Journal
BIOCHEMICAL JOURNAL
Volume 453, Issue -, Pages 115-123Publisher
PORTLAND PRESS LTD
DOI: 10.1042/BJ20130131
Keywords
calcium and diacylglycerol-regulated guanine-nucleotide-exchange factor I (CalDAG-GEFI); cAMP; platelet; platelet inhibition; protein phosphorylation; Rap1b; signal transduction
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Funding
- Ministero Dell'Universita e della Ricerca Scientifica (PRIN), Italy
- Cariplo Foundation [2011-0436]
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In blood platelets the small GTPase Rap1b is activated by cytosolic Ca2+ and promotes integrin alpha(IIb)beta(3) inside-out activation and platelet aggregation. cAMP is the major inhibitor of platelet function and antagonizes Rap1b stimulation through a mechanism that remains unclear. In the present study we demonstrate that the Ca2+-dependent exchange factor for Rap1b, CalDAG-GEFI (calcium and diacylglycerol-regulated guanine-nucleotide-exchange factor I), is a novel substrate for the cAMP-activated PKA (protein kinase A). CalDAG-GEFI phosphorylation occurred in intact platelets treated with the cAMP-increasing agent forskolin and was inhibited by the PKA inhibitor H89. Purified recombinant CalDAG-GEFI was also phosphorylated in vitro by the PKA catalytic subunit. By screening a panel of specific serine to alanine residue mutants, we identified Ser(116) and Ser(586) as PKA phosphorylation sites in CalDAG-GEFI. In transfected HEK (human embryonic kidney)-293 cells, as well as in platelets, forskolin-induced phosphorylation of CalDAG-GEFI prevented the activation of Rap1b induced by the Ca2+ ionophore A23187. In platelets this effect was associated with the inhibition of aggregation. Moreover, cAMP-mediated inhibition of Rap1b was lost in HEK-293 cells transfected with a double mutant of CalDAG-GEFI unable to be phosphorylated by PKA. The results of the present study demonstrate that phosphorylation of CalDAG-GEFI by PKA affects its activity and represents a novel mechanism for cAMP-mediated inhibition of Rap1b in platelets.
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