title: Inhibition of MLC20 phosphorylation downstream of Ca2+ and RhoA: A novel mechanism involving phosphorylation of myosin phosphatase interacting protein (M-RIP) by PKG and stimulation of MLC phosphatase activity.
abstract: Previous studies have shown that cGMP-dependent protein kinase (PKG) act on several targets in the contractile pathway to reduce intracellular Ca(2+) and/or augment RhoA-regulated myosin light chain phosphatase (MLCP) activity and cause muscle relaxation. Recent studies have identified a novel protein M-RIP that associates with MYPT1, the regulatory subunit of MLCP. Herein, we examine whether PKG enhance MLCP activity downstream of Ca(2+) and RhoA via phosphorylation of M-RIP in gastric smooth muscle cells. Treatment of permeabilized muscle cells with 10 μM Ca(2+) caused an increase in MLC20 phosphorylation and muscle contraction, but had no effect on Rho kinase activity. Activators of PKG (GSNO or cGMP) decreased MLC20 phosphorylation and contraction in response to 10 μM Ca(2+), implying existence of inhibitory mechanism independent of Ca(2+) and RhoA. The effect of PKG on Ca(2+)-induced MLC20 phosphorylation was attenuated by M-RIP siRNA. Both GSNO and 8-pCPT-cGMP induced phosphorylation of M-RIP; phosphorylation was accompanied by an increase in the association of M-RIP with MYPT1 and MLCP activity. Taken together, these results provide evidence that PKG induces phosphorylation of M-RIP and enhances its association with MYPT1 to augment MLCP activity and MLC20 dephosphorylation and inhibits muscle contraction, downstream of Ca(2+)- or RhoA-dependent pathways.
authors.affiliation: Department of Physiology and Biophysics, Virginia Commonwealth University, Richmond, VA, 23298-0711, USA.
authors.forename: Sunila
authors.initials: S
authors.lastname: Mahavadi
authors.forename: Ancydimpy
authors.initials: A
authors.lastname: Nalli
authors.forename: Othman
authors.initials: O
authors.lastname: Al-Shboul
authors.forename: Karnam S
authors.initials: KS
authors.lastname: Murthy
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000577:Amides / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000494:pharmacology; D017869:Cyclic GMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D009119:Muscle Contraction / Q000187:drug effects; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D043263:Myosin-Light-Chain Phosphatase / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011725:Pyridines / Q000494:pharmacology; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D011817:Rabbits; D026422:S-Nitrosoglutathione / Q000494:pharmacology; D013270:Stomach / Q000166:cytology; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000577:Amides; D011725:Pyridines; D034741:RNA, Small Interfering; C108830:Y 27632; D026422:S-Nitrosoglutathione; D054460:rho-Associated Kinases; D017869:Cyclic GMP-Dependent Protein Kinases; D043263:Myosin-Light-Chain Phosphatase; D020742:rhoA GTP-Binding Protein; D006152:Cyclic GMP; D002118:Calcium