Inhibitors of protein phosphatase type 1 and 2A attenuate phosphatidylinositol metabolism and Ca(2+)-transients in human platelets. Role of a cdc2-related protein kinase.
The addition of either okadaic acid or calyculin A desensitizes human platelets to thrombin. One objective of this study was to determine which step(s) leading to secretion reactions may be affected by these protein phosphatase inhibitors. In a dose-dependent manner, okadaic acid or calyculin A inhibits phosphatidylinositol metabolism and Ca(2+)-transients. In all cases, calyculin A was approximately 10-fold more potent than okadaic acid, and it had maximal effects at a concentration of 1 microM. Although thrombin-induced rises in [Ca2+]i were diminished, an increase in the phosphorylation state of myosin light chains (MLC) was still observed. Changes in this phosphorylation were diminished, however, following the addition of thrombin to calyculin A-treated platelets that were loaded with dimethyl-BAPTA. These data demonstrate that calyculin A and okadaic acid lower agonist-induced Ca(2+)-transients, which in turn prevents responses such as secretion reactions. Calyculin A/okadaic acid-induced phosphorylation events were not diminished in BAPTA-loaded platelets, suggesting that these phosphorylations are Ca(2+)-insensitive. Thus, a second objective of this study was to identify the protein kinase(s) that was(were) responsible for the calyculin A-induced phosphorylations. In a platelet lysate system, calyculin A caused an increase in the incorporation of [32P]phosphate into p50. This phosphorylation event was identical to that observed in the intact platelet and was not mimicked by cAMP, cGMP, Ca2+, or a Ca2+/phospholipid/diacylglycerol mixture. Kinase activity was removed after the lysate was incubated with p13suc1-Sepharose. This suggests that a p13suc1-sensitive protein kinase, e.g., a cell cycle-dependent protein kinase, is responsible for the calyculin A-sensitive phosphorylation events.(ABSTRACT TRUNCATED AT 250 WORDS)
Lerea
K M
KM
Department of Cell Biology and Anatomy, New York Medical College, Valhalla 10595.
D001792:Blood Platelets / Q000378:metabolism*; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D002118:Calcium / Q000097:blood*; D015180:Electrophoresis, Gel, Two-Dimensional; D004988:Ethers, Cyclic / Q000494:pharmacology*; D006801:Humans; D066298:In Vitro Techniques; D008387:Marine Toxins; D019319:Okadaic Acid; D010080:Oxazoles / Q000494:pharmacology*; D010449:Peptide Mapping; D010716:Phosphatidylinositols / Q000097:blood*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors* / Q000145:classification; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D012636:Secretory Rate / Q000187:drug effects; D012701:Serotonin / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology
D004988:Ethers, Cyclic; D008387:Marine Toxins; D010080:Oxazoles; D010716:Phosphatidylinositols; D010750:Phosphoproteins; D019319:Okadaic Acid; D012701:Serotonin; C059041:calyculin A; D016203:CDC2 Protein Kinase; D010749:Phosphoprotein Phosphatases; D013917:Thrombin; D002118:Calcium