Ligand-induced lysosomal epidermal growth factor receptor (EGFR) degradation is preceded by proteasome-dependent EGFR de-ubiquitination

Studies on the differential routing of internalized epidermal growth factor receptors (EGFRs) induced by EGF, TGF alpha, and the superagonist EGF-TGF alpha chimera E4T suggested a correlation between receptor recycling and their mitogenic potency. EGFR sorting to lysosomes depends on its kinase domain and its ubiquitination by Cbl proteins. Proteasomes have also been proposed to regulate EGFR degradation, but the underlying mechanism remains obscure. Here we evaluated EGFR activation, Cbl recruitment, EGFR ubiquitination and degradation in response to EGF, TGF alpha, and E4T. We also determined the fate of activated EGFRs and Cbl proteins by using v-ATPase (bafilomycin A1) and proteasome (lactacystin) inhibitors. Our results demonstrate that E4T and TGF alpha provoke decreased Cbl recruitment, EGFR ubiquitination and EGFR degradation compared with EGF. Furthermore, bafilomycin treatment blocks EGFR but not c-Cbl degradation. In contrast, lactacystin treatment blocks EGF-induced c-Cbl degradation but does not block EGFR degradation, even though lactacystin causes a minor delay in EGFR degradation. Surprisingly, even though bafilomycin completely blocks EGFR degradation, it does not prevent EGFR de-ubiquitination upon prolonged EGF stimulation. Strikingly, when combined with bafilomycin, lactacystin treatment stabilizes the ubiquitinated EGFR and prevents its de-ubiquitination. We conclude that the enhanced EGFR recycling that has been observed in HER-14 cells following TGF alpha or E4T stimulation correlates with decreased EGFR ubiquitination and EGFR degradation, and that proteasomal activity is required for de-ubiquitination of the EGFR prior to its lysosomal degradation.     

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.     

Endocytosis of receptor tyrosine kinases is driven by monoubiquitylation,not polyubiquitylation

Growth factors stimulate specific receptor tyrosine kinases, but subsequent receptor endocytosis terminates signaling. The ubiquitin ligase c-Cbl targets epidermal growth factor receptors (EGFRs) to endocytosis by tagging them with multiple ubiquitin molecules. However, the type of ubiquitylation is unknown; whereas polyubiquitin chains signal proteasomal degradation, ubiquitin monomers control other processes. We report that in isolation c-Cbl mediates monoubiquitylation rather than polyubiquitylation of EGFRs. Consistent with the sufficiency of monoubiquitylation, when fused to the tail of EGFR, a single ubiquitin induces receptor endocytosis and degradation in cells. By using receptor and ubiquitin mutants, we infer that c-Cbl attaches a founder monoubiquitin to the kinase domain of EGFR and this is complemented by the conjugation of additional monoubiquitins. Hence, receptor tyrosine kinases are desensitized through conjugation of multiple monoubiquitins, which is distinct from polyubiquitin-dependent proteasomal degradation.     

c-Cbl ubiquitin ligase regulates focal adhesion protein turnover and myofibril degeneration induced by neutrophil protease cathepsin G

The neutrophil-derived serine protease, cathepsin G (Cat.G), has been shown to induce myocyte detachment and apoptosis by anoikis through down-regulation of focal adhesion (FA) signaling. However, the mechanisms that control FA protein stability and turnover in myocytes are not well understood. Here, we have shown that the Casitas b-lineage lymphoma (c-Cbl), adaptor protein with an intrinsic E3 ubiquitin ligase activity, is involved in FA and myofibrillar protein stability and turnover in myocytes. Cat.G treatment induced c-Cbl activation and its interaction with FA proteins. Deletion of c-Cbl using c-Cbl knock-out derived myocytes or inhibition of c-Cbl ligase activity significantly reduced FA protein degradation, myofibrillar degeneration, and myocyte apoptosis induced by Cat.G. We also found that inhibition of the proteasome activity, but not the lysosome or the calpain activity, markedly attenuated FA and myofibrillar protein degradation induced by Cat.G. Interestingly, c-Cbl activation induced by Cat.G was mediated through epidermal growth factor receptor (EGFR) transactivation as inhibition of EGFR kinase activity markedly attenuated c-Cbl phosphorylation and FA protein degradation induced by Cat.G. These findings support a model in which neutrophil protease Cat.G promotes c-Cbl interaction with FA proteins, resulting in enhanced c-Cbl-mediated FA protein ubiquitination and degradation, myofibril degradation, and subsequent down-regulation of myocyte survival signaling.     

