Insulin stimulates tyrosine phosphorylation of the proto-oncogene product of c-Cbl in 3T3-L1 adipocytes

Abstract

We report here that the product of the c-Cbl proto-oncogene is prominently tyrosine phosphorylated in response to insulin in 3T3-L1 adipocytes. The tyrosine phosphorylation of c-Cbl reaches a maximum within 1-2 min after stimulation by insulin and gradually declines thereafter. The tyrosine phosphorylation of c-Cbl was also observed after treatment of 3T3-L1 adipocytes with epidermal growth factor, whereas platelet-derived growth factor had no effect. After insulin-dependent tyrosine phosphorylation, c-Cbl specifically associates with fusion proteins containing the Src homology 2 (SH2) domains of Crk and the Fyn tyrosine kinase, but not with fusion proteins containing the SH2 domains of either the p85 subunit of phosphatidylinositol 3'-kinase or the tyrosine phosphatase SHPTP2/Syp. Furthermore insulin stimulates the association of c-Cbl with endogenous c-Crk and Fyn in intact 3T3-L1 adipocytes. The tyrosine phosphorylation of c-Cbl is regulated during adipocyte differentiation. Neither insulin-like growth factor 1 nor insulin stimulated the tyrosine phosphorylation of c-Cbl in 3T3-L1 fibroblasts. Moreover, c-Cbl is not tyrosine phosphorylated in response to insulin in cells expressing high levels of the human insulin receptor, or in hepatocytes, despite comparable levels of c-Cbl expression. These results suggest that c-Cbl might have a novel function in the regulation of insulin receptor intracellular signalling in 3T3-L1 adipocytes.

Insulin-stimulated Tyrosine Phosphorylation of Caveolin Is Specific for the Differentiated Adipocyte Phenotype in 3T3-L1 Cells

“…In contrast, the proto-oncogene product c-Cbl shares many properties with this presumed substrate protein. Insulin stimulates the phosphorylation of c-Cbl in adipocytes, and this phosphorylation shows specificity for insulin in this cell type (53). As has been observed in lymphocytes, Cbl constitutively binds to Fyn in unstimulated adipocytes through an SH3 domain-mediated interaction, and insulin-stimulated tyrosine phosphorylation of Cbl increases this association.…”

“…Cbl is expressed at comparable levels in both the preadipocytes and adipocytes. Unlike IRS-1/-2 and Shc, however, Cbl does not directly interact with the insulin receptor (53). We hypothesize that tyrosine phosphorylation of Cbl may require a specific adapter protein (56), which allows for the interaction of Cbl with the insulin receptor (53), and that it is the regulation of the expression of this protein that accounts for the coordinate regulation of the phosphorylations of Cbl and caveolin in response to insulin in adipocytes.…”

“…8). Most interestingly, unlike the other known substrates of the insulin receptor such as IRS-1/-2 and Shc, Cbl phosphorylation is specific for the differentiated adipocyte phenotype (53).…”

“…Previous work suggested that the insulinstimulated tyrosine phosphorylation of Cbl may be involved in the activation of Fyn in adipocytes (53). Cbl is rapidly and specifically phosphorylated on tyrosine in response to insulin in adipocytes.…”

Ins (endocytosis) and outs (exocytosis) of GLUT4 trafficking

“…In addition to IRS proteins serving as important targets to scaffold various signaling effectors, the insulin receptor is also well established to induce the tyrosine phosphorylation of the Cbl proteins [81]. Cbl proteins containing several protein modulatory regions and most notably a ring finger domain that functions as an E3 ligase in the ubiquitation and rapid down-regulation of various receptors including the EGF, PDGF, T and B cell receptors [82][83][84][85].…”