Protein kinase C-catalyzed phosphorylation of an inhibitory phosphoprotein of myosin phosphatase is involved in human platelet secretion

Protein kinase C (PKC)-potentiated inhibitory phosphoprotein of myosin phosphatase (CPI) was detected in human platelets, Like smooth muscle CPI-17, in vitro phosphorylation of platelet CPI by PKC inhibited the activity of myosin phosphatase containing the PP1 delta catalytic subunit and the 130-kd myosin-binding subunit (MBS), Treatment of intact platelets with thrombin or the stable thromboxane Aa analog STA(2) resulted in increased phosphorylation of both CPI and MBS at Thr-696, whereas phorbol myristate acetate (PMA) and the Ca++ ionophore ionomycin only induced CPI phosphorylation. PMA induced slow adenosine triphosphate (ATP) secretion of fura 2 platelets with no change in cytosolic Ca++, The PMA-induced increase in CPI phosphorylation preceded phosphorylation of 20-kd myosin light chain (MLC20) at Ser-19 and ATP secretion. The PKC inhibitor, GF109203X, inhibited PMA-induced phosphorylation of CPI and MLC20 with similar IC50 values. These findings suggest that the activation of PKC by PMA induces MLC20 phosphorylation by inhibiting myosin phosphatase through phosphorylation of CPI, STA(2)-induced MLC20 phosphorylation was also diminished but not abolished by GF109203X, even at high concentrations that completely inhibited STA(2)-induced CPI phosphorylation. A combination of the Rho-kinase inhibitor-loaded Y-27632 and GF109203X led to a further decrease in STA(2)-induced MLC20 phosphorylation, mainly because of a significant inhibition of MBS phosphorylation at Thr-696, Inhibition of STA(2)-induced ATP release by Y-27632, GF109203X, or both appeared to correlate with the extent of MLC20 phosphorylation, Thus, CPI phosphorylation by PKC may participate in inhibiting myosin phosphatase, in addition to the Rho-kinase-mediated regulation of myosin phosphatase, during agonist-induced platelet secretion.