A chimeric pre-ubiquitinated EGF receptor is constitutively endocytosed in a clathrin-dependent, but kinase-independent manner

The roles of EGF receptor (EGFR) kinase activity and ubiquitination in EGFR endocytosis have been controversial. The adaptor protein and ubiquitin ligase Cbl has reportedly been required. Consistently, we now report that siRNA-mediated knock-down of c-Cbl and Cbl-b significantly slowed clathrin-dependent internalization of activated wild-type (wt) EGFR by inhibiting recruitment of the EGFR to clathrin-coated pits. However, a chimeric protein consisting of wt-EGFR, a C-terminal linker and four linearly connected ubiquitins was found to interact with Eps15 and epsin 1 and to be constitutively endocytosed in a clathrin-dependent manner. Interestingly, endocytosis of this fusion protein did not require binding of EGF. Nor was kinase activity required, and the fusion protein was endocytosed in the presence of an EGFR kinase inhibitor, which efficiently counteracted tyrosine phosphorylation. This demonstrates that ubiquitination over-rides the requirement for kinase activity in recruitment of the EGFR to clathrin-coated pits.     

A tale of two Cbls: interplay of c-Cbl and Cbl-b in epidermal growth factor receptor downregulation

The precise role of Cbl in epidermal growth factor (EGF) receptor (EGFR) endocytosis and trafficking remains to be fully uncovered. Here, we showed that mutant EGFR1044, which was truncated after residue 1044, did not associate with c-Cbl and was not ubiquitinated initially in response to EGF but was internalized with kinetics similar to those of wild-type EGFR. This finding indicates that c-Cbl-mediated ubiquitination is not required for EGF-induced EGFR endocytosis. We also showed that the previously identified internalization-deficient mutant receptor EGFR1010LL/AA bound to c-Cbl and was fully ubiquitinated in response to EGF, which indicates that c-Cbl binding and ubiquitination are not sufficient for EGFR internalization. We next investigated EGFR trafficking following EGFR internalization. We found that c-Cbl disassociation from EGFR occurred well in advance of EGFR degradation and that this event was concurrent with the selective dephosphorylation of EGFR at Y1045. This finding suggests that once EGFR is ubiquitinated, continual Cbl association is not required for EGFR degradation. Because EGFR1044 is ubiquitinated and degraded similarly to wild-type EGFR, we examined the role of another prominent Cbl homologue, Cbl-b, and found that Cbl-b was associated with both EGFR and EGFR1044. Further study showed that Cbl-b bound to EGFR at two regions: one in the C-terminal direction from residue 1044 and one in the N-terminal direction from residue 958. Moreover, Cbl-b association with EGFR rose markedly following a decrease in c-Cbl association, corresponding to a second peak of EGFR ubiquitination occurring later in EGFR trafficking. Using RNA interference to knock down both c-Cbl and Cbl-b, we were able to abolish EGFR downregulation. This knockdown had no affect on the rate of EGF-induced EGFR internalization. We found that the two Cbls accounted for total receptor ubiquitination and that while c-Cbl and Cbl-b are each alone sufficient to effect EGFR degradation, both are involved in the physiological, EGF-mediated process of receptor downregulation. Furthermore, these data ultimately reveal a previously unacknowledged temporal interplay of two major Cbl homologues with the trafficking of EGFR.     

