Inhibition of interleukin-2-mediated DNA synthesis in activated human T-lymphoblasts by okadaic acid is accompanied by hyperphosphorylation of lck.
We have previously shown that, during interleukin-2-driven G1/S transition in activated human T-lymphoblasts, a restricted group of cellular proteins become tyrosine phosphorylated de novo, and that p56lck is the major active tyrosine kinase at this stage of the cell cycle. We now report that okadaic acid, a potent and specific inhibitor of protein phosphatases type 1 and type 2A, inhibits S-phase entry, and that this occurs with the simultaneous disappearance of a 56 kDa tyrosine phosphoprotein. We show that this protein is the lck tyrosine kinase and that okadaic acid stimulates a mobility shift to 59 and 64 kDa forms. These two forms of lck were found to have decreased autocatalytic activity, as judged by immune-complex kinase assays. Two-dimensional phosphopeptide mapping and phosphoamino-acid analyses revealed that, in the presence of okadaic acid, lck becomes phosphorylated mainly on serine and to a lesser extent threonine, and that phosphorylation occurs at novel sites. These results show that the kinase activity of lck is at least partly regulated by protein phosphatases, and suggest a role for lck in directing growth-factor-mediated DNA synthesis during T-cell proliferation.
Churcher
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Y
Department of Physiology, University College London, UK.
Moss
S E
SE
D015153:Blotting, Western; D002453:Cell Cycle / Q000187:drug effects; D002455:Cell Division / Q000235:genetics; D004247:DNA / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D015180:Electrophoresis, Gel, Two-Dimensional; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007376:Interleukin-2 / Q000494:pharmacology*; D007527:Isoenzymes / Q000378:metabolism; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D008970:Molecular Weight; D019319:Okadaic Acid / Q000494:pharmacology*; D010748:Phosphopeptides / Q000032:analysis; D010749:Phosphoprotein Phosphatases / Q000378:metabolism / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D010768:Phosphoserine / Q000378:metabolism; D010769:Phosphothreonine / Q000378:metabolism; D013601:T-Lymphocytes / Q000378:metabolism*; D019061:src-Family Kinases / Q000378:metabolism*
D004791:Enzyme Inhibitors; D007376:Interleukin-2; D007527:Isoenzymes; D010748:Phosphopeptides; D010769:Phosphothreonine; D010768:Phosphoserine; D019319:Okadaic Acid; D004247:DNA; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D019061:src-Family Kinases; D010749:Phosphoprotein Phosphatases