Cloning and characterization of a novel adaptor protein, CIN85, that interacts with c-Cbl

The c-Cbl protooncogene product is a prominent substrate of protein tyrosine kinases and is rapidly tyrosine-phosphorylated upon stimulation of a wide variety of cell-surface receptors. We have identified a novel c-Cbl-interacting protein termed CIN85 with a molecular mass of 85 kDa which shows similarity to adaptor proteins, CMS and CD2AP. CIN85 mRNA is expressed ubiquitously in normal human tissues and cancer cell lines analyzed. CIN85 was basally associated with c-Cbl. For interaction of CIN85 with c-Cbl, the second SH3 domain of CIN85 was shown to serve as a central player. The CIN85-c-Cbl association was enhanced shortly after stimulation of 293 cells with epidermal growth factor (EGF) and gradually diminished to a basal level, which correlated with a tyrosine phosphorylation level of c-Cbl. Our results suggest that CIN85 may play a specific role in the EGF receptor-mediated signaling cascade via its interaction with c-Cbl.     

Leukocyte Ig-like Receptor B4 (LILRB4) Is a Potent Inhibitor of Fc��RI-mediated Monocyte Activation via Dephosphorylation of Multiple Kinases

The leukocyte immunoglobulin-like receptor (LILR) B4 belongs to a family of cell surface receptors that possesses cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). LILRB4 is believed to down-regulate activation signals mediated by non-receptor tyrosine kinase cascades through the recruitment of SHP-1. However, the exact mechanisms of LILRB4-mediated inhibition are not fully elucidated. In this study, we demonstrate high level surface expression of LILRB4 on THP-1 cells and primary peripheral blood monocytes, which profoundly inhibited production of a key pro-inflammatory cytokine (TNFα) induced by FcγRI (CD64). We also report that LILRB4 aggregated to sites of activation upon co-ligation with CD64 and that this may enhance its inhibitory effects. Cross-linking of CD64 on THP-1 cells markedly increased phosphorylation of multiple proteins including tyrosine kinases and signaling molecules (Lck, Syk, LAT, and Erk), an adaptor protein that targets protein-tyrosine kinases for degradation (c-Cbl) and a protein involved in the formation of actin cytoskeletal rearrangement (α-actinin-4). Co-ligation of LILRB4 considerably reduced CD64-mediated phosphorylation of Lck, Syk, LAT, Erk, and c-Cbl but not α-actinin-4, suggesting selective inhibition of signaling molecules. Treatment of cells with a broad-spectrum phosphatase inhibitor, sodium pervanadate (SP), significantly reversed LILRB4-mediated inhibition of TNFα production and protein tyrosine phosphorylation. In comparison, treatment with an SHP-1 specific inhibitor, sodium stibogluconate (SS) has no effects indicating involvement of phosphatase(s) other than SHP-1 in LILRB4 signaling. Collectively, our data show LILRB4 is a potent inhibitor of monocytes activation. This may provide a new potential therapeutic strategy for inflammatory conditions characterized by excessive TNFα production.     

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.     

Inhibition of CIN85-Mediated Invasion by a Novel SH3 Domain Binding Motif in the Lysyl Oxidase Propeptide

The lysyl oxidase gene inhibits Ras signaling in transformed fibroblasts and breast cancer cells. Its activity was mapped to the 162 amino acid propeptide domain (LOX-PP) of the lysyl oxidase precursor protein. LOX-PP inhibited the Her-2/Ras signaling axis in breast cancer cells, and reduced the Her-2-driven breast tumor burden in a xenograft model. Since its mechanism of action is largely unknown, co-affinity-purification/mass spectrometry was performed and the “Cbl-interacting protein of 85-kDa” (CIN85) identified as an associating protein. CIN85 is an SH3-containing adapter protein that is overexpressed in invasive breast cancers. The CIN85 SH3 domains interact with c-Cbl, an E3 ubiquitin ligase, via an unconventional PxxxPR ligand sequence, with the highest affinity displayed by the SH3-B domain. Interaction with CIN85 recruits c-Cbl to the AMAP1 complex where its ubiquitination activity is necessary for cancer cells to develop an invasive phenotype and to degrade the matrix. Direct interaction of LOX-PP with CIN85 was confirmed using co-immunoprecipitation analysis of lysates from breast cancer cells and of purified expressed proteins. CIN85 interaction with c-Cbl was reduced by LOX-PP. Domain specific CIN85 regions and deletion mutants of LOX-PP were prepared and used to map the sites of interaction to the SH3-B domain of CIN85 and to an epitope encompassing amino acids 111 to 116 of LOX-PP. Specific LOX-PP point mutant proteins P111A and R116A failed to interact with CIN85 or to compete for CIN85 binding with c-Cbl. Structural modeling identified a new atypical PxpxxRh SH3-binding motif in this region of LOX-PP. The LOX-PP interaction with CIN85 was shown to reduce the invasive phenotype of breast cancer cells, including their ability to degrade the surrounding extracellular matrix and for Matrigel outgrowth. Thus, LOX-PP interacts with CIN85 via a novel SH3-binding motif and this association reduces CIN85-promoted invasion by breast cancer cells.     

