title: Phosphorylation of CalDAG-GEFI by protein kinase A prevents Rap1b activation.
abstract: BACKGROUND
  Signaling via protein kinase A (PKA) and protein kinase G (PKG) is critical for maintaining platelets in the resting state. Both kinases down-regulate the activity of the small GTPase Rap1b, a critical signaling switch for integrin activation and platelet aggregation. However, the mechanism of Rap1b regulation by PKA and PKG is largely unknown.
  
  
  OBJECTIVE
  To identify the PKA phosphorylation sites in calcium and diacylglycerol-regulated guanine nucleotide exchange factor I (CalDAG-GEFI), the main GEF for Rap1b in platelets, and the effect of CalDAG-GEFI phosphorylation in Rap1b activation.
  
  
  METHODS
  The phosphorylation sites in CalDAG-GEFI were identified by radio-active phosphate incorporation assay and mass spectrometry. Phospho-antibody was developed to detect CalDAG-GEFI phosphorylation in Western blots. Rap1b activation was detected by Rap1-GTP pull-down assay.
  
  
  RESULTS
  S587 was identified as the major PKA phosphorylation site in CalDAG-GEFI, while S116/117 was weakly phosphorylated. Phosphorylation of S587 correlated with the inhibitory effect of PKA on Rap1b activation in platelets. In HEK293 cells, expression of a phospho-mimetic mutant of CalDAG-GEFI (S587D) abolished agonist-induced Rap1b activation. Mutation of S587 to alanine partially reversed the inhibitory effect of PKA signaling on Rap1b activation, while mutation of S116, S117 and S587 to alanine completely abolished the inhibitory effect of PKA on Rap1b activation.
  
  
  CONCLUSIONS
  Our study strongly suggests that phosphorylation of CalDAG-GEFI is a critical mechanism by which PKA controls Rap1b-dependent platelet aggregation.
authors.affiliation: Institute of Clinical Biochemistry and Pathobiochemistry, University of Wuerzburg, Wuerzburg, Germany.
authors.forename: H
authors.initials: H
authors.lastname: Subramanian
authors.forename: R P
authors.initials: RP
authors.lastname: Zahedi
authors.forename: A
authors.initials: A
authors.lastname: Sickmann
authors.forename: U
authors.initials: U
authors.lastname: Walter
authors.forename: S
authors.initials: S
authors.lastname: Gambaryan
mesh_terms: D000409:Alanine / Q000378:metabolism; D000595:Amino Acid Sequence; D001792:Blood Platelets / Q000378:metabolism; D020013:Calcium Signaling; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004789:Enzyme Activation; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D010974:Platelet Aggregation; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D020761:rab1 GTP-Binding Proteins / Q000378:metabolism*
chemical_list: D020662:Guanine Nucleotide Exchange Factors; C415483:RASGRP2 protein, human; D006160:Guanosine Triphosphate; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; C529587:Rab1B protein, human; D020761:rab1 GTP-Binding Proteins; D000409:Alanine
keywords: calcium signaling; cyclic AMP; human Rap1b protein; human RasGRP2 protein; protein kinase A