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PHI-1 induced enhancement of myosin phosphorylation in chicken smooth muscle

Amr El-Touhky1, Allison M Given, Audrey Cochard

  • 1Department of Physiology and Biophysics, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106, USA.

FEBS Letters
|August 6, 2005
PubMed

Insights

G-protein stimulation in chicken smooth muscle increases myosin light chain phosphorylation via the Rho-kinase pathway. PHI-1, a CPI-17 analog, is phosphorylated and may inhibit myosin phosphatase, causing calcium sensitization.

Area of Science:

  • Biochemistry
  • Cellular Biology
  • Muscle Physiology

Background:

  • G-protein signaling pathways regulate smooth muscle contraction.
  • Rho-kinase (ROCK) and protein kinase C (PKC) are key regulators of smooth muscle function.
  • CPI-17 is a known inhibitor of myosin phosphatase in mammalian smooth muscle.

Purpose of the Study:

  • To investigate the role of G-protein stimulation and Rho-kinase in chicken smooth muscle.
  • To identify potential analogs of CPI-17 in chicken smooth muscle.
  • To elucidate the mechanism of calcium sensitization in chicken smooth muscle.

Main Methods:

  • Stimulation of chicken smooth muscle with agonists and GTP(gamma)S.
  • Western blot analysis to detect protein phosphorylation.
  • Inhibition studies using Rho-kinase (Y-27632) and PKC (GF109203x) inhibitors.

Main Results:

  • G-protein stimulation increased myosin light chain phosphorylation in chicken smooth muscle.
  • MYPT1 phosphorylation at Thr695 or Thr850 was not affected by G-protein stimulation.
  • PHI-1, but not CPI-17, was expressed in chicken smooth muscle and its phosphorylation increased upon stimulation.
  • PHI-1 phosphorylation was inhibited by Y-27632 and GF109203x, suggesting involvement of Rho-kinase and PKC.
  • PHI-1 phosphorylation increased with agonist and GTP(gamma)S stimulation.

Conclusions:

  • PHI-1 acts as a functional analog of CPI-17 in chicken smooth muscle.
  • G-protein stimulation leads to PHI-1 phosphorylation, inhibiting myosin phosphatase activity.
  • This mechanism contributes to calcium sensitization in chicken smooth muscle contraction.

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