In situ phosphorylation of platelet actin-binding protein by cAMP-dependent protein kinase stabilizes it against proteolysis by calpain.
To identify the protein kinase that is responsible for catalyzing phosphorylation of actin-binding protein (ABP) in platelets, we have examined the effects of protein kinase C and cAMP-dependent protein kinase on this process. We found that purified platelet protein kinase C from platelets was unable to phosphorylate ABP in vitro. However, a crude platelet kinase preparation phosphorylated ABP in the presence of cAMP, but not in the presence of Ca2+/phosphatidylserine. Fresh platelet plasma membranes incubated with [gamma-32P]ATP phosphorylated ABP in the presence of cAMP and the process was blocked by a cAMP-dependent protein kinase inhibitor; ABP phosphorylation induced by prostaglandin E1 (PGE1) appeared to be reduced by the subsequent addition of thrombin. These results strongly suggest that in situ ABP is phosphorylated by activated cAMP-dependent protein kinase when platelet function is inhibited by PGE1. Furthermore, in the PGE1-treated platelets, ABP was proteolyzed at a slower rate than in control platelets when they were lysed with Triton in the absence of EGTA. Partially purified ABP was proteolyzed by calpain in vitro at a slower rate as well. It was demonstrated that ABP from PGE1-treated platelets recovered its sensitivity to calpain after ABP was incubated with a protein phosphatase that had been purified from platelets. We postulate that ABP is stabilized against proteolysis in response to cAMP-elevating agents and that this blocks cytoskeleton reorganization.
Chen
M
M
Department of Biochemistry, State University of New York Health Science Center, Brooklyn 11203.
Stracher
A
A
D001792:Blood Platelets / Q000378:metabolism*; D002154:Calpain / Q000494:pharmacology*; D006801:Humans; D006868:Hydrolysis; D066298:In Vitro Techniques; D008840:Microfilament Proteins / Q000097:blood*; D010749:Phosphoprotein Phosphatases / Q000097:blood / Q000302:isolation & purification; D010766:Phosphorylation; D011493:Protein Kinase C / Q000097:blood / Q000302:isolation & purification; D011494:Protein Kinases / Q000097:blood / Q000502:physiology*
D008840:Microfilament Proteins; D011494:Protein Kinases; D011493:Protein Kinase C; D010749:Phosphoprotein Phosphatases; D002154:Calpain