Cbl-mediated ubiquitinylation is required for lysosomal sorting of epidermal growth factor receptor but is dispensable for endocytosis

Ligand-induced down-regulation controls the signaling potency of the epidermal growth factor receptor (EGFR/ErbB1). Overexpression studies have identified Cbl-mediated ubiquitinylation of EGFR as a mechanism of ligand-induced EGFR down-regulation. However, the role of endogenous Cbl in EGFR down-regulation and the precise step in the endocytic pathway regulated by Cbl remain unclear. Using Cbl-/- mouse embryonic fibroblast cell lines, we demonstrate that endogenous Cbl is essential for ligand-induced ubiquitinylation and efficient degradation of EGFR. Further analyses using Chinese hamster ovary cells with a temperature-sensitive defect in ubiquitinylation confirm a crucial role of the ubiquitin machinery in Cbl-mediated EGFR degradation. However, internalization into early endosomes did not require Cbl function or an intact ubiquitin pathway. Confocal immunolocalization studies indicated that Cbl-dependent ubiquitinylation plays a critical role at the early endosome to late endosome/lysosome sorting step of EGFR down-regulation. These findings establish Cbl as the major endogenous ubiquitin ligase responsible for EGFR degradation, and show that the critical role of Cbl-mediated ubiquitinylation is at the level of endosomal sorting, rather than at the level of internalization.     

Cbl-mediated negative regulation of platelet-derived growth factor receptor-dependent cell proliferation. A critical role for Cbl tyrosine kinase-binding domain.

The Cbl proto-oncogene product has emerged as a novel negative regulator of receptor and non-receptor tyrosine kinases. Our previous observations that Cbl overexpression in NIH3T3 cells enhanced the ubiquitination and degradation of the platelet-derived growth factor receptor-alpha (PDGFRalpha) and that the expression of oncogenic Cbl mutants up-regulated the PDGFRalpha signaling machinery strongly suggested that Cbl negatively regulates PDGFRalpha signaling. Here, we show that, similar to PDGFRalpha, selective stimulation of PDGFRbeta induces Cbl phosphorylation, and its physical association with the receptor. Overexpression of wild type Cbl in NIH3T3 cells led to an enhancement of the ligand-dependent ubiquitination and subsequent degradation of the PDGFRbeta, as observed with PDGFRalpha. We show that Cbl-dependent negative regulation of PDGFRalpha and beta results in a reduction of PDGF-induced cell proliferation and protection against apoptosis. A point mutation (G306E) that inactivates the tyrosine kinase binding domain in the N-terminal transforming region of Cbl compromised the PDGF-inducible tyrosine phosphorylation of Cbl although this mutant could still associate with the PDGFR. More importantly, the G306E mutation abrogated the ability of Cbl to enhance the ligand-induced ubiquitination and degradation of the PDGFR and to inhibit the PDGF-dependent cell proliferation and protection from apoptosis. These results demonstrate that Cbl can negatively regulate PDGFR-dependent biological responses and that this function requires the conserved tyrosine kinase binding domain of Cbl.     

Biochemical basis for the requirement of kinase activity for Cbl-dependent ubiquitinylation and degradation of a target tyrosine kinase

Members of the Cbl family of ubiquitin ligases have emerged as crucial negative regulators of tyrosine kinase signaling. These proteins preferentially interact with and target activated tyrosine kinases for ubiquitinylation, thereby facilitating the lysosomal sorting of receptor tyrosine kinases or proteasomal degradation of nonreceptor tyrosine kinases. Recent work has indicated a crucial role of the target kinase activity in Cbl-dependent ubiquitinylation and degradation, but the biochemical basis for this requirement is not understood. Here, we have used the Src-family kinase Fyn, a well characterized Cbl target, to address this issue. Using defined Fyn mutants, we demonstrate that the kinase activity of Fyn is crucial for its Cbl-dependent ubiquitinylation and degradation, but a low level of ubiquitinylation and degradation of kinase-inactive Fyn mutants was consistently observed. Mutational induction of an open conformation enhanced the susceptibility of kinase-active Fyn to Cbl but was insufficient to promote the ubiquitinylation and degradation of kinase-inactive Fyn. Notably, the Cbl-dependent degradation of Fyn did not require the Fyn-mediated phosphorylation of Cbl. Finally, we show that the major determinant of the susceptibility of Fyn protein to Cbl-dependent ubiquitinylation and degradation is the extent to which it physically associates with Cbl; kinase activity of Fyn serves as a critical determinant to promote its association with Cbl, which we demonstrate is mediated by multiple protein-protein interactions. Our results strongly suggest that promotion of association with Cbl is the primary mechanism by which the kinase activity of the targets of Cbl contributes to their susceptibility to Cbl.     

