title: Phosphorylation of the guanine-nucleotide-exchange factor CalDAG-GEFI by protein kinase A regulates Ca(2+)-dependent activation of platelet Rap1b GTPase.
abstract: In blood platelets the small GTPase Rap1b is activated by cytosolic Ca2+ and promotes integrin αIIbβ3 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 Ser116 and Ser586 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.
authors.affiliation: Department of Biology and Biotechnology, University of Pavia, via Bassi 21, 27100 Pavia, Italy.
authors.forename: Gianni Francesco
authors.initials: GF
authors.lastname: Guidetti
authors.forename: Daria
authors.initials: D
authors.lastname: Manganaro
authors.forename: Alessandra
authors.initials: A
authors.lastname: Consonni
authors.forename: Ilaria
authors.initials: I
authors.lastname: Canobbio
authors.forename: Cesare
authors.initials: C
authors.lastname: Balduini
authors.forename: Mauro
authors.initials: M
authors.lastname: Torti
mesh_terms: D000818:Animals; D001792:Blood Platelets / Q000187:drug effects; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology*; D005576:Colforsin / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology; D010766:Phosphorylation; D015539:Platelet Activation / Q000187:drug effects; D051381:Rats; D013449:Sulfonamides / Q000494:pharmacology; D020668:rap GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; D020662:Guanine Nucleotide Exchange Factors; D007546:Isoquinolines; C491789:Rasgrp1 protein, rat; D013449:Sulfonamides; D005576:Colforsin; D000001:Calcimycin; D017868:Cyclic AMP-Dependent Protein Kinases; C116753:RAP1B protein, human; D020668:rap GTP-Binding Proteins; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; D002118:Calcium