CIN85 regulates ubiquitination and degradative endosomal sorting of the EGF receptor

CIN85 has been demonstrated to interact with a number of proteins involved in endocytosis and intracellular sorting. However, the exact functional role of CIN85 in endocytosis remains unclear. We have investigated whether CIN85 plays a role in EGF-induced EGF receptor (EGFR) internalization, as previously suggested, or whether CIN85 is rather involved in endosomal sorting of the EGFR. When over-expressing a dominant negative interfering CIN85 mutant consisting of three SH3 domains only, we found that internalization of EGF was inhibited. However, when knocking down CIN85 by RNAi, the EGF–EGFR uptake appeared similar to in control cells. Furthermore, in CIN85 depleted cells, EGF-induced ubiquitination of the EGFR was decreased, and degradation of EGF–EGFR complexes was delayed. Our data further demonstrated that depletion of CIN85 increased the recycling of EGF, suggesting that CIN85 plays a role in endosomal sorting of the ubiquitinated EGFR. Our data also demonstrated that CIN85 was constitutively associated with Hrs, and this strengthens the hypothesis of a functional role of CIN85 in endosomal EGFR sorting.     

Suppression of PPARγ through MKRN1-mediated ubiquitination and degradation prevents adipocyte differentiation

The central regulator of adipogenesis, PPARγ, is a nuclear receptor that is linked to obesity and metabolic diseases. Here we report that MKRN1 is an E3 ligase of PPARγ that induces its ubiquitination, followed by proteasome-dependent degradation. Furthermore, we identified two lysine sites at 184 and 185 that appear to be targeted for ubiquitination by MKRN1. Stable overexpression of MKRN1 reduced PPARγ protein levels and suppressed adipocyte differentiation in 3T3-L1 and C3H10T1/2 cells. In contrast, MKRN1 depletion stimulated adipocyte differentiation in these cells. Finally, MKRN1 knockout MEFs showed an increased capacity for adipocyte differentiation compared with wild-type MEFs, with a concomitant increase of PPARγ and adipogenic markers. Together, these data indicate that MKRN1 is an elusive PPARγ E3 ligase that targets PPARγ for proteasomal degradation by ubiquitin-dependent pathways, and further depict MKRN1 as a novel target for diseases involving PPARγ.     

Interactions between the three CIN85 SH3 domains and ubiquitin: implications for CIN85 ubiquitination

CIN85 is an adaptor protein linking the ubiquitin ligase Cbl and clathrin-binding proteins in clathrin-mediated receptor endocytosis. The SH3 domains of CIN85 bind to a proline-rich region of Cbl. Here we show that all three SH3 domains of CIN85 bind to ubiquitin. We also present a data-based structural model of the CIN85 SH3-C domain in complex with ubiquitin. In this complex, ubiquitin binds to the canonical interaction surface of the SH3 domain for proline-rich ligands and mimics the PPII helix, and we provide evidence that ubiquitin competes with these ligands for binding. We demonstrate that disruption of ubiquitin binding results in constitutive ubiquitination of CIN85 and an increased level of ubiquitination of EGFR in the absence of EGF stimulation. These results suggest that competition between Cbl and ubiquitin binding to CIN85 regulates Cbl function and EGFR endocytosis.     

Atypical polyproline recognition by the CMS N-terminal Src homology 3 domain

The CIN85/CMS (human homologs of mouse SH3KBP1/CD2AP) family of endocytic adaptor proteins has the ability to engage multiple effectors and couple cargo trafficking with the cytoskeleton. CIN85 and CMS (Cas ligand with multiple Src homology 3 (SH3) domains) facilitate the formation of large multiprotein complexes required for an efficient internalization of cell surface receptors. It has recently been shown that c-Cbl/Cbl-b could mediate the formation of a ternary complex between one c-Cbl/Cbl-b molecule and two SH3 domains of CIN85, important for the ability of Cbl to promote epidermal growth factor receptor down-regulation. To further investigate whether multimerization is conserved within the family of adaptor proteins, we have solved the crystal structures of the CMS N-terminal SH3 domain-forming complexes with Cbl-b- and CD2-derived peptides. Together with biochemical evidence, the structures support the notion that, despite clear differences in the interaction surface, both Cbl-b and CD2 can mediate multimerization of N-terminal CMS SH3 domains. Detailed analyses on the interacting surfaces also provide the basis for a differential Cbl-b molecular recognition of CMS and CIN85.     