Intersectin 1 enhances Cbl ubiquitylation of epidermal growth factor receptor through regulation of Sprouty2-Cbl interaction

Ubiquitylation of receptor tyrosine kinases plays a critical role in regulating the trafficking and lysosomal degradation of these important signaling molecules. We identified the multidomain scaffolding protein intersectin 1 (ITSN1) as an important regulator of this process (N. P. Martin et al., Mol. Pharmacol. 70:1463-1653, 2006) ITSN1 stimulates ubiquitylation of the epidermal growth factor receptor (EGFR) through enhancing the activity of the Cbl E3 ubiquitin ligase. However, the precise mechanism through which ITSN1 enhances Cbl activity was unclear. In this study, we found that ITSN1 enhances Cbl activity through disrupting the interaction of Cbl with the Sprouty2 (Spry2) inhibitory protein. We demonstrate that ITSN1 binds Pro-rich regions in both Cbl and Spry2 and that interaction of ITSN1 with Spry2 disrupts Spry2-Cbl interaction, resulting in enhanced ubiquitylation of the EGFR. Disruption of ITSN1 binding to Spry2 through point mutation of the Pro-rich ITSN1 binding site in Spry2 results in enhanced Cbl-Spry2 interaction and inhibition of receptor ubiquitylation. This study demonstrates that ITSN1 enhances Cbl activity by modulating the interaction of Cbl with Spry2. In addition, our results reveal a new level of complexity in the regulation of Cbl through the interaction with ITSN1 and Spry2.     

Phosphotyrosine binding domain-dependent upregulation of the platelet-derived growth factor receptor alpha signaling cascade by transforming mutants of Cbl: implications for Cbl's function and oncogenicity.

Recent studies have demonstrated that Cbl, the 120-kDa protein product of the c-cbl proto-oncogene, serves as a substrate of a number of receptor-coupled tyrosine kinases and forms complexes with SH3 and SH2 domain-containing proteins, pointing to its role in signal transduction. Based on genetic evidence that the Caenorhabditis elegans Cbl homolog, SLI-1, functions as a negative regulator of the LET-23 receptor tyrosine kinase and our demonstration that Cbl's evolutionarily conserved N-terminal transforming region (Cbl-N; residues 1 to 357) harbors a phosphotyrosine binding (PTB) domain that binds to activated ZAP-70 tyrosine kinase, we examined the possibility that oncogenic Cbl mutants may activate mitogenic signaling by deregulating cellular tyrosine kinase machinery. Here, we show that expression of Cbl-N and two other transforming Cbl mutants (CblY368 delta and Cbl366-382 delta or Cb170Z), but not wild-type Cbl, in NIH 3T3 fibroblasts leads to enhancement of endogenous tyrosine kinase signaling. We identified platelet-derived growth factor receptor alpha (PDGFR alpha) as one target of mutant Cbl-induced deregulation. In mutant Cbl transfectants, PDGFR alpha was hyperphosphorylated and constitutively complexed with a number of SH2 domain-containing proteins. PDGFR alpha hyperphosphorylation and enhanced proliferation of mutant Cbl-transfected NIH 3T3 cells were drastically reduced upon serum starvation, and PDGF-AA substituted for the maintenance of these traits. PDGF-AA stimulation of serum-starved Cbl transfectants induced the in vivo association of transfected Cbl proteins with PDGFR alpha. In vitro, Cbl-N directly bound to PDGFR alpha derived from PDGF-AA-stimulated cells but not to that from unstimulated cells, and this binding was abrogated by a point mutation (G306E) corresponding to a loss-of-function mutation in SLI-1. The Cbl-N/G306E mutant protein, which failed to induce enhanced growth and transformation of NIH 3T3 cells, also failed to induce hyperphosphorylation of PDGFR alpha. Altogether, these findings identify a novel mechanism of Cbl's physiological function and oncogenesis, involving its PTB domain-dependent direct interaction with cellular tyrosine kinases.     

