Threonine phosphorylation of integrin beta3 in calyculin A-treated platelets is selectively sensitive to 5'-iodotubercidin.
Exposure of platelets to toxins (calyculin A or okadaic acid) that inhibit protein serine/threonine phosphatases types 1 and 2A, at concentrations that block aggregatory and secretory responses, results in the phosphorylation of several platelet proteins including integrin beta(3). Since protein phosphorylation represents a balance between kinase and phosphatase activities, this increase in phosphorylation reflects either the removal of phosphatases that oppose constitutively active kinases known to reside in the platelet (e.g., casein kinase 2) or the activation of endogenous kinases. In this study, we demonstrate that the addition of calyculin A promotes the activation of several endogenous platelet protein kinases, including p42/44(mapk), p38(mapk), Akt/PKB, and LKB1. Using a pharmacologic approach, we assessed whether inhibition of these and other enzymes block phosphorylation of beta(3). Inhibitors of p38(mapk), casein kinase, AMP kinase, protein kinase C, and calcium-calmodulin-dependent kinases did not block phosphorylation of beta(3) on thr(753). In contrast, 5'-iodotubercidin, at 50 muM, blocks beta(3) phosphorylation without affecting the efficacy of calyculin A to inhibit platelet aggregation and spreading. These data dissociate threonine phosphorylation of beta(3) molecules and inhibition of platelet responses by protein phosphatase inhibitors.
Lerea
Kenneth M
KM
Department of Cell Biology and Anatomy, New York Medical College, Valhalla, NY 10595, USA. ken_lerea@nymc.edu
Venjara
Aysha Y
AY
Olson
Susan C
SC
Kelly
Melissa R
MR
D064413:3-Phosphoinositide-Dependent Protein Kinases; D000091162:AMP-Activated Protein Kinase Kinases; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001792:Blood Platelets / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D017124:Conserved Sequence; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D039661:Integrin beta3 / Q000737:chemistry / Q000378:metabolism*; D008387:Marine Toxins; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D008969:Molecular Sequence Data; D010080:Oxazoles / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D010769:Phosphothreonine / Q000378:metabolism*; D010974:Platelet Aggregation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D013379:Substrate Specificity / Q000187:drug effects; D013917:Thrombin / Q000494:pharmacology; D014372:Tubercidin / Q000031:analogs & derivatives* / Q000494:pharmacology
C040541:5'-deoxy-5-iodotubercidin; D039661:Integrin beta3; D008387:Marine Toxins; D010080:Oxazoles; D047428:Protein Kinase Inhibitors; D010769:Phosphothreonine; C059041:calyculin A; D064413:3-Phosphoinositide-Dependent Protein Kinases; D017346:Protein Serine-Threonine Kinases; C109790:STK11 protein, human; D020928:Mitogen-Activated Protein Kinases; D000091162:AMP-Activated Protein Kinase Kinases; D013917:Thrombin; D014372:Tubercidin