Significant data had linked activation of Src-family kinases to caveolin phosphor-ylation. Therefore, it was initially hypothesized that a Src-family kinase was the insulin-stimulated caveolin-tyrosine kinase in adipocytes. Consistent with this, treatment of isolated caveolar fractions with Src-family kinase inhibitors blocked caveolin phosphorylation in vitro, indicating that the caveolin kinase activity that co-purifies with caveolin is a Src-family kinase (CCM, unpublished observation; ). (These inhibitors did not block Src-family kinase activation in intact adipocytes as measured by kinase autophosphorylation. In contrast, inhibition of autophosphorylation was readily detectable in fibroblasts. This is a common problem in adipocytes. Compounds that are sufficiently lipophilic to cross membranes are often sequestered within the prominent fat droplets in these cells.) The most abundant Src-family kinase that co-purifies with caveolin-1 in adipocytes is Fyn [14,15,69]. Fyn is activated in response to insulin via Src-homology 2 (SH2) domain-mediated binding to IRS-1 . To determine if Fyn was involved in insulin-stimulated caveolin tyrosine phosphorylation, Fyn was overexpressed in adipocytes . Overexpression of Fyn was sufficient to induce caveolin tyrosine phosphorylation under basal conditions, and hyperphosphorylation of caveolin-1 in response to insulin. These results verify that Fyn is activated in response to insulin, and that it is part of the signaling cascade leading to caveolin tyrosine phosphorylation in response to insulin. However, differentiation does not change the level of expression of Fyn, and Fyn co-localizes with caveolin-1 in both pre-adipocytes and adipocytes. Furthermore, overexpression of Fyn in fibroblasts did not increase basal caveolin phosphorylation or reconstitute insulin-stimulated phosphorylation. These data indicate that, while Fyn is a part of the insulin-stimulated caveolin phosphorylation pathway in adipocytes, differentiation induces the expression of an additional protein or proteins required for caveolin tyrosine phosphorylation.
An additional line of evidence suggested that a second non-receptor tyrosine kinase may be involved in caveolin phosphorylation. The phosphorylation site on caveolin-1 (Tyr14, L-Y-T-V-P) is not in the context of a consensus Fyn phosphorylation site (I/L-Y-D/E-X-L). Tyr14 lies within the motif I/L-Y-X-X-P, a consensus Abl phosphorylation site [71,72]. Tyr27 in caveolin-2 falls within a similar sequence (E-Y-A-D-P). It has not been determined whether insulin activates Abl in adipocytes, although several known Abl substrates are phosphorylated in response to insulin, including Crk, Cbl, and Dok [73,74]. In addition, the Crk binding partner CAS is dephosphorylated in response to insulin, consistent with Abl activation . Importantly, differentiation of 3T3-L1 fibroblasts into adipocytes leads to a large increase in Abl expression (CCM, unpublished observation), which may account for the cell type dependence of insulin-stimulated caveolin phosphorylation.
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