Protein Kinase C-mediated Phosphorylation and Activation of PDE3A Regulate cAMP Levels in Human Platelets^*

Abstract

The elevation of [cAMP]_i is an important mechanism of platelet inhibition and is regulated by the opposing activity of adenylyl cyclase and phosphodiesterase (PDE). In this study, we demonstrate that a variety of platelet agonists, including thrombin, significantly enhance the activity of PDE3A in a phosphorylation-dependent manner. Stimulation of platelets with the PAR-1 agonist SFLLRN resulted in rapid and transient phosphorylation of PDE3A on Ser³¹², Ser⁴²⁸, Ser⁴³⁸, Ser⁴⁶⁵, and Ser⁴⁹², in parallel with the PKC (protein kinase C) substrate, pleckstrin. Furthermore, phosphorylation and activation of PDE3A required the activation of PKC, but not of PI3K/PKB, mTOR/p70S6K, or ERK/RSK. Activation of PKC by phorbol esters also resulted in phosphorylation of the same PDE3A sites in a PKC-dependent, PKB-independent manner. This was further supported by the finding that IGF-1, which strongly activates PI3K/PKB, but not PKC, did not regulate PDE3A. Platelet activation also led to a PKC-dependent association between PDE3A and 14-3-3 proteins. In contrast, cAMP-elevating agents such as PGE₁ and forskolin-induced phosphorylation of Ser³¹² and increased PDE3A activity, but did not stimulate 14-3-3 binding. Finally, complete antagonism of PGE₁-evoked cAMP accumulation by thrombin required both G_i and PKC activation. Together, these results demonstrate that platelet activation stimulates PKC-dependent phosphorylation of PDE3A on Ser³¹², Ser⁴²⁸, Ser⁴³⁸, Ser⁴⁶⁵, and Ser⁴⁹² leading to a subsequent increase in cAMP hydrolysis and 14-3-3 binding.