WORTMANNIN INHIBITS THE EFFECTS OF INSULIN AND SERUM ON THE ACTIVITIES OF GLYCOGEN-SYNTHASE KINASE-3 AND MITOGEN-ACTIVATED PROTEIN-KINASE

We have previously shown that insulin causes inactivation of glycogen synthase kinase-3 (GSK-3) in Chinese hamster ovary cells over-expressi ng the human insulin receptor (CHO.T cells). We now show that serum an d phorbol ester also cause rapid inactivation of GSK-3, both in CHO.T cells and in the nontransfected parental cell line, CHO.K1 cells. Rapa mycin was without effect on the inactivation of GSK-3 by insulin, seru m or phorbol ester, indicating that the p70 S6 kinase pathway is not i nvolved. In contrast, wortmannin, a potent inhibitor of phosphatidylin ositol 3-kinase, blocked the effects of both insulin and serum on GSK- 3 activity, and also substantially reduced the activation of both p90 S6 kinase (by insulin) and mitogen-activated protein (MAP) kinase (by insulin and serum). These findings imply (i) that GSK-3 activity is re gulated by a cascade involving MAP kinase and p90 S6 kinase and (ii) t hat wortmannin affects an early step in the MAP kinase pathway. One ca n infer from this that GSK-3 may be an important regulatory enzyme for the control of several biosynthetic pathways, key enzymes in which ar e regulated by GSK-3-mediated phosphorylation. Wortmannin had a smalle r effect on the activation of MAP kinase by phorbol ester, indicating that phorbol esters may stimulate MAP kinase by a different or additio nal mechanism to that employed by insulin or serum. Wortmannin had ver y little effect on the inactivation of GSK-3 by phorbol ester: possibl e reasons for this are discussed.