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Cell Biochemistry and Biophysics

, Volume 68, Issue 1, pp 1–8 | Cite as

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

  • Sunila Mahavadi
  • Ancydimpy Nalli
  • Othman Al-Shboul
  • Karnam S. Murthy
Original Paper

Abstract

Previous studies have shown that cGMP-dependent protein kinase (PKG) act on several targets in the contractile pathway to reduce intracellular Ca2+ 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 Ca2+ and RhoA via phosphorylation of M-RIP in gastric smooth muscle cells. Treatment of permeabilized muscle cells with 10 μM Ca2+ 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 Ca2+, implying existence of inhibitory mechanism independent of Ca2+ and RhoA. The effect of PKG on Ca2+-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 Ca2+- or RhoA-dependent pathways.

Keywords

Smooth muscle  Relaxation  cGMP-dependent protein kinase  cGMP 

Notes

Acknowledgments

This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant to KSM (DK28300).

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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Sunila Mahavadi
    • 1
  • Ancydimpy Nalli
    • 1
  • Othman Al-Shboul
    • 1
  • Karnam S. Murthy
    • 1
  1. 1.Department of Physiology and BiophysicsVirginia Commonwealth UniversityRichmondUSA

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