Negative regulation of EGFR-Vav2 signaling axis by Cbl ubiquitin ligase controls EGF receptor-mediated epithelial cell adherens junction dynamics and cell migration

The E3 ubiquitin ligase Casitas B lymphoma protein (Cbl) controls the ubiquitin-dependent degradation of EGF receptor (EGFR), but its role in regulating downstream signaling elements with which it associates and its impact on biological outcomes of EGFR signaling are less clear. Here, we demonstrate that stimulation of EGFR on human mammary epithelial cells disrupts adherens junctions (AJs) through Vav2 and Rac1/Cdc42 activation. In EGF-stimulated cells, Cbl regulates the levels of phosphorylated Vav2 thereby attenuating Rac1/Cdc42 activity. Knockdown of Cbl and Cbl-b enhanced the EGF-induced disruption of AJs and cell motility. Overexpression of constitutively active Vav2 activated Rac1/Cdc42 and reorganized junctional actin cytoskeleton; these effects were suppressed by WT Cbl and enhanced by a ubiquitin ligase-deficient Cbl mutant. Cbl forms a complex with phospho-EGFR and phospho-Vav2 and facilitates phospho-Vav2 ubiquitinylation. Cbl can also interact with Vav2 directly in a Cbl Tyr-700-dependent manner. A ubiquitin ligase-deficient Cbl mutant enhanced the morphological transformation of mammary epithelial cells induced by constitutively active Vav2; this effect requires an intact Cbl Tyr-700. These results indicate that Cbl ubiquitin ligase plays a critical role in the maintenance of AJs and suppression of cell migration through down-regulation of EGFR-Vav2 signaling.     

Cbl-mediated ubiquitinylation and negative regulation of Vav

The Cbl ubiquitin ligase has emerged as a negative regulator of receptor and non-receptor tyrosine kinases. Cbl is known to associate with the proto-oncogene product Vav, a hematopoietic-restricted Rac guanine nucleotide exchange factor, but the consequences of this interaction remain to be elucidated. Using immortalized T cell lines from Cbl(+/+) and Cbl(-/-) mice, and transfection analyses in 293T cells, we demonstrate that Vav undergoes Cbl-dependent ubiquitinylation under conditions that promote Cbl and Vav phosphorylation. Interaction with Cbl also induced the loss of phosphorylated Vav. In addition, we show that an activated Vav mutant (Vav-Y174F) is more sensitive to Cbl-dependent ubiquitinylation. We demonstrate that the Cbl-dependent ubiquitinylation of Vav requires Cbl/Vav association through phosphorylated Tyr-700 on Cbl, and also requires an intact Cbl RING finger domain. Finally, using transfection analyses in the Jurkat T cell line, we show that Cbl, but not its ubiquitin ligase mutant, can inhibit Vav-dependent signaling. Thus, our findings strongly support the role of Cbl, via its ubiquitin ligase activity, as a negative regulator of activated Vav.     

