Phosphorylation results in activation of a cAMP phosphodiesterase in human platelets.
Agents such as prostaglandins E1 and I2 which elevate cAMP levels in platelets also increase cAMP phosphodiesterase activity. Since much of the cAMP phosphodiesterase activity in human platelets is due to the cGMP-inhibited isozyme (Macphee, C. H., Harrison, S. A., and Beavo, J. A. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 6600-6663), we examined the regulation of this isozyme by prostaglandins E1 and I2 in intact platelets. Because this isozyme is a minor component of platelet protein, normally requiring several thousand-fold purification to achieve homogeneity, a specific monoclonal antibody (CGI-5) was utilized to identify and isolate the cGMP-inhibited phosphodiesterase activity. Treatment of intact platelets with the prostaglandins promoted an increase in the phosphorylation state of the cGMP-inhibited phosphodiesterase and a corresponding increase in phosphodiesterase activity. The effect on activity and phosphorylation of the cGMP-inhibited phosphodiesterase was observed within 2 min after intact platelets were exposed to the prostaglandins. The half-maximal effective dose for prostaglandin I2 (10 nM) was approximately 10-fold lower than that for prostaglandin E1. The phosphorylated, cGMP-inhibited isozyme migrated as a 110-kDa peptide following sodium dodecyl sulfate gel electrophoresis. Direct in vitro phosphorylation of the platelet cGMP-inhibited phosphodiesterase by the catalytic subunit of cAMP-dependent protein kinase caused a similar increase in phosphodiesterase activity. Treatment with PKI peptide, a specific inhibitor of cAMP-dependent protein kinase, blocked the phosphorylation and the effect on activity. Taken together, the data strongly suggest that the effects of prostaglandins E1 and I2 on platelet phosphodiesterase activity are mediated by a direct cAMP-dependent protein kinase-catalyzed phosphorylation of the cGMP-inhibited phosphodiesterase isozyme.
Macphee
C H
CH
Department of Pharmacology, School of Medicine, University of Washington, Seattle 98195.
Reifsnyder
D H
DH
Moore
T A
TA
Lerea
K M
KM
Beavo
J A
JA
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000097:blood* / Q000302:isolation & purification; D000527:Alprostadil / Q000494:pharmacology; D001792:Blood Platelets / Q000201:enzymology*; D004789:Enzyme Activation; D011464:Epoprostenol / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D010766:Phosphorylation
D011464:Epoprostenol; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D000527:Alprostadil