Smad7 Modulates Epidermal Growth Factor Receptor Turnover through Sequestration of c-Cbl

Epidermal growth factor (EGF) regulates various cellular events, including proliferation, differentiation, migration, and tumorigenesis. For the maintenance of homeostasis, EGF signaling should be tightly regulated to prevent the aberrant activation. Smad7 has been known as inhibitory Smad that blocks the signal transduction of transforming growth factor β. In the process of cell proliferation or transformation, Smad7 has been shown the opposite activities as a promoter or suppressor depending on cell types or microenvironments. We found that the overexpression of Smad7 in human HaCaT keratinocyte cells and mouse skin tissues elevated EGF receptor (EGFR) activity by impairing ligand-induced ubiquitination and degradation of activated receptor, which is induced by the E3 ubiquitin ligase c-Cbl. The C-terminal MH2 region but not MH1 region of Smad7 is critical for interaction with c-Cbl to inhibit the ubiquitination of EGFR. Interestingly, wild-type Smad7, but not Smad6 or mutant Smad7, destabilized the EGF-induced complex formation of c-Cbl and EGFR. These data suggest a novel role for Smad7 as a promoter for prolonging the EGFR signal in keratinocyte and skin tissue by reducing its ligand-induced ubiquitination and degradation.     

The APS adapter protein couples the insulin receptor to the phosphorylation of c-Cbl and facilitates ligand-stimulated ubiquitination of the insulin receptor

The APS adapter protein is rapidly tyrosine-phosphorylated following insulin stimulation. In insulin-stimulated 3T3-L1 adipocytes, APS co-precipitated with phosphorylated c-Cbl. In CHO.T-APS cells overexpressing the insulin receptor and APS, APS co-precipitated with c-Cbl but not in CHO.T cells which do not express APS. APS-mediated recruitment of c-Cbl to the insulin receptor led to rapid ubiquitination of the insulin receptor beta-subunit in CHO. T-APS but not in parental CHO.T cells. These results suggest that the function of APS is to facilitate coupling of the insulin receptor to c-Cbl in order to catalyse the ubiquitination of the receptor and initiation of internalisation or degradation.     

Ubiquitination and proteasomal activity is required for transport of the EGF receptor to inner membranes of multivesicular bodies

EGF, but not TGF alpha, efficiently induces degradation of the EGF receptor (EGFR). We show that EGFR was initially polyubiquitinated to the same extent upon incubation with EGF and TGF alpha, whereas the ubiquitination was more sustained by incubation with EGF than with TGF alpha. Consistently, the ubiquitin ligase c-Cbl was recruited to the plasma membrane upon activation of the EGFR with EGF and TGF alpha, but localized to endosomes only upon activation with EGF. EGF remains bound to the EGFR upon endocytosis, whereas TGF alpha dissociates from the EGFR. Therefore, the sustained polyubiquitination is explained by EGF securing the kinase activity of endocytosed EGFR. Overexpression of the dominant negative N-Cbl inhibited ubiquitination of the EGFR and degradation of EGF and EGFR. This demonstrates that EGF-induced ubiquitination of the EGFR as such is important for lysosomal sorting. Both lysosomal and proteasomal inhibitors blocked degradation of EGF and EGFR, and proteasomal inhibitors inhibited translocation of activated EGFR from the outer limiting membrane to inner membranes of multivesicular bodies (MVBs). Therefore, lysosomal sorting of kinase active EGFR is regulated by proteasomal activity. Immuno-EM showed the localization of intact EGFR on internal membranes of MVBs. This demonstrates that the EGFR as such is not the proteasomal target.     

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.     

