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Phosphorylation of CalDAG-GEFI by protein kinase A prevents Rap1b activation

H Subramanian1, R P Zahedi, A Sickmann

  • 1Institute of Clinical Biochemistry and Pathobiochemistry, University of Wuerzburg, Wuerzburg, Germany.

Journal of Thrombosis and Haemostasis : JTH
|April 25, 2013
PubMed

Insights

Protein kinase A (PKA) regulates platelet aggregation by phosphorylating CalDAG-GEFI at S587, inhibiting Rap1b activation. This phosphorylation is key to PKA

Area of Science:

  • Platelet signaling
  • Molecular cell biology
  • Signal transduction

Background:

  • Protein kinase A (PKA) and protein kinase G (PKG) signaling are crucial for maintaining resting platelets.
  • These kinases inhibit Rap1b, a key regulator of integrin activation and platelet aggregation.
  • The precise mechanism of Rap1b regulation by PKA and PKG remains unclear.

Purpose of the Study:

  • To pinpoint PKA phosphorylation sites on calcium and diacylglycerol-regulated guanine nucleotide exchange factor I (CalDAG-GEFI), the primary Rap1b GEF in platelets.
  • To investigate how CalDAG-GEFI phosphorylation influences Rap1b activation.

Main Methods:

  • Radioactive phosphate incorporation and mass spectrometry were used to identify CalDAG-GEFI phosphorylation sites.
  • A phospho-antibody was generated for Western blot detection of CalDAG-GEFI phosphorylation.
  • Rap1b activation was assessed using Rap1-GTP pull-down assays.

Main Results:

  • Serine 587 (S587) was identified as the main PKA phosphorylation site on CalDAG-GEFI; S116/117 showed weak phosphorylation.
  • S587 phosphorylation correlated with PKA's inhibitory effect on Rap1b activation in platelets.
  • In HEK293 cells, a phospho-mimetic CalDAG-GEFI mutant (S587D) blocked agonist-induced Rap1b activation, while alanine mutations at S116, S117, and S587 abolished PKA's inhibitory effect.

Conclusions:

  • PKA controls Rap1b-dependent platelet aggregation through CalDAG-GEFI phosphorylation.
  • Phosphorylation of CalDAG-GEFI at S587 is a critical regulatory mechanism in platelet function.
Abstract

Keywords:
calcium signalingcyclic AMPhuman Rap1b proteinhuman RasGRP2 proteinprotein kinase A

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