G protein-mediated Ca2+-sensitization of CPI-17 phosphorylation in arterial smooth muscle

CPI-17 is a unique phosphoprotein that specifically inhibits myosin light chain phosphatase in smooth muscle and plays an essential role in agonist-induced contraction. To elucidate the in situ mechanism for G protein-mediated Ca2+-sensitization of CPI-17 phosphorylation, α-toxin-permeabilized arterial smooth muscle strips were used to monitor both force development and CPI-17 phosphorylation in response to GTPγS with varying Ca2+ concentrations. CPI-17 phosphorylation increased at unphysiologically high Ca2+ levels of pCa ⩽ 6. GTPγS markedly enhanced the Ca2+ sensitivity of CPI-17 steady-state phosphorylation but had no enhancing effect under Ca2+-free conditions, while the potent PKC activator PDBu increased CPI-17 phosphorylation regardless of Ca2+ concentration. CPI-17 phosphorylation induced by pCa 4.5 alone was markedly inhibited by the presence of PKC inhibitor but not ROCK inhibitor. In the presence of calyculin A, a potent PP1/PP2A phosphatase inhibitor, CPI-17 phosphorylation increased with time even under Ca2+-free conditions. Furthermore, as Ca2+ concentration increased, so did CPI-17 phosphorylation rate. GTPγS markedly enhanced the rate of phosphorylation of CPI-17 at a given Ca2+. In the absence of calyculin A, either steady-state phosphorylation of CPI-17 under Ca2+-free conditions in the presence of GTPγS or at pCa 6.7 in the absence of GTPγS was negligible, suggesting a high intrinsic CPI-17 phosphatase activity. In conclusion, cooperative increases in Ca2+ and G protein activation are required for a significant activation of total kinases that phosphorylate CPI-17, which together overcome CPI-17 phosphatase activity and effectively increase the Ca2+ sensitivity of CPI-17 phosphorylation and smooth muscle contraction.

Research highlights
► Phosphorylation of smooth muscle-specific CPI-17 increases at only high Ca2+.
► G protein activation increases Ca2+ sensitivity of CPI-17 phosphorylation and smooth muscle contraction, but has no enhancing effect under Ca2+-free conditions.
► When phosphatase is blocked, CPI-17 phosphorylation increases with time even at low Ca2+.
► Because of high intrinsic CPI-17 phosphatase activity, cooperative increases in Ca2+ and G protein activation are required for a significant activation of CPI-17 kinases and smooth muscle contraction.