The Casitas B lineage lymphoma (Cbl) mutant G306E enhances osteogenic differentiation in human mesenchymal stromal cells in part by decreased Cbl-mediated platelet-derived growth factor receptor alpha and fibroblast growth factor receptor 2 ubiquitination

Human bone marrow-derived mesenchymal stromal cells (hMSCs) have the capacity to differentiate into several cell types including osteoblasts and are therefore an important cell source for bone tissue regeneration. A crucial issue is to identify mechanisms that trigger hMSC osteoblast differentiation to promote osteogenic potential. Casitas B lineage lymphoma (Cbl) is an E3 ubiquitin ligase that ubiquitinates and targets several molecules for degradation. We hypothesized that attenuation of Cbl-mediated degradation of receptor tyrosine kinases (RTKs) may promote osteogenic differentiation in hMSCs. We show here that specific inhibition of Cbl interaction with RTKs using a Cbl mutant (G306E) promotes expression of osteoblast markers (Runx2, alkaline phosphatase, type 1 collagen, osteocalcin) and increases osteogenic differentiation in clonal bone marrow-derived hMSCs and primary hMSCs. Analysis of molecular mechanisms revealed that the Cbl mutant increased PDGF receptor α and FGF receptor 2 but not EGF receptor expression in hMSCs, resulting in increased ERK1/2 and PI3K signaling. Pharmacological inhibition of FGFR or PDGFR abrogated in vitro osteogenesis induced by the Cbl mutant. The data reveal that specific inhibition of Cbl interaction with RTKs promotes the osteogenic differentiation program in hMSCs in part by decreased Cbl-mediated PDGFRα and FGFR2 ubiquitination, providing a novel mechanistic approach targeting Cbl to promote the osteogenic capacity of hMSCs.     

Abl tyrosine kinase regulates endocytosis of the epidermal growth factor receptor

Signal attenuation from ligand-activated epidermal growth factor receptor (EGFR) is mediated in part by receptor endocytosis and trafficking to the lysosomal degradative compartment. Uncoupling the activated EGFR from endocytosis and degradation has emerged as a mechanism for oncogenic activation of the EGFR. The Abl nonreceptor tyrosine kinase is activated by ligand-stimulated EGFR, but the role of Abl in EGFR signaling has not been defined. Here we uncovered a novel role for the activated Abl kinase in the regulation of EGFR endocytosis. We show that activated Abl impairs EGFR internalization. Moreover, we show that activated Abl phosphorylates the EGFR primarily on tyrosine 1173, and that mutation of this site to phenylalanine restores ligand-dependent endocytosis of the EGFR in the presence of activated Abl. Furthermore, we show that activated Abl allows the ligand-activated EGFR to escape Cbl-dependent down-regulation by inhibiting the accumulation of Cbl at the plasma membrane in response to epidermal growth factor stimulation and disrupting the formation of the EGFR.Cbl complex without affecting Cbl protein stability. These findings reveal a novel role for Abl in promoting increased cell-surface expression of the EGFR and suggest that Abl/EGFR signaling may cooperate in human tumors.     

Direct interaction of Cbl with pTyr 1045 of the EGF receptor (EGFR) is required to sort the EGFR to lysosomes for degradation

Mutation of the binding site for Cbl (Tyr1045) in the EGF receptor (EGFR) results in impaired ubiquitination but does not affect EGFR internalization. However, the Y1045F mutation resulted in strongly decreased degradation of the EGFR, as well as efficient recycling of EGFR to the plasma membrane. Significantly, more wild-type EGFR than Y1045F EGFR was found localizing to multivesicular late endosomes. Ubiquitination of the EGFR was in HeLa cells inhibited both upon overexpressing the N-terminal part of Cbl and upon overexpressing a double mutant Grb2 incapable of interacting with Cbl and thereby being incapable of indirectly recruiting Cbl to the EGFR. Collectively, these data suggest that the ubiquitination resulting from direct binding of Cbl to pTyr1045 of the EGFR is critical for lysosomal sorting of the EGFR in contrast to ubiquitination resulting from Grb2-mediated binding of Cbl to the EGFR.     

