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-expressing the human insulin receptor (CHO.T cells). We now show that serum and phorbol ester also cause rapid inactivation of GSK-3, both in CHO.T cells and in the nontransfected parental cell line, CHO.K1 cells. Rapamycin was without effect on the inactivation of GSK-3 by insulin, serum or phorbol ester, indicating that the p70 S6 kinase pathway is not involved. In contrast, wortmannin, a potent inhibitor of phosphatidylinositol 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 regulated by a cascade involving MAP kinase and p90 S6 kinase and (ii) that wortmannin affects an early step in the MAP kinase pathway. One can infer from this that GSK-3 may be an important regulatory enzyme for the control of several biosynthetic pathways, key enzymes in which are regulated by GSK-3-mediated phosphorylation. Wortmannin had a smaller effect on the activation of MAP kinase by phorbol ester, indicating that phorbol esters may stimulate MAP kinase by a different or additional mechanism to that employed by insulin or serum. Wortmannin had very little effect on the inactivation of GSK-3 by phorbol ester: possible reasons for this are discussed.
Welsh
G I
GI
Department of Biochemistry, School of Medical Sciences, University of Bristol, U.K.
Foulstone
E J
EJ
Young
S W
SW
Tavaré
J M
JM
Proud
C G
CG
D000595:Amino Acid Sequence; D000730:Androstadienes / Q000494:pharmacology*; D000818:Animals; D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D001769:Blood; D016466:CHO Cells; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D006224:Cricetinae; D003470:Culture Media; D004789:Enzyme Activation; D038362:Glycogen Synthase Kinase 3; D038341:Glycogen Synthase Kinases; D020662:Guanine Nucleotide Exchange Factors; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D007329:Insulin Antagonists / Q000494:pharmacology*; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D011972:Receptor, Insulin / Q000096:biosynthesis / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D013379:Substrate Specificity; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection; D000077191:Wortmannin
D000730:Androstadienes; D000893:Anti-Inflammatory Agents; D003470:Culture Media; D020662:Guanine Nucleotide Exchange Factors; D007328:Insulin; D007329:Insulin Antagonists; D010455:Peptides; D011506:Proteins; D011994:Recombinant Proteins; D011494:Protein Kinases; D011972:Receptor, Insulin; D038341:Glycogen Synthase Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D038362:Glycogen Synthase Kinase 3; D013755:Tetradecanoylphorbol Acetate; D000077191:Wortmannin