Induction of Gsk3β-β-TrCP Interaction Is Required for Late Phase Stabilization of β-Catenin in Canonical Wnt Signaling

A pivotal step in canonical Wnt signaling is Wnt-induced β-catenin stabilization. In the absence of Wnt, β-catenin is targeted for β-transducin repeats-containing proteins (β-TrCP)-mediated degradation due to phosphorylation by glycogen synthase kinase 3 (Gsk3). How canonical Wnt signaling regulates Gsk3 to inhibit β-catenin proteolysis remains largely elusive. This study reveals novel key molecular events in Wnt signaling: induction of Gsk3β ubiquitination and Gsk3β-β-TrCP binding. We found that Wnt stimulation induced prolonged monoubiquitination of Gsk3β and Gsk3β-β-TrCP interaction. Monoubiquitination did not cause Gsk3β degradation nor affects its enzymatic activity. Rather, increased monoubiquitination of Gsk3β/Gsk3β-β-TrCP association suppressed β-catenin recruitment of β-TrCP, leading to long-term inhibition of β-catenin ubiquitination and degradation.     

Inducible Priming Phosphorylation Promotes Ligand-independent Degradation of the IFNAR1 Chain of Type I Interferon Receptor

Phosphorylation-dependent ubiquitination and ensuing down-regulation and lysosomal degradation of the interferon α/β receptor chain 1 (IFNAR1) of the receptor for Type I interferons play important roles in limiting the cellular responses to these cytokines. These events could be stimulated either by the ligands (in a Janus kinase-dependent manner) or by unfolded protein response (UPR) inducers including viral infection (in a manner dependent on the activity of pancreatic endoplasmic reticulum kinase). Both ligand-dependent and -independent pathways converge on phosphorylation of Ser⁵³⁵ within the IFNAR1 degron leading to recruitment of β-Trcp E3 ubiquitin ligase and concomitant ubiquitination and degradation. Casein kinase 1α (CK1α) was shown to directly phosphorylate Ser⁵³⁵ within the ligand-independent pathway. Yet given the constitutive activity of CK1α, it remained unclear how this pathway is stimulated by UPR. Here we report that induction of UPR promotes the phosphorylation of a proximal residue, Ser⁵³², in a pancreatic endoplasmic reticulum kinase-dependent manner. This serine serves as a priming site that promotes subsequent phosphorylation of IFNAR1 within its degron by CK1α. These events play an important role in regulating ubiquitination and degradation of IFNAR1 as well as the extent of Type I interferon signaling.     

Development of a Cell-Based Assay Measuring the Activation of FcγRIIa for the Characterization of Therapeutic Monoclonal Antibodies

Antibody-dependent cellular cytotoxicity (ADCC) is one of the important mechanisms of action of the targeting of tumor cells by therapeutic monoclonal antibodies (mAbs). Among the human Fcγ receptors (FcγRs), FcγRIIIa is well known as the only receptor expressed in natural killer (NK) cells, and it plays a pivotal role in ADCC by IgG1-subclass mAbs. In addition, the contributions of FcγRIIa to mAb-mediated cytotoxicity have been reported. FcγRIIa is expressed in myeloid effector cells including neutrophils and macrophages, and it is involved in the activation of these effector cells. However, the measurement of the cytotoxicity via FcγRIIa-expressing effector cells is complicated and inconvenient for the characterization of therapeutic mAbs. Here we report the development of a cell-based assay using a human FcγRIIa-expressing reporter cell line. The FcγRIIa reporter cell assay was able to estimate the activation of FcγRIIa by antigen-bound mAbs by a very simple method in vitro. The usefulness of this assay for evaluating the activity of mAbs with different abilities to activate FcγRIIa was confirmed by the examples including the comparison of the activity of the anti-CD20 mAb rituximab and its Fc-engineered variants, and two anti-EGFR mAbs with different IgG subclasses, cetuximab (IgG1) and panitumumab (IgG2). We also applied this assay to the characterization of a force-oxidized mAb, and we observed that oxidation significantly decreased the FcγRIIa activation by EGFR-bound cetuximab. These results suggest that our FcγRIIa reporter assay is a promising tool for the characterization of therapeutic mAbs, including Fc-engineered mAbs, IgG2-subclass mAbs, and their product-related variants.     