The Relative Expression of Mig6 and EGFR Is Associated with Resistance to EGFR Kinase Inhibitors

The sensitivity of only a few tumors to anti-epidermal growth factor receptor EGFR tyrosine kinase inhibitors (TKIs) can be explained by the presence of EGFR tyrosine kinase (TK) domain mutations. In addition, such mutations were rarely found in tumor types other than lung, such as pancreatic and head and neck cancer. In this study we sought to elucidate mechanisms of resistance to EGFR-targeted therapies in tumors that do not harbor TK sensitizing mutations in order to identify markers capable of guiding the decision to incorporate these drugs into chemotherapeutic regimens. Here we show that EGFR activity was markedly decreased during the evolution of resistance to the EGFR tyrosine kinase inhibitor (TKI) erlotinib, with a concomitant increase of mitogen-inducible gene 6 (Mig6), a negative regulator of EGFR through the upregulation of the PI3K-AKT pathway. EGFR activity, which was more accurately predicted by the ratio of Mig6/EGFR, highly correlated with erlotinib sensitivity in panels of cancer cell lines of different tissue origins. Blinded testing and analysis in a prospectively followed cohort of lung cancer patients treated with gefitinib alone demonstrated higher response rates and a marked increased in progression free survival for patients with a low Mig6/EGFR ratio (approximately 100 days, P = 0.01).     

Alix facilitates the interaction between c-Cbl and platelet-derived growth factor beta-receptor and thereby modulates receptor down-regulation

Alix (ALG-2-interacting protein X) is an adaptor protein involved in down-regulation and sorting of cell surface receptors through the endosomal compartments toward the lysosome. In this study, we show that Alix interacts with the C-terminal region of the platelet-derived growth factor (PDGF) beta-receptor (PDGFRbeta) and becomes transiently tyrosine-phosphorylated in response to PDGF-BB stimulation. Increased expression levels of Alix resulted in a reduced rate of PDGFRbeta removal from the cell surface following receptor activation, and this was associated with decreased receptor degradation. Furthermore, Alix was found to co-immunoprecipitate with the ubiquitin ligase c-Cbl, and elevated Alix levels increased the interaction between c-Cbl and PDGFRbeta. Interestingly, Alix interacted constitutively with both c-Cbl and PDGFRbeta. Moreover, c-Cbl was found to be hyperphosphorylated in cells engineered to overexpress Alix compared with control cells. The increased c-Cbl phosphorylation correlated with enhanced proteasomal degradation of c-Cbl, which in turn correlated with a decreased ubiquitination of PDGFRbeta. Our data suggest that Alix inhibits down-regulation of PDGFRbeta by modulating the interaction between c-Cbl and the receptor, thereby affecting the ubiquitination of the receptor.     

GAPex-5 mediates ubiquitination, trafficking, and degradation of epidermal growth factor receptor

Upon ligand stimulation, epidermal growth factor receptor (EGFR) is rapidly ubiquitinated, internalized, and sorted to lysosomes for degradation. Rab5 has been shown to play an important role in the early stages of EGFR trafficking. GAPex-5 is a newly described Rab5 exchange factor. Herein, we investigate the role of GAPex-5 on EGFR trafficking and degradation. Down-regulation of GAPex-5 by RNA interference decreases epidermal growth factor-stimulated EGFR degradation. Moreover, ubiquitination of EGFR is impaired by depletion of GAPex-5. This inhibitory effect is due to a decrease in the interaction between the adapter protein c-Cbl and EGFR, but not the phosphorylation state of EGFR. Consistently, when examined by immunofluorescence microscopy in cells depleted of GAPex-5, ligand-bound EGFR appeared trapped in early endosomes and the trafficking of internalized receptor from early to late endosomes was impaired. In agreement with the depletion studies, EGFR degradation is enhanced by overexpressing GAPex-5 wild type, but not GAPex-5DeltaGAP, a mutant lacking the Ras GTPase-activating protein (GAP) domain. This is consistent with the finding that c-Cbl binds specifically to the Ras GAP domain. Finally, overexpression of dominant negative Rab5a or depletion of all three isoforms of Rab5 does not inhibit ubiquitination of EGFR, which suggests that GAPex-5-mediated EGFR ubiquitination is independent of Rab5 activation. Collectively, the results suggest a novel mechanism by which EGF-stimulated receptor ubiquitination and trafficking are mediated via GAPex-5.     

