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.
Kitazawa
Toshio
T
Boston Biomedical Research Institute, 64 Grove Street, Watertown, MA 02472, USA. Kitazawa@bbri.org
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002199:Capillary Permeability; D002478:Cells, Cultured; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D009119:Muscle Contraction; D009124:Muscle Proteins / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000201:enzymology / Q000502:physiology*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011817:Rabbits
C491521:CPI-17 protein, Oryctolagus cuniculus; D009124:Muscle Proteins; D010750:Phosphoproteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019204:GTP-Binding Proteins; D002118:Calcium