Insulin-stimulated protein phosphorylation in 3T3-L1 preadipocytes

A protein of molecular weight 31,000 became labeled with ³²P within 5 min after addition of insulin to differentiated 3T3-L1 preadipocytes previously incubated for 55 min, with ³²P(i). The effect was mimicked by antisera directed against the insulin receptor and was eliminated by anti-insulin antiserum. Incorporation of ³²P into this protein was more than 20-fold greater in insulin-treated cells than in cells not exposed to the hormone. At concentrations greater than required with insulin, epidermal growth factor and serum (1-5%) also stimulated phosphorylation whereas l-isoproterenol, a β-adrenergic agonist that increases intracellular accumulation of cyclic AMP, was without effect. The 31,000-dalton protein has been tentatively identified as ribosomal protein S6 by two-dimensional polyacrylamide gel electrophoresis. Incorporation of ³²P into S6 could be detected within the same time period (5 min) and at the same insulin concentrations (0.1-1.0 nM) as are required to stimulate hexose uptake in both 3T3-L1 cells and mature mammalian adipocytes. The mechanism by which this phosphorylation either mediates or reflects the intracellular actions of insulin remains to be elucidated.