Protein Kinase C-mediated Phosphorylation and activation of PDE3A Regulate cAMP levels in human platelets

Roger Hunter, Carol MacKintosh, Ingeborg Hers

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    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 Ser(312), Ser(428), Ser(438), Ser(465), and Ser(492), 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 PDE3Aand 14-3-3 proteins. In contrast, cAMP-elevating agents such as PGE(1) and forskolin-induced phosphorylation of Ser312 and increased PDE3A activity, but did not stimulate 14-3-3 binding. Finally, complete antagonism of PGE(1)-evoked cAMP accumulation by thrombin required bothGi andPKCactivation. Together, these results demonstrate that platelet activation stimulates PKC-dependent phosphorylation of PDE3A on Ser(312), Ser(428), Ser(438), Ser(465), and Ser(492) leading to a subsequent increase in cAMP hydrolysis and 14-3-3 binding.

    Original languageEnglish
    Pages (from-to)12339-12348
    Number of pages10
    JournalJournal of Biological Chemistry
    Volume284
    Issue number18
    DOIs
    Publication statusPublished - 2009

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