The gradient of Gurken, a long-range morphogen, is directly regulated by Cbl-mediated endocytosis

The asymmetric localization of gurken mRNA and post-translational sorting mechanisms are responsible for the polar distribution of Gurken protein in Drosophila. However, endocytosis of Egfr, the receptor for Gurken in the follicle cells, also plays a role in shaping the extracellular gradient of the Gurken morphogen. Previously, we have found that mutation in the Cbl gene caused elevated Egfr signaling along the dorsoventral axis, and resulted in dorsalization phenotypes in embryos and egg shells. Here, we report that overexpression of the Cbl long isoform significantly changed Gurken distribution. Using an HRP-Gurken fusion protein, we demonstrate that internalization of the Gurken-Egfr complex depends on the activity of Cbl. Increased levels of CblL promote the internalization of this complex, leading to the reduction of free ligands. The Gurken-Egfr complex trafficks through the Rab5/Rab7 associated endocytic pathway to the lysosomal degradation compartment for signaling termination. We observe endocytic Gurken not only in the dorsal but also in the ventral follicle cells, which is, to our knowledge, the first visualization of Gurken on the ventral side of egg chambers. Our results show that Gurken travels towards the lateral/posterior of the egg chamber in the absence of Cbl, suggesting that Cbl actively regulates Gurken distribution through promoting endocytosis and subsequent degradation.     

p38 kinase regulates epidermal growth factor receptor downregulation and cellular migration

Internalization and proteolytic degradation of epidermal growth factor (EGF) receptor (R) following ligand binding is an important mechanism for regulating EGF-stimulated signals. Using pharmacological and RNA interference inhibition of p38 mitogen-activated protein kinase, we show that p38 is required for efficient EGF-induced EGFR destruction but not internalization. In the absence of p38 activity, EGF fails to stimulate the ubiquitin ligase Cbl or ubiquitinylation of EGFR, and internalized EGFR accumulates in intracellular vesicles containing caveolin-1. These effects are accompanied by loss of EGFR phosphorylation on Y1045, a phosphorylation site required for Cbl activation. Furthermore, similar to cells treated with p38 inhibitors, intestinal epithelial cells expressing Y1045F EGFR mutants show increased proliferation but not migration in response to EGF, thus uncoupling these biological responses. Together these data position p38 as a modulator of ligand-stimulated EGFR processing and demonstrate that this processing has a profound impact on the cellular outcome of EGFR signaling.     

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.     

Receptor Tyrosine Kinase Ubiquitylation Involves the Dynamic Regulation of Cbl-Spry2 by Intersectin 1 and the Shp2 Tyrosine Phosphatase

Ubiquitylation of receptor tyrosine kinases (RTKs) regulates their trafficking and lysosomal degradation. The multidomain scaffolding protein intersectin 1 (ITSN1) is an important regulator of this process. ITSN1 stimulates ubiquitylation of the epidermal growth factor receptor (EGFR) through enhancing the activity of the Cbl E3 ubiquitin ligase. However, the precise mechanism through which ITSN1 enhances Cbl activity is unclear. Here, we demonstrate that ITSN1 interacts with and recruits the Shp2 tyrosine phosphatase to Spry2 to enhance its dephosphorylation, thereby disrupting the inhibitory effect of Spry2 on Cbl and enhancing EGFR ubiquitylation. In contrast, expression of a catalytically inactive Shp2 mutant reversed the effect of ITSN1 on Spry2 dephosphorylation and decreased Cbl-mediated EGFR ubiquitylation. In addition, disruption of ITSN1 binding to Spry2 through point mutation of the Pro-rich ITSN1 binding site in Spry2 resulted in decreased Shp2-Spry2 interaction and enhanced Spry2 tyrosine phosphorylation. This study demonstrates that ITSN1 enhances Cbl activity, in part, by modulating the interaction of Cbl with Spry2 through recruitment of Shp2 phosphatase to the Cbl-Spry2 complex. These findings reveal a new level of complexity in the regulation of RTKs by Cbl through ITSN1 binding with Shp2 and Spry2.     

