Ribon V, Printen J A, Hoffman N G, Kay B K, Saltiel A R
Department of Physiology, University of Michigan School of Medicine, Ann Arbor 48109, USA.
Mol Cell Biol. 1998 Feb;18(2):872-9. doi: 10.1128/MCB.18.2.872.
The protein product of the c-Cbl proto-oncogene is prominently tyrosine phosphorylated in response to insulin in 3T3-L1 adipocytes and not in 3T3-L1 fibroblasts. After insulin-dependent tyrosine phosphorylation, c-Cbl specifically associates with endogenous c-Crk and Fyn. These results suggest a role for tyrosine-phosphorylated c-Cbl in 3T3-L1 adipocyte activation by insulin. A yeast two-hybrid cDNA library prepared from fully differentiated 3T3-L1 adipocytes was screened with full-length c-Cbl as the target protein in an attempt to identify adipose-specific signaling proteins that interact with c-Cbl and potentially are involved in its tyrosine phosphorylation in 3T3-L1 adipocytes. Here we describe the isolation and the characterization of a novel protein that we termed CAP for c-Cbl-associated protein. CAP contains a unique structure with three adjacent Src homology 3 (SH3) domains in the C terminus and a region showing significant sequence similarity with the peptide hormone sorbin. Both CAP mRNA and proteins are expressed predominately in 3T3-L1 adipocytes and not in 3T3-L1 fibroblasts. CAP associates with c-Cbl in 3T3-L1 adipocytes independently of insulin stimulation in vivo and in vitro in an SH3-domain-mediated manner. Furthermore, we detected the association of CAP with the insulin receptor. Insulin stimulation resulted in the dissociation of CAP from the insulin receptor. Taken together, these data suggest that CAP represents a novel c-Cbl binding protein in 3T3-L1 adipocytes likely to participate in insulin signaling.
c-Cbl原癌基因的蛋白质产物在3T3-L1脂肪细胞中对胰岛素产生显著的酪氨酸磷酸化反应，而在3T3-L1成纤维细胞中则不会。在胰岛素依赖性酪氨酸磷酸化后，c-Cbl特异性地与内源性c-Crk和Fyn结合。这些结果表明酪氨酸磷酸化的c-Cbl在胰岛素激活3T3-L1脂肪细胞中发挥作用。以全长c-Cbl作为靶蛋白，对从完全分化的3T3-L1脂肪细胞制备的酵母双杂交cDNA文库进行筛选，试图鉴定与c-Cbl相互作用并可能参与其在3T3-L1脂肪细胞中酪氨酸磷酸化的脂肪特异性信号蛋白。在此，我们描述了一种新型蛋白质的分离和特性，我们将其命名为c-Cbl相关蛋白（CAP）。CAP在C末端含有一个独特的结构，具有三个相邻的Src同源3（SH3）结构域，以及一个与肽激素索宾具有显著序列相似性的区域。CAP的mRNA和蛋白质主要在3T3-L1脂肪细胞中表达，而不在3T3-L1成纤维细胞中表达。在体内和体外，CAP在3T3-L1脂肪细胞中以SH3结构域介导的方式与c-Cbl结合，且不依赖胰岛素刺激。此外，我们检测到CAP与胰岛素受体的结合。胰岛素刺激导致CAP从胰岛素受体上解离。综上所述，这些数据表明CAP是3T3-L1脂肪细胞中一种新型的c-Cbl结合蛋白，可能参与胰岛素信号传导。
