Cyclic Nucleotide-dependent Protein Kinases Target ARHGAP17 and ARHGEF6 Complexes in Platelets.
Endothelial cells release prostacyclin (PGI2) and nitric oxide (NO) to inhibit platelet functions. PGI2 and NO effects are mediated by cyclic nucleotides, cAMP- and cGMP-dependent protein kinases (PKA, PKG), and largely unknown PKA and PKG substrate proteins. The small G-protein Rac1 plays a key role in platelets and was suggested to be a target of cyclic nucleotide signaling. We confirm that PKA and PKG activation reduces Rac1-GTP levels. Screening for potential mediators of this effect resulted in the identification of the Rac1-specific GTPase-activating protein ARHGAP17 and the guanine nucleotide exchange factor ARHGEF6 as new PKA and PKG substrates in platelets. We mapped the PKA/PKG phosphorylation sites to serine 702 on ARHGAP17 using Phos-tag gels and to serine 684 on ARHGEF6. We show that ARHGAP17 binds to the actin-regulating CIP4 protein in platelets and that Ser-702 phosphorylation interferes with this interaction. Reduced CIP4 binding results in enhanced inhibition of cell migration by ARHGAP17. Furthermore, we show that ARHGEF6 is constitutively linked to GIT1, a GAP of Arf family small G proteins, and that ARHGEF6 phosphorylation enables binding of the 14-3-3 adaptor protein to the ARHGEF6/GIT1 complex. PKA and PKG induced rearrangement of ARHGAP17- and ARHGEF6-associated protein complexes might contribute to Rac1 regulation and platelet inhibition.
From the UCD Conway Institute and the School of Medicine, University College Dublin, Belfield, Dublin 4, Ireland.
Zoltan
Z
Nagy
Mass Spectrometry Resource, UCD Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland.
Kieran
K
Wynne
Systems Biology Ireland, University College Dublin, Belfield, Dublin 4, Ireland, and.
Alexander
A
von Kriegsheim
Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, 44 Thorez Prospect, St. Petersburg, 194223 Russia.
Stepan
S
Gambaryan
From the UCD Conway Institute and the School of Medicine, University College Dublin, Belfield, Dublin 4, Ireland, albert.smolenski@ucd.ie.
Albert
A
Smolenski
D001792:Blood Platelets / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000097:blood*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D010766:Phosphorylation; D064067:Rho Guanine Nucleotide Exchange Factors / Q000097:blood; D013379:Substrate Specificity
C504466:ARHGAP17 protein, human; C576603:ARHGEF6 protein, human; D020690:GTPase-Activating Proteins; D064067:Rho Guanine Nucleotide Exchange Factors; D017868:Cyclic AMP-Dependent Protein Kinases; D017869:Cyclic GMP-Dependent Protein Kinases
Rac (Rac GTPase); cyclic AMP (cAMP); cyclic GMP (cGMP); phosphorylation; platelet