Synergy between the ESCRT-III complex and Deltex defines a ligand-independent Notch signal

The Notch signaling pathway defines a conserved mechanism that regulates cell fate decisions in metazoans. Signaling is modulated by a broad and multifaceted genetic circuitry, including members of the endocytic machinery. Several individual steps in the endocytic pathway have been linked to the positive or negative regulation of the Notch receptor. In seeking genetic elements involved in regulating the endosomal/lysosomal degradation of Notch, mediated by the molecular synergy between the ubiquitin ligase Deltex and Kurtz, the nonvisual β-arrestin in Drosophila, we identified Shrub, a core component of the ESCRT-III complex as a key modulator of this synergy. Shrub promotes the lysosomal degradation of the receptor by mediating its delivery into multivesicular bodies (MVBs). However, the interplay between Deltex, Kurtz, and Shrub can bypass this path, leading to the activation of the receptor. Our analysis shows that Shrub plays a pivotal rate-limiting step in late endosomal ligand-independent Notch activation, depending on the Deltex-dependent ubiquitinylation state of the receptor. This activation mode of the receptor emphasizes the complexity of Notch signal modulation in a cell and has significant implications for both development and disease.     

Met kinase-dependent loss of the E3 ligase Cbl in gastric cancer

Strict regulation of signaling by receptor tyrosine kinases (RTKs) is essential for normal biological processes, and disruption of this regulation can lead to tumor initiation and progression. Signal duration by the Met RTK is mediated in part by the E3 ligase Cbl. Cbl is recruited to Met upon kinase activation and promotes ubiquitination, trafficking, and degradation of the receptor. The Met RTK has been demonstrated to play a role in various types of cancer. Here, we show that Met-dependent loss of Cbl protein in MET-amplified gastric cancer cell lines represents another mechanism contributing to signal dysregulation. Loss of Cbl protein is dependent on Met kinase activity and is partially rescued with a proteasome inhibitor, lactacystin. Moreover, Cbl loss not only uncouples Met from Cbl-mediated negative regulation but also releases other Cbl targets, such as the EGF receptor, from Cbl-mediated signal attenuation. Thus, Met-dependent Cbl loss may also promote cross-talk through indirect enhancement of EGF receptor signaling.     

Met/Hepatocyte growth factor receptor ubiquitination suppresses transformation and is required for Hrs phosphorylation

The Met receptor tyrosine kinase (RTK) regulates epithelial remodeling, dispersal, and invasion and is deregulated in many human cancers. It is now accepted that impaired down-regulation, as well as sustained activation, of RTKs could contribute to their deregulation. Down-regulation of the Met receptor involves ligand-induced internalization, ubiquitination by Cbl ubiquitin ligases, and lysosomal degradation. Here we report that a ubiquitination-deficient Met receptor mutant (Y1003F) is tumorigenic in vivo. The Met Y1003F mutant is internalized, and undergoes endosomal trafficking with kinetics similar to the wild-type Met receptor, yet is inefficiently targeted for degradation. This results in sustained activation of Met Y1003F and downstream signals involving the Ras-mitogen-activated protein kinase pathway, cell transformation, and tumorigenesis. Although Met Y1003F undergoes endosomal trafficking and localizes with the cargo-sorting protein Hrs, it is unable to induce phosphorylation of Hrs. Fusion of monoubiquitin to Met Y1003F is sufficient to decrease Met receptor stability and prevent sustained MEK1/2 activation. In addition, this rescues Hrs tyrosine phosphorylation and decreases transformation in a focus-forming assay. These results demonstrate that Cbl-dependent ubiquitination is dispensable for Met internalization but is critical to target the Met receptor to components of the lysosomal sorting machinery and to suppress its inherent transforming activity.     