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.     

Ligand-induced ubiquitination of the epidermal growth factor receptor involves the interaction of the c-Cbl RING finger and UbcH7.

c-Cbl plays a negative regulatory role in tyrosine kinase signaling by an as yet undefined mechanism. We demonstrate here, using the yeast two-hybrid system and an in vitro binding assay, that the c-Cbl RING finger domain interacts with UbcH7, a ubiquitin-conjugating enzyme (E2). UbcH7 interacted 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 was enhanced by sequences on both the N- and C-terminal sides of the RING finger. In vivo and in vitro experiments revealed that c-Cbl and UbcH7 synergistically promote the ligand-induced ubiquitination of the epidermal growth factor receptor (EGFR). In contrast, 70Z-Cbl markedly reduced the ligand-induced, UbcH7-mediated ubiquitination of the EGFR. MG132, a proteasome inhibitor, significantly prolonged 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.     

Ubiquitin ligase activity of c-Cbl guides the epidermal growth factor receptor into clathrin-coated pits by two distinct modes of Eps15 recruitment

We have demonstrated previously that c-Cbl requires the presence of a functional ubiquitin interacting motif (UIM) in Eps15 to mediate epidermal growth factor receptor (EGFR) endocytosis. Both the ubiquitin ligase activity of c-Cbl and the UIM of Eps15 were necessary for plasma membrane recruitment of Eps15 and entry of ligand-bound EGFR into coated pits and vesicles containing Eps15. This is consistent with a scenario in which ubiquitin moieties appended to activated EGFR complexes act as docking sites for Eps15 and thereby recruit receptors into clathrin coated pits. Here, we have investigated which additional structural features of c-Cbl are required for this process. We find that c-Cbl can guide ligand-bound EGFR into the Eps15 internalization route by two distinct mechanisms. These are either dependent on the phosphotyrosine binding domain of c-Cbl that directly binds to the EGFR or on the region C-terminal of the Ring finger, which allows for indirect binding to an alternative site on the receptor. No strict requirement exists for either ubiquitin modified EGFR or the Cbl binding ubiquitination substrate CIN85 as docking site for the UIM of Eps15. Only in the phosphotyrosine binding-dependent pathway, the EGFR is ubiquitinated and may serve as a site of recruitment for Eps15. Only in this pathway, Eps15 is tyrosine-phosphorylated, but this appears unrelated to its capacity to participate in EGFR internalization.     

Epidermal growth factor receptor fate is controlled by Hrs tyrosine phosphorylation sites that regulate Hrs degradation

Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is an endosomal protein essential for the efficient sorting of activated growth factor receptors into the lysosomal degradation pathway. Hrs undergoes ligand-induced tyrosine phosphorylation on residues Y329 and Y334 downstream of epidermal growth factor receptor (EGFR) activation. It has been difficult to investigate the functional roles of phosphoHrs, as only a small proportion of the cellular Hrs pool is detectably phosphorylated. Using an HEK 293 model system, we found that ectopic expression of the protein Cbl enhances Hrs ubiquitination and increases Hrs phosphorylation following cell stimulation with EGF. We exploited Cbl's expansion of the phosphoHrs pool to determine whether Hrs tyrosine phosphorylation controls EGFR fate. In structure-function studies of Cbl and EGFR mutants, the level of Hrs phosphorylation and rapidity of apparent Hrs dephosphorylation correlated directly with EGFR degradation. Differential expression of wild-type versus Y329,334F mutant Hrs in Hrs-depleted cells revealed that one or both tyrosines regulate ligand-dependent Hrs degradation, as well as EGFR degradation. By modulating Hrs ubiquitination, phosphorylation, and protein levels, Cbl may control the composition of the endosomal sorting machinery and its ability to target EGFR for lysosomal degradation.     

