Phosphorylation of the guanine-nucleotide exchange factor CalDAG-GEFI by protein kinase A regulates Ca2{+}-dependent activation of platelet Rap1b GTPase

In blood platelets, the small GTPase Rap1b is activated by cytosolic Ca2+, and promotes integrin aIIbb3 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 this work we demonstrate that the Ca2+-dependent exchange factor for Rap1b, CalDAG-GEFI, is a novel substrate for the cAMP-activated protein kinase A (PKA). 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 mutants, we identified serine 116 and serine 586 as PKA phosphorylation sites in CalDAG-GEFI. In transfected HEK293 cells, as well as in platelets, forskolin-induced phosphorylation of CalDAG-GEFI prevented activation of Rap1b induced by the Ca2+ ionophore A23187. In platelets, this effect was associated to the inhibition of aggregation. Moreover, cAMP-mediated inhibition of Rap1b was lost in HEK293 cell transfected with a double mutant of CalDAG-GEFI unable to be phosphorylated by PKA. These results demonstrate that phosphorylation of CalDAG-GEFI by PKA affects its activity, and represents a novel mechanism for cAMP-mediated inhibition of Rap1b in platelets.

Source: BJ Cell - April 19, 2013 Category: Biochemistry Authors: G Francesco Guidetti, D Manganaro, A Consonni, I Canobbio, C Balduini, M Torti Tags: BJ Signal Source Type: research