APS facilitates c-Cbl tyrosine phosphorylation and GLUT4 translocation in response to insulin in 3T3-L1 adipocytes.

c-Cbl is the cellular homolog of the transforming v-Cbl oncogene (4, 17). Cbl contains numerous tyrosine residues, which could serve as docking sites for multiple SH2-containing signaling molecules upon phosphorylation. Cbl consists of an N-terminal variant SH2 domain, a Ring finger domain, multiple proline-rich stretches, several potential tyrosine phosphorylation sites, and a conserved ubiquitin-associated domain. Cbl is tyrosine phosphorylated in response to epidermal growth factor (EGF), platelet-derived growth factor (PDGF), various antigens, integrins, and cytokines (21, 41). Src family kinases may catalyze c-Cbl tyrosine phosphorylation in the antigen and integrin signaling pathways (26, 30, 40), and the EGF and PDGF receptors appear to phosphorylate c-Cbl directly upon ligand binding (5, 9). Tyrosine-phosphorylated Cbl can bind the SH2 domains of the non-receptor tyrosine kinases Fyn and Syk, the p85 subunit of phosphatidylinositol (PI) 3-kinase, and the adapter protein Crk (2, 7, 8, 22, 36). c-Cbl contains an E2-dependent ubiquitin ligase activity and can function as a negative regulator of tyrosine kinase signaling (13, 14, 18, 24, 43). However, there is increasing evidence that c-Cbl can also serve a positive role. For example, the protein enhances proliferation and survival through PI 3-kinase-dependent pathways after cytokine simulation (11, 42) and enhances mitogen-activated protein (MAP) kinase activation in response to stimulation of the Met receptor (10). We previously showed that insulin stimulates the tyrosine phosphorylation of Cbl in 3T3-L1 adipocytes, inducing its association with Crk (36). The phosphorylation of Cbl in response to insulin correlates with expression of the Cbl-associated protein (CAP). CAP contains three SH3 domains in its C terminus and a region of homology to the gut peptide sorbin (SoHo domain) in its N terminus. CAP interacts with Cbl via its C-terminal SH3 domain (15, 35). Upon Cbl phosphorylation, the CAP/Cbl complex migrates to caveolin-enriched lipid rafts, due to the interaction of the SoHo domain on CAP with the lipid raft-associated protein flotillin (3, 15). This leads to the recruitment of the Crk/C3G complex to this microdomain of the plasma membrane, where C3G, a guanyl nucleotide exchange factor, activates the small G protein TC10 (6). Both translocation of the CAP/Cbl complex and activation of TC10 have been shown to occur independently of the PI 3-kinase pathway and, more importantly, to be crucial to insulin-stimulated GLUT4 translocation (3, 6). In 3T3-L1 adipocytes, overexpression of a CAP mutant in which either the SH3 domains or the SoHo domain have been deleted inhibited insulin-stimulated recruitment of Cbl to lipid rafts and subsequently blocked the translocation of GLUT4 in response to insulin (3, 15). However, it remained unclear whether CAP directly targeted Cbl to insulin receptor for phosphorylation or if a separate protein existed to serve this function. One such candidate is an adapter protein containing PH and SH2 domains, called APS. APS is a member of the Lnk family of adapter proteins that is highly expressed in insulin-responsive tissues such as fat, skeletal muscle, and heart (28). Recently APS was identified as a substrate for the PDGF and insulin receptors, both of which catalyze the tyrosine phosphorylation of APS at a C-terminal phosphorylation site (28, 45). Ectopic expression of APS in Chinese hamster ovary (CHO) cells overexpressing insulin receptor led to concomitant ubiquitination of the insulin receptor, possibly through recruitment of Cbl (1). However, the function of APS in cells metabolically responsive to insulin has not been evaluated. We report here that APS is necessary for the insulin-stimulated tyrosine phosphorylation of Cbl and its subsequent association with Crk in differentiated adipocytes. We also demonstrate that APS specifically interacts and colocalizes with CAP in an insulin-independent manner. Moreover, APS-facilitated Cbl phosphorylation is crucial for the translocation of GLUT4 in response to insulin. Thus, APS is an essential component of the CAP/Cbl pathway in the control of insulin-stimulated glucose transport.