LIM zinc coordinating domain.