ALIX binds a YPX(3)L motif of the GPCR PAR1 and mediates ubiquitin-independent ESCRT-III/MVB sorting

The sorting of signaling receptors to lysosomes is an essential regulatory process in mammalian cells. During degradation, receptors are modified with ubiquitin and sorted by endosomal sorting complex required for transport (ESCRT)-0, -I, -II, and -III complexes into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs). However, it remains unclear whether a single universal mechanism mediates MVB sorting of all receptors. We previously showed that protease-activated receptor 1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is internalized after activation and sorted to lysosomes independent of ubiquitination and the ubiquitin-binding ESCRT components hepatocyte growth factor-regulated tyrosine kinase substrate and Tsg101. In this paper, we report that PAR1 sorted to ILVs of MVBs through an ESCRT-III-dependent pathway independent of ubiquitination. We further demonstrate that ALIX, a charged MVB protein 4-ESCRT-III interacting protein, bound to a YPX(3)L motif of PAR1 via its central V domain to mediate lysosomal degradation. This study reveals a novel MVB/lysosomal sorting pathway for signaling receptors that bypasses the requirement for ubiquitination and ubiquitin-binding ESCRTs and may be applicable to a subset of GPCRs containing YPX(n)L motifs.     

Cbl promotes clustering of endocytic adaptor proteins

The ubiquitin ligases c-Cbl and Cbl-b play a crucial role in receptor downregulation by mediating multiple monoubiquitination of receptors and promoting their sorting for lysosomal degradation. Their function is modulated through interactions with regulatory proteins including CIN85 and PIX, which recognize a proline-arginine motif in Cbl and thus promote or inhibit receptor endocytosis. We report the structures of SH3 domains of CIN85 and beta-PIX in complex with a proline-arginine peptide from Cbl-b. Both structures reveal a heterotrimeric complex containing two SH3 domains held together by a single peptide. Trimerization also occurs in solution and is facilitated by the pseudo-symmetrical peptide sequence. Moreover, ternary complexes of CIN85 and Cbl are formed in vivo and are important for the ability of Cbl to promote epidermal growth factor receptor (EGFR) downregulation. These results provide molecular explanations for a novel mechanism by which Cbl controls receptor downregulation.     

ErbB2 degradation mediated by the co-chaperone protein CHIP

ErbB2 overexpression contributes to the evolution of a substantial group of human cancers and signifies a poor clinical prognosis. Thus, down-regulation of ErbB2 signaling has emerged as a new anti-cancer strategy. Ubiquitinylation, mediated by the Cbl family of ubiquitin ligases, has emerged as a physiological mechanism of ErbB receptor down-regulation, and this mechanism appears to contribute to ErbB2 down-regulation induced by therapeutic anti-ErbB2 antibodies. Hsp90 inhibitory ansamycin antibiotics such as geldanamycin (GA) induce rapid ubiquitinylation and down-regulation of ErbB2. However, the ubiquitin ligase(s) involved has not been identified. Here, we show that ErbB2 serves as an in vitro substrate for the Hsp70/Hsp90-associated U-box ubiquitin ligase CHIP. Overexpression of wild type CHIP, but not its U-box mutant H260Q, induced ubiquitinylation and reduction in both cell surface and total levels of ectopically expressed or endogenous ErbB2 in vivo, and this effect was additive with that of 17-allylamino-geldanamycin (17-AAG). The CHIP U-box mutant H260Q reduced 17-AAG-induced ErbB2 ubiquitinylation. Wild type ErbB2 and a mutant incapable of association with Cbl (ErbB2 Y1112F) were equally sensitive to CHIP and 17-AAG, implying that Cbl does not play a major role in geldanamycin-induced ErbB2 down-regulation. Both endogenous and ectopically expressed CHIP and ErbB2 coimmunoprecipitated with each other, and this association was enhanced by 17-AAG. Notably, CHIP H260Q induced a dramatic elevation of ErbB2 association with Hsp70 and prevented the 17-AAG-induced dissociation of Hsp90. Our results demonstrate that ErbB2 is a target of CHIP ubiquitin ligase activity and suggest a role for CHIP E3 activity in controlling both the association of Hsp70/Hsp90 chaperones with ErbB2 and the down-regulation of ErbB2 induced by inhibitors of Hsp90.