Ubc4/5 and c-Cbl continue to ubiquitinate EGF receptor after internalization to facilitate polyubiquitination and degradation

c-Cbl is the E3 ubiquitin ligase that ubiquitinates the epidermal growth factor (EGF) receptor (EGFR). On the basis of localization, knockdown, and in vitro activity analyses, we have identified the E2 ubiquitin-conjugating enzyme that cooperates with c-Cbl as Ubc4/5. Upon EGF stimulation, both Ubc4/5 and c-Cbl were relocated to the plasma membrane and then to Hrs-positive endosomes, strongly suggesting that EGFR continues to be ubiquitinated after internalization. Our time-course experiment showed that EGFR undergoes polyubiquitination, which seemed to be facilitated during the transport to Hrs-positive endosomes. Use of a conjugation-defective ubiquitin mutant suggested that receptor polyubiquitination is required for efficient interaction with Hrs and subsequent sorting to lysosomes. Abrupt inhibition of the EGFR kinase activity resulted in dissociation of c-Cbl from EGFR. Concomitantly, EGFR was rapidly deubiquitinated and its degradation was delayed. We propose that sustained tyrosine phosphorylation of EGFR facilitates its polyubiquitination in endosomes and counteracts rapid deubiquitination, thereby ensuring Hrs-dependent lysosomal sorting.     

Ligand-induced downregulation of TrkA is partly regulated through ubiquitination by Cbl

Nerve growth factor (NGF) binding to its receptor TrkA, which belongs to the family of receptor tyrosine kinases (RTKs), is known to induce its internalization, endosomal trafficking and subsequent lysosomal degradation. The Cbl family of ubiquitin ligases plays a major role in mediating ubiquitination and degradation of RTKs. However, it is not known whether Cbl participates in mediating ubiquitination of TrkA. Here we report that c-Cbl mediates ligand-induced ubiquitination and degradation of TrkA. TrkA ubiquitination and degradation required direct interactions between c-Cbl and phosphorylated TrkA. c-Cbl and ubiquitinated TrkA are found in a complex after NGF stimulation and are degraded in lysosomes. Taken together, our data demonstrate that c-Cbl can induce downregulation of NGF-TrkA complexes through ubiquitination and degradation of TrkA.     

PEST motif serine and tyrosine phosphorylation controls vascular endothelial growth factor receptor 2 stability and downregulation

The internalization and degradation of vascular endothelial growth factor receptor 2 (VEGFR-2), a potent angiogenic receptor tyrosine kinase, is a central mechanism for the regulation of the coordinated action of VEGF in angiogenesis. Here, we show that VEGFR-2 is ubiquitinated in response to VEGF, and Lys 48-linked polyubiquitination controls its degradation via the 26S proteosome. The degradation and ubiquitination of VEGFR-2 is controlled by its PEST domain, and the phosphorylation of Ser1188/Ser1191 is required for the ubiquitination of VEGFR-2. F-box-containing β-Trcp1 ubiquitin E3 ligase is recruited to S1188/S1191 VEGFR-2 and mediates the ubiquitination and degradation of VEGFR-2. The PEST domain also controls the activation of p38 mitogen-activated protein kinase (MAPK) through phospho-Y1173. The activation of p38 stabilizes VEGFR-2, and its inactivation accelerates VEGFR-2 downregulation. The VEGFR-2-mediated activation of p38 is established through the protein kinase A (PKA)/MKK6 pathway. PKA is recruited to VEGFR-2 through AKAP1/AKAP149, and its phosphorylation requires Y1173 of VEGFR-2. The study has identified a unique mechanism in which VEGFR-2 stability and degradation is modulated. The PEST domain acts as a dual modulator of VEGFR-2; the phosphorylation of S1188/S1191 controls ubiquitination and degradation via β-Trcp1, where the phosphorylation of Y1173 through PKA/p38 MAPK controls the stability of VEGFR-2.