TY - JOUR T1 - Essential Role for Class II Phosphoinositide 3-kinase α-Isoform in Ca<sup>2+</sup>-Induced, Rho- and Rho Kinase-Dependent Regulation of Myosin Phosphatase and Contraction in Isolated Vascular Smooth Muscle Cells JF - Molecular Pharmacology JO - Mol Pharmacol SP - 912 LP - 920 DO - 10.1124/mol.106.032599 VL - 71 IS - 3 AU - Kazuaki Yoshioka AU - Naotoshi Sugimoto AU - Noriko Takuwa AU - Yoh Takuwa Y1 - 2007/03/01 UR - http://molpharm.aspetjournals.org/content/71/3/912.abstract N2 - The laser confocal fluorescent microscope-based observation of contractile responses in green fluorescent protein-expressing differentiated vascular smooth muscle cells, combined with the RNA interference-mediated gene-silencing technique, allowed us to determine the role of phosphoinositide 3-kinase (PI3K) class II α-isoform (PI3K-C2α) as a novel, Ca2+-dependent regulator of myosin light-chain phosphatase (MLCP) and contraction. The Ca2+-ionophore ionomycin induced a robust contractile response with an increase in the intracellular free Ca2+ concentration ([Ca2+]i). The PI3K-C2α-specific short interfering RNA (siRNA) induced a selective and marked reduction in PI3K-C2α protein expression. The siRNA-mediated knockdown of PI3K-C2α, but not class I PI3K p110α, suppressed ionomycin-induced contraction without altering Ca2+-mobilization. PI3K-C2α is uniquely less sensitive to the PI3K inhibitor 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) than the other PI3K members, including p110α. Ionomycin-induced contraction was inhibited only by a relatively high concentration of LY294002. Consistent with our previous observations showing that ionomycin and membrane depolarization induced Rho activation in vascular smooth muscle tissues in a Ca2+-dependent manner, ionomycin-induced contraction was dependent on Rho and Rho-kinase. Ionomycin induced phosphorylation of the MLCP-regulatory subunit myosin targeting protein 1(MYPT1) at Thr850 and the 20-kDa myosin light chain (MLC) in a Rho kinase-dependent manner. Knockdown of PI3K-C2α suppressed phosphorylation of both MYPT1 and MLC. The receptor agonist noradrenaline, which induced a rapid increase in the [Ca2+]i and Ca2+-dependent contraction, stimulated phosphorylation of MYPT1 and MLC, which was also dependent on Ca2+, PI3K-C2α, and Rho-kinase. These observations indicate that PI3K-C2α is necessary for Ca2+-induced Rho- and Rho kinase-dependent negative regulation of MLCP and consequently MLC phosphorylation and contraction. The American Society for Pharmacology and Experimental Therapeutics ER -