Insulin stimulates the tyrosine phosphorylation of caveolin.

Abstract

Abstract. The specialized plasma membrane structures termed caveolae and the caveolar-coat protein caveolin are highly expressed in insulin-sensitive cells such as adipocytes and muscle. Stimulation of 3T3-L1 adipocytes with insulin significantly increased the tyrosine phosphorylation of caveolin and a 29-kD caveolin-associated protein in caveolin-enriched Triton-insoluble complexes. Maximal phosphorylation occurred within 5 min, and the levels of phosphorylation remained elevated for at least 30 min. The insulin-dose responses for the tyrosine phosphorylation of caveolin and the 29-kD caveolin-associated protein paralleled those for the phosphorylation of the insulin receptor. The stimulation of caveolin tyrosine phosphorylation was specific for insulin and was not observed with PDGF or EGF, although PDGF stimulated the tyrosine phosphorylation of the 29-kD caveolin-associated protein. Increased tyrosine phosphorylation of caveolin, its associated 29-kD protein, and a 60-kD protein was observed in an in vitro kinase assay after incubation of the caveolin-enriched Triton-insoluble complexes with Mg-ATP, suggesting the presence of an intrinsic tyrosine kinase in these complexes. These fractions contain only trace amounts of the activated insulin receptor. In addition, these complexes contain a 60-kD kinase detected in an in situ gel kinase assay and an ~60 kD protein that cross-reacts with an antibody against the Src-family kinase p59 Fy". Thus, the insulin-dependent tyrosine phosphorylation of caveolin represents a novel, insulinspecific signal transduction pathway that may involve activation of a tyrosine kinase downstream of the insulin receptor.

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

“…The cells were lysed in MES/NaCl/ Triton X-100; caveolin was immunoprecipitated, and tyrosine phosphorylation was determined by anti-phosphotyrosine Western blotting. As reported previously (36), in response to insulin there was a significant increase in the tyrosine phosphorylation of two forms of caveolin (22 and 24 kDa) and a 29-kDa caveolin-associated protein in adipocytes (Fig. 1A).…”

“…Monoclonal anti-insulin receptor antiserum was purchased from Oncogene Sciences (Cambridge, MA), and polyclonal anti-IRS-1 antisera was prepared as described (37). Polyclonal anti-Fyn antiserum (␣-Fyn-268 -389) was prepared as described (36) or was purchased from Santa Cruz Biotechnology (Santa Cruz, CA) (␣-Fyn-29 -48); monoclonal anti-Fyn antiserum was purchased from Transduction Laboratories. Polyclonal anti-Src antiserum was purchased from Santa Cruz; this antibody cross-reacts with Fyn and Yes.…”

“…Preparation of Triton-insoluble Complexes-Caveolin-enriched Triton-insoluble complexes were prepared essentially as described (36). Adipocytes were incubated overnight in DME supplemented with 0.5% calf serum.…”

“…In Vitro Kinase Assays-Assays were performed essentially as described (36). Briefly, Triton-insoluble pellets or low density Tritoninsoluble complexes were resuspended in 5 l of MES/NaCl/Triton X-100 and 20 l of kinase assay buffer (22.5 mM Hepes, pH 7.5, 12.5 mM MgCl 2 , 1.25 mM MnCl 2 , 1.25 mM EGTA).…”

“…Caveolins-1 and -2 are highly expressed in adipocytes (33)(34)(35), and caveolin-1 expression increases upon adipocyte differentiation (34). Recently, we reported that caveolin is tyrosinephosphorylated in response to insulin in 3T3-L1 adipocytes (36). Consistent with an important role for caveolae in insulin action, phosphorylation of caveolin is specific for insulin and does not occur in response to other growth factors such as PDGF or EGF, suggesting that caveolae may be involved in an insulin-specific signaling pathway.…”