Protein tyrosine phosphorylation is an early event in insulin-initiated intracellular signaling cascades (34
). Recently, we have demonstrated that insulin stimulates the tyrosine phosphorylation of the c-Cbl proto-oncogene product, and this phosphorylation shows specificity for the differentiated adipocyte phenotype. Following insulin stimulation, the tyrosine-phosphorylated c-Cbl specifically associates with the adapter c-Crk and the Fyn tyrosine kinase in vivo and in vitro (30
). Because we could not detect c-Cbl tyrosine phosphorylation in response to insulin in a variety of other cell types expressing functional insulin receptor (30
), we suspected that 3T3-L1 adipocytes might express a specific adapter protein that may be involved in the tyrosine phosphorylation of c-Cbl.
To search for such novel proteins we used the full-length c-Cbl as a target protein to screen a 3T3-L1 adipocyte cDNA library in the yeast two-hybrid system. Multiple, independent c-DNA inserts, which encoded different fragments of the same protein were cloned from this library and designated CAP for c-Cbl-associated protein. These interactions were then verified in several experiments by using GST fusion proteins and coimmunoprecipitation. Analysis of the predicted amino acid sequences of these clones revealed three adjacent SH3 domains at the carboxyl terminus and a novel putative sorbin homology domain at the N terminus. The structure of CAP suggests that it may participate in multiple signaling cascades. cDNA sequencing and Northern blot analysis indicated that there are multiple splice variants of CAP that may result in a family of different isoforms. Interestingly, both CAP mRNA and proteins are expressed predominately in 3T3-L1 adipocytes and not in 3T3-L1 fibroblasts, implying a restricted signaling role for CAP in these cells.
The three SH3 domains of CAP were cloned as a fragment from the yeast two-hybrid library, suggesting that the association of CAP with c-Cbl was SH3 mediated. This was further suggested by an examination of the proline-rich regions of c-Cbl, revealing multiple potential SH3 binding motifs (6
). In vitro binding assays with GST fusion proteins containing the individual SH3 domains of CAP confirmed that the functional association of CAP with c-Cbl is mediated primarily by the carboxyl-terminal SH3 domain of CAP. Interestingly, in 3T3-L1 adipocytes, which normally express both CAP and c-Cbl, CAP and c-Cbl associate in a constitutive manner independent of insulin stimulation. The specific tyrosine phosphorylation of c-Cbl and CAP expression in the differentiated adipocytes suggest that the CAP-c-Cbl complex may have a specialized signaling function in insulin action in these cells. The observation that one major CAP isoform associated with the insulin receptor suggests that at least one role of CAP might be to facilitate the interaction of c-Cbl with the insulin receptor, allowing for the phosphorylation of c-Cbl and its association with c-Crk and Fyn. Indeed, c-Cbl tyrosine phosphorylation is observed only in 3T3-L1 adipocytes, which uniquely express CAP. Moreover, receptor activation leads to rapid dissociation of the insulin receptor-CAP complex. The kinetics of the insulin receptor-CAP dissociation parallels the time course of insulin-stimulated tyrosine phosphorylation of c-Cbl in these cells (30
). The structural requirements for this interaction remain to be determined.
gene was cloned as the cellular homolog of the v-cbl
oncogene, which is transforming in early B-lineage and myeloid cells (5
). However, overexpression of c-Cbl does not induce cellular transformation, and the function of the proto-oncogene product remains unclear. The potential signaling function of c-Cbl downstream of tyrosine kinases depends on the association of its proline-rich region with SH3 domain-containing signaling proteins such as Grb2 and Nck (8
) and the tyrosine phosphorylation sites with SH2 domain-containing proteins (8
). Since Grb2 and Nck are ubiquitous in their expression, CAP with its specific expression in 3T3-L1 adipocytes may represent a specialized component in the signal transduction role of c-Cbl in these cells. Thus, different pools of c-Cbl may associate with different proteins forming various signaling complexes, each with distinct cellular function.
The ability of insulin to stimulate glucose uptake and storage of glucose as glycogen and lipids is significantly increased after 3T3-L1 differentiation. The expression of many fat cell-specific genes critical to insulin action is increased during adipocyte differentiation, such as GLUT4 (10
). Thus, changes in the level of CAP expression and c-Cbl tyrosine phosphorylation might contribute to increased insulin responsiveness observed in differentiated 3T3-L1 adipocytes. CAP was also found to associate with the nucleotide exchange protein Sos. Insulin treatment of 3T3-L1 adipocytes induces a dissociation of the CAP-SH3 domain from Sos similar to the dissociation of the Grb2/Sos complex (44
). CAP, CAP/c-Cbl, and CAP/Sos may represent specific endogenous regulators in insulin-activated signal transduction pathways in 3T3-L1 adipocytes.