Insulin‐induced S6 kinase activation in HeLa cells and its reversal by hyperthermic stress

Abstract

Insulin treatment of HeLa S3 cells activates an S6-phosphorylating protein kinase. Although this enzyme has chromatographic properties resembling those of described proteolytic fragments of other protein kinases, namely protein kinase C, protease-activated kinase 11 and histone-4 protein kinase, and although insulin has been proposed by others to cause S6 phosphorylation via proteolytic protein kinase activation, the insulin-induced increase in S6-kinase activity described here is probably not due to proteolysis. Rather, the activity indicates the existence, in HeLa cells, of an interconvertible S6 kinase, since the insulin-induced activity increase was rapidly reversed under hyperthermic stress, and since this effect of hyperthermia was itself reversible. The S6-kinase activities from serum-and from insulin-stimulated HeLa cells resemble each other closely and are likely to represent the same enzyme. The enzyme may therefore mediate both signals delivered by mitogens and the insulin signal. Analysed at an in vitvo transfer of 1 mol phosphate/mol S6, this S6 kinase activity does not phosphoryhte the (principal) S6 site recognized by the CAMP-dependent protein kinase.A characteristic facet of the very early phase of the response of cells to serum or growth factors, including insulin, is the multiple phosphorylation of the ribosomal protein S6 (for serum: see references in [I, 21; for growth factors [3-71; for insulin [8 -131). We have described an S6-phosphorylating protein kinase which is activated in serum-treated cells and therefore a candidate mediator of serum-induced S6 phosphorylation [l, 141. As noted previously [14], the serum effect could be mimicked with a high dose (5 x M) of insulin. We have since observed S6 kinase activation at lower insulin doses. This suggests that insulin-responsive S6 kinase from 3T3-Ll cells [15, 161 and other systems [17-211 may be homologues of the human mitogen-responsive S6 kinase, and furthermore, that both insulin and a non-insulin signal(s) contained in serum (the insulin content of sera was low) may activate the same or a very closely related S6-phosphorylating enzyme(s).As shown in [l], chromatographic criteria allowed us to distinguish the S6 kinase from other S6-phosphorylating enzymes implicated in the transfer of anabolic signals [22 -261.