Okadaic acid mimics the action of insulin in stimulating protein kinase activity in isolated adipocytes. The role of protein phosphatase 2a in attenuation of the signal.
Treatment of adipocytes with okadaic acid (a specific inhibitor of type 1 and 2a protein phosphatases) resulted in a rapid 8-10-fold stimulation of cell extract myelin basic protein (MBP) kinase activity (t1/2 = 10 min) and kinase activity toward a synthetic peptide RRLSSLRA (S6 peptide) (t1/2 = 5 min). Insulin brought about a smaller stimulation of these two activities (t1/2 = 2.5 min). MBP kinase activity from cells treated with okadaic acid or insulin was resolved by anion exchange chromatography into two well defined peaks; S6 peptide kinase activity was less well resolved. The two partially purified MBP kinases were inactivated by the protein tyrosine phosphatase CD45 or by protein phosphatase 2a (PP-2a). In contrast, partially purified S6 peptide kinase activity was inactivated only by PP-2a or protein phosphatase 1 (PP-1). Furthermore, a 38-kDa protein which co-eluted with one peak of MBP kinase and a 42-kDa protein which co-eluted with the other peak of MBP kinase were phosphorylated on tyrosine after treatment with okadaic acid. These findings illustrate several important points concerning regulation of MBP and S6 peptide kinases. First, these protein kinases are regulated by phosphorylation, and, second, in the absence of hormonal stimuli their activities are strongly suppressed by protein phosphatases. Lastly, the increased tyrosine phosphorylation accompanying the activation of MBP kinases following okadaic acid treatment suggests a role for PP-2a in events that are mediated by tyrosine phosphorylation.
Haystead
T A
TA
Department of Pharmacology, University of Washington, Seattle 98195.
Weiel
J E
JE
Litchfield
D W
DW
Tsukitani
Y
Y
Fischer
E H
EH
Krebs
E G
EG
D000273:Adipose Tissue / Q000201:enzymology*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002478:Cells, Cultured; D004988:Ethers, Cyclic / Q000494:pharmacology*; D038362:Glycogen Synthase Kinase 3; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D007476:Ionophores / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010750:Phosphoproteins / Q000302:isolation & purification; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2; D051381:Rats; D011919:Rats, Inbred Strains; D011994:Recombinant Proteins / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects*; D013379:Substrate Specificity
D000596:Amino Acids; D004988:Ethers, Cyclic; D007328:Insulin; D007476:Ionophores; D010750:Phosphoproteins; D011994:Recombinant Proteins; D019319:Okadaic Acid; D011494:Protein Kinases; D038362:Glycogen Synthase Kinase 3; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2