Peroxynitrite causes phosphorylation of vasodilator-stimulated phosphoprotein through a PKC dependent mechanism.
Peroxynitrite is a potent nitrating and oxidizing agent that exerts differential effects on platelets. In the present study we investigated the influence of peroxynitrite on vasodilator-stimulated phosphoprotein (VASP), a protein that plays a key role in inhibition of platelet adhesion and spreading. In platelets, VASP is a substrate for protein kinase A (PKA), PKC and PKG and phosphorylation by these kinases is thought to block VASP-mediated actin cytoskeletal rearrangement. In the present study, we demonstrate that peroxynitrite phosphorylates VASP by a PKC-dependent mechanism. Peroxynitrite (0-100 microM) induced a concentration and time-dependent increase in phosphorylation of VASP at serine(157) (Ser(157)) and Ser(239). Inhibition of soluble guanylyl cyclase (sGC) did not significantly reduce peroxynitrite-mediated phosphorylation, indicating a cGMP-independent pathway for VASP phosphorylation. In contrast nitric oxide-mediated VASP phosphorylation was abolished under conditions of sGC inhibition. Further exploration of the mechanisms underlying VASP phosphorylation indicated a requirement for Ca2+ mobilization, but was independent of protein kinase A, Src kinases and protein nitration. Consistent with previous reports phorbol 12-myristate 13-acetate (PMA; 300 nM) induced phosphorylation of VASP at Ser(157), but not Ser(239), which was blocked by general protein kinase C (PKC) inhibitors, Ro31-8220 and Bisindolylmaleimide I (BIM-1), and Gö6976, an inhibitor of conventional PKC isoforms. Interestingly, treatment of platelets with these PKC inhibitors significantly reduced peroxynitrite-mediated phosphorylation of both sites, indicating that phosphorylation occurred through PKC-dependent mechanism. Consistent with these findings peroxynitrite caused a small increase in PKC activity as evidenced by increased phosphorylation of PKC substrates. Together these data indicate that peroxynitrite may inhibit platelet function by inducing the phosphorylation of VASP through a mechanism that requires the activation of PKC.
Aburima
Ahmed
A
Hull York Medical School, University of Hull, Castle Hill Hospital, Castle Road, Cottingham HU67RX, UK.
Riba
Rocio
R
Naseem
Khalid M
KM
D002118:Calcium / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D006801:Humans; D008840:Microfilament Proteins / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D010084:Oxidation-Reduction; D030421:Peroxynitrous Acid / Q000494:pharmacology*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism*; D000098662:Vasodilator-Stimulated Phosphoprotein
D015815:Cell Adhesion Molecules; D008840:Microfilament Proteins; D010750:Phosphoproteins; D000098662:Vasodilator-Stimulated Phosphoprotein; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; D011493:Protein Kinase C; D002118:Calcium