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Targeting of type I protein kinase A to lipid rafts is required for platelet inhibition by the 3′,5′‐cyclic adenosine monophosphate‐signaling pathway
Author(s) -
Raslan Z.,
Magwenzi S.,
Aburima A.,
Taskén K.,
Naseem K. M.
Publication year - 2015
Publication title -
journal of thrombosis and haemostasis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.947
H-Index - 178
eISSN - 1538-7836
pISSN - 1538-7933
DOI - 10.1111/jth.13042
Subject(s) - phosphorylation , protein kinase a , microbiology and biotechnology , lipid raft , cyclic adenosine monophosphate , platelet activation , chemistry , signal transduction , proximity ligation assay , kinase , biochemistry , platelet , biology , receptor , immunology
Summary Background Platelet adhesion to von Willebrand factor ( VWF ) is modulated by 3′,5′‐cyclic adenosine monophosphate ( cAMP ) signaling through protein kinase A ( PKA )‐mediated phosphorylation of glycoprotein ( GP )Ibβ. A‐kinase anchoring proteins ( AKAP s) are proposed to control the localization and substrate specificity of individual PKA isoforms. However, the role of PKA isoforms in regulating the phosphorylation of GPI bβ and platelet response to VWF is unknown. Objectives We wished to determine the role of PKA isoforms in the phosphorylation of GPI bβ and platelet activation by VWF as a model for exploring the selective partitioning of cAMP signaling in platelets. Results The two isoforms of PKA in platelets, type I ( PKA ‐I) and type II ( PKA ‐ II ), were differentially localized, with a small pool of PKA ‐I found in lipid rafts. Using a combination of Far Western blotting, immunoprecipitation, proximity ligation assay and cAMP pull‐down we identified moesin as an AKAP that potentially localizes PKA ‐I to rafts. Introduction of cell‐permeable anchoring disruptor peptide, RI anchoring disruptor ( RIAD ‐Arg 11 ), to block PKA ‐I/ AKAP interactions, uncoupled PKA ‐ RI from moesin, displaced PKA ‐ RI from rafts and reduced kinase activity in rafts. Examination of GPI bβ demonstrated that it was phosphorylated in response to low concentrations of PGI 2 in a PKA ‐dependent manner and occurred primarily in lipid raft fractions. RIAD ‐Arg 11 caused a significant reduction in raft‐localized phospho GPI bβ and diminished the ability of PGI 2 to regulate VWF ‐mediated aggregation and thrombus formation in vitro . Conclusion We propose that PKA ‐I‐specific AKAP s in platelets, including moesin, organize a selective localization of PKA ‐I required for phosphorylation of GPI bβ and contribute to inhibition of platelet VWF interactions.