EphrinA2 Regulates Clathrin Mediated KSHV Endocytosis in Fibroblast Cells by Coordinating Integrin-Associated Signaling and c-Cbl Directed Polyubiquitination

Kaposi''s sarcoma-associated herpesvirus (KSHV) interacts with human dermal endothelial cell surface tyrosine kinase EphrinA2 (EphA2) and integrins (α3β1 and αVβ3) in the lipid raft (LR) region, and EphA2 regulates macropinocytic virus entry by coordinating integrin-c-Cbl associated signaling. In contrast, KSHV enters human foreskin fibroblast (HFF) cells by LR-independent clathrin mediated endocytosis. The present studies conducted to identify the key molecules regulating KSHV entry in HFF cells showed that KSHV induces association with integrins (αVβ5, αVβ3 and α3β1) and EphA2 in non-LR regions early during infection and activates EphA2, which in turn associates with phosphorylated c-Cbl, myosin IIA, FAK, Src, and PI3-K, as well as clathrin and its adaptor AP2 and effector Epsin-15 proteins. EphA2 knockdown significantly reduced these signal inductions, virus internalization and gene expression. c-Cbl knockdown ablated the c-Cbl mediated K63 type polyubiquitination of EphA2 and clathrin association with EphA2 and KSHV. Mutations in EphA2''s tyrosine kinase domain (TKD) or sterile alpha motif (SAM) abolished its interaction with c-Cbl. Mutations in tyrosine kinase binding (TKB) or RING finger (RF) domains of c-Cbl resulted in very poor association of c-Cbl with EphA2 and decreased EphA2 polyubiquitination. These studies demonstrated the contributions of these domains in EphA2 and c-Cbl association, EphA2 polyubiquitination and virus-EphA2 internalization. Collectively, these results revealed for the first time that EphA2 influences the tyrosine phosphorylation of clathrin, the role of EphA2 in clathrin mediated endocytosis of a virus, and c-Cbl mediated EphA2 polyubiquitination directing KSHV entry in HFF cells via coordinated signal induction and progression of endocytic events, all of which suggest that targeting EphA2 and c-Cbl could block KSHV entry and infection.     

c-Cbl Facilitates Endocytosis and Lysosomal Degradation of Cystic Fibrosis Transmembrane Conductance Regulator in Human Airway Epithelial Cells

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated Cl⁻ channel expressed in the apical membrane of fluid-transporting epithelia. The apical membrane density of CFTR channels is determined, in part, by endocytosis and the postendocytic sorting of CFTR for lysosomal degradation or recycling to the plasma membrane. Although previous studies suggested that ubiquitination plays a role in the postendocytic sorting of CFTR, the specific ubiquitin ligases are unknown. c-Cbl is a multifunctional molecule with ubiquitin ligase activity and a protein adaptor function. c-Cbl co-immunoprecipitated with CFTR in primary differentiated human bronchial epithelial cells and in cultured human airway cells. Small interfering RNA-mediated silencing of c-Cbl increased CFTR expression in the plasma membrane by inhibiting CFTR endocytosis and increased CFTR-mediated Cl⁻ currents. Silencing c-Cbl did not change the expression of the ubiquitinated fraction of plasma membrane CFTR. Moreover, the c-Cbl mutant with impaired ubiquitin ligase activity (FLAG-70Z-Cbl) did not affect the plasma membrane expression or the endocytosis of CFTR. In contrast, the c-Cbl mutant with the truncated C-terminal region (FLAG-Cbl-480), responsible for protein adaptor function, had a dominant interfering effect on the endocytosis and plasma membrane expression of CFTR. Moreover, CFTR and c-Cbl co-localized and co-immunoprecipitated in early endosomes, and silencing c-Cbl reduced the amount of ubiquitinated CFTR in early endosomes. In summary, our data demonstrate that in human airway epithelial cells, c-Cbl regulates CFTR by two mechanisms: first by acting as an adaptor protein and facilitating CFTR endocytosis by a ubiquitin-independent mechanism, and second by ubiquitinating CFTR in early endosomes and thereby facilitating the lysosomal degradation of CFTR.     

Intersectin 1 forms a complex with adaptor protein Ruk/CIN85 in vivo independently of epidermal growth factor stimulation

Intersectin 1 (ITSN1) is an adaptor protein involved in clathrin-mediated endocytosis, apoptosis, signal transduction and cytoskeleton organization. Here, we show that ITSN1 forms a complex with adaptor protein Ruk/CIN85, implicated in downregulation of receptor tyrosine kinases. The interaction is mediated by the SH3A domain of ITSN1 and the third or fourth proline-rich blocks of Ruk/CIN85, and does not depend on epidermal growth factor stimulation, suggesting a constitutive association of ITSN1 with Ruk/CIN85. Moreover, both proteins colocalize in MCF-7 cells with their common binding partner, the ubiquitin ligase c-Cbl. The possible biological role of the interaction between ITSN1 and Ruk/CIN85 is discussed.     

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.