Protein kinase C-mediated phosphorylation and activation of PDE3A regulate cAMP levels in human platelets.
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 PDE3A and 14-3-3 proteins. In contrast, cAMP-elevating agents such as PGE(1) and forskolin-induced phosphorylation of Ser(312) and increased PDE3A activity, but did not stimulate 14-3-3 binding. Finally, complete antagonism of PGE(1)-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 Ser(312), Ser(428), Ser(438), Ser(465), and Ser(492) leading to a subsequent increase in cAMP hydrolysis and 14-3-3 binding.
Hunter
Roger W
RW
Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, UK.
Mackintosh
Carol
C
Hers
Ingeborg
I
D048948:14-3-3 Proteins / Q000378:metabolism; D000527:Alprostadil / Q000494:pharmacology; D001792:Blood Platelets / Q000201:enzymology*; D001798:Blood Proteins / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism*; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3 / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D006490:Hemostatics / Q000494:pharmacology; D006801:Humans; D006868:Hydrolysis / Q000187:drug effects; D010446:Peptide Fragments / Q000494:pharmacology; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D015539:Platelet Activation / Q000187:drug effects / Q000502:physiology*; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D011493:Protein Kinase C / Q000378:metabolism*; D044463:Receptor, PAR-1 / Q000819:agonists / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology
D048948:14-3-3 Proteins; D001798:Blood Proteins; D006490:Hemostatics; D010446:Peptide Fragments; D010750:Phosphoproteins; D010975:Platelet Aggregation Inhibitors; D044463:Receptor, PAR-1; C039077:platelet protein P47; C082835:thrombin receptor peptide (42-47); D000242:Cyclic AMP; D011493:Protein Kinase C; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; C517287:PDE3A protein, human; D013917:Thrombin; D000527:Alprostadil