title: Protein kinase C phosphorylation of syntaxin 4 in thrombin-activated human platelets.
abstract: We postulated that the syntaxins, because of their key role in SNARE complex formation and exocytosis, could be important targets for signaling by intracellular kinases involved in secretion. We found that syntaxin 4 was phosphorylated in human platelets treated with a physiologic agent that induces secretion (thrombin) but not when they were treated with an agent that prevents secretion (prostacyclin). Syntaxin 4 phosphorylation was blocked by inhibitors of activated protein kinase C (PKC), and, in parallel assays, PKC inhibitors also blocked secretion from thrombin-activated platelets. In platelets, cellular activation by thrombin or phorbol 12-myristate 13-acetate decreased the binding of syntaxin 4 with SNAP-23, another platelet t-SNARE. Phosphatase inhibitors increased syntaxin 4 phosphorylation and further decreased syntaxin 4-SNAP-23 binding induced by cell activation. Conversely, a PKC inhibitor blocked syntaxin 4 phosphorylation and returned binding of syntaxin 4-SNAP-23 to that seen in nonstimulated platelets. In vitro, PKC directly phosphorylated platelet syntaxin 4 and recombinant syntaxin 4. PKC phosphorylation in vitro inhibited (71 +/- 8%) the binding of syntaxin 4 to SNAP-23. These results provide evidence that extracellular activation can be coupled through intracellular PKC signaling so as to modulate SNARE protein interactions involved in platelet exocytosis.
authors.affiliation: Cardiovascular Biology Laboratory, Harvard School of Public Health, Boston, MA 02115, USA.
authors.forename: S H
authors.initials: SH
authors.lastname: Chung
authors.forename: J
authors.initials: J
authors.lastname: Polgar
authors.forename: G L
authors.initials: GL
authors.lastname: Reed
mesh_terms: D001792:Blood Platelets / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D011464:Epoprostenol / Q000494:pharmacology; D005089:Exocytosis; D006801:Humans; D015151:Immunoblotting; D008565:Membrane Proteins / Q000378:metabolism*; D010766:Phosphorylation; D015539:Platelet Activation; D011233:Precipitin Tests; D011485:Protein Binding; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000502:physiology; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D011994:Recombinant Proteins / Q000378:metabolism; D012701:Serotonin / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013917:Thrombin / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D002352:Carrier Proteins; D008565:Membrane Proteins; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D011994:Recombinant Proteins; C100493:SNAP23 protein, human; D012701:Serotonin; D011464:Epoprostenol; D011493:Protein Kinase C; D013917:Thrombin; D013755:Tetradecanoylphorbol Acetate