Abstract
Background Under normal conditions circulating platelets are maintained in a resting state by the paracrine signaling molecules, prostacyclin (PGI2) and nitric oxide (NO), released from vascular endothelial cells. PGI2 and NO trigger the synthesis of cAMP and cGMP, respectively, which in turn leads to the activation of Protein Kinase A (PKA) and Protein Kinase G (PKG). These cyclic nucleotide-dependent protein kinases phosphorylate numerous substrate proteins involved in several aspects of platelet activation. Both, PKA and PKG can phosphorylate the same substrates including VASP, LASP, RAP1GAP2, Rap1B, HSP27, and some others. Only few substrates are known to be specific for PKA (PDE3) and PKG (PDE5) [1,2]. Here we report the identification and characterization of a new PKA specific substrate, CalDAG-GEFI (calcium and diacylglycerol regulated guanine nucleotide exchange factor I), a guanine exchange factor for Rap1b in platelets.
Highlights
Under normal conditions circulating platelets are maintained in a resting state by the paracrine signaling molecules, prostacyclin (PGI2) and nitric oxide (NO), released from vascular endothelial cells
Using radioactive phosphate incorporation assay, we identified S587 as the major Protein Kinase A (PKA) phosphorylation site in CalDAG-GEFI, which was confirmed by mass spectrometry
PKA stimulation leads to strong phosphorylation of CalDAG-GEFI and inhibition of Rap1b activation triggered by thrombin, as well as calcium ionophore, which indicates a potential role of CalDAG-GEFI phosphorylation in the inhibition of Rap1b activation
Summary
Under normal conditions circulating platelets are maintained in a resting state by the paracrine signaling molecules, prostacyclin (PGI2) and nitric oxide (NO), released from vascular endothelial cells. Results Using radioactive phosphate incorporation assay, we identified S587 as the major PKA phosphorylation site in CalDAG-GEFI, which was confirmed by mass spectrometry.
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