Activation of S6 kinase activity in 3T3-L1 cells by insulin and phorbol ester.

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Abstract
Treatment of 3T3-L1 cells with 0.1-1.0 nM insulin results in rapid (5-15 min) activation of a soluble protein kinase that phosphorylases Ser residues in ribosomal protein S6. The insulin-stimulated kinase activity is detectable in confluent, nongrowing preadipocytes and adipocytes. In the presence of 2 .mu.g of cycloheximide per ml, preconfluent 3T3-L1 cells also respond to insulin by acquiring an S6 kinase activity whose properties are the same as those of the enzyme activity elicited by insulin alone in growth-inhibited cells. The principal insulin-stimulated S6 kinase has a MW of .simeq. 50,000-60,000; there is a variable amount of activity that sediments with a MW of .apprx. 80,000. The soluble enzyme exhibits optimal activity between pH 8 and pH 9, requires Mg2+ (10-20 mM), and is inhibited by Ca2+ (0.5 mM), Mn2+ (0.5 mM) and NaF (30 mM). GTP cannot substitute for ATP in the phosphotransferase reaction; cAMP, cGMP, phosphatidylserine plus diolein, the cAMP-dependent protein kinase inhibitor, and heparin (0.7 .mu.g/ml) are without effect. Although treatment of 3T3-L1 cells with insulin does not influence the activity or the subcellular distribution of the phospholipid and Ca2+-dependent protein kinase C, exposure to the phorbol tumor promoter phorbol 12-myristate-13-acetate (PMA) results in translocation of protein kinase C to the membrane and activation of a soluble phospholipid and Ca2+-independent S6 protein kinase that has the same magnitude of activity and sedimentation behavior as the insulin-induced activity. Trypsin treatment of either 3T3-L1 cytosolic extracts or partially purified 3T3-L1 protein kinase C generates a small amount of S6 kinase activity of MW 50,000. This activity, resolved by sucrose gradient centrifugation, is less active than that elicited by either insulin or PMA and, unlike the activities generated by insulin and PMA, is associated with histone kinase activity. Evidently, the S6 kinase elicited by either insulin or PMA is neither protein kinase C, its phospholipid and Ca2+-independent proteolytic derivative nor the rsult of proteolytic activation of an inactive proenzyme that can be reproduced by trypsin treatment of cell extracts in vitro.