REGULATION OF PLATELET PLASMA-MEMBRANE CA2-ATPASE BY CAMP-DEPENDENT AND TYROSINE PHOSPHORYLATION()

As a consequence of its central role in the regulation of calcium meta bolism in the platelet, the plasma membrane Ca2+-ATPase (PMCA) was ass essed for cAMP dependent and tyrosine phosphorylation, Addition of for skolin or prostaglandin El, agents known to elevate platelet cAMP and calcium efflux, to platelets pre-labeled with [P-32]PO4 resulted in th e direct phosphorylation of platelet PMCA. Similarly, addition of the catalytic subunit of protein kinase A to platelet plasma membranes res ulted in a 1.4-fold stimulation of activity, Thus, the previously repo rted inhibition of platelet activation by elevated intracellular cAMP may be accomplished in part by stimulation of PMCA, likely resulting i n a decrease in intracellular calcium. Treatment with thrombin evoked tyrosine phosphorylation of platelet PMCA, while PMCA from resting pla telets exhibited little tyrosine phosphorylation. Phosphorylation of p latelet plasma membranes by pp60(src) resulted in 75% inhibition of PM CA activity within 15 min, Similarly, membranes isolated from thrombin treated platelets exhibited 40% lower PMCA activity than those from r esting platelets, Phosphorylation of erythrocyte ghosts and purified P MCA by pp60(src) also resulted in up to 75% inhibition of Ca2+-ATPase activity, and inhibition was correlated with tyrosine phosphorylation, Sequencing of a peptide obtained after P-32 labeling of purified eryt hrocyte PMCA in vitro showed that tyrosine 1176 of PMCA4b is phosphory lated by pp60(src). These results indicate that tyrosine phosphorylati on of platelet PMCA may serve as positive feedback to inhibit PMCA and increase intracellular calcium during platelet activation.