Par-4: a new activator of myosin phosphatase

Mol Biol Cell. 2010 Apr 1;21(7):1214-24. doi: 10.1091/mbc.e09-08-0711. Epub 2010 Feb 3.

Abstract

Myosin phosphatase (MP) is a key regulator of myosin light chain (LC20) phosphorylation, a process essential for motility, apoptosis, and smooth muscle contractility. Although MP inhibition is well studied, little is known about MP activation. We have recently demonstrated that prostate apoptosis response (Par)-4 modulates vascular smooth muscle contractility. Here, we test the hypothesis that Par-4 regulates MP activity directly. We show, by proximity ligation assays, surface plasmon resonance and coimmunoprecipitation, that Par-4 interacts with the targeting subunit of MP, MYPT1. Binding is mediated by the leucine zippers of MYPT1 and Par-4 and reduced by Par-4 phosphorylation. Overexpression of Par-4 leads to increased phosphatase activity of immunoprecipitated MP, whereas small interfering RNA knockdown of endogenous Par-4 significantly decreases MP activity and increases MYPT1 phosphorylation. LC20 phosphorylation assays demonstrate that overexpression of Par-4 reduces LC20 phosphorylation. In contrast, a phosphorylation site mutant, but not wild-type Par-4, interferes with zipper-interacting protein kinase (ZIPK)-mediated MP inhibition. We conclude from our results Par-4 operates through a "padlock" model in which binding of Par-4 to MYPT1 activates MP by blocking access to the inhibitory phosphorylation sites, and inhibitory phosphorylation of MYPT1 by ZIPK requires "unlocking" of Par-4 by phosphorylation and displacement of Par-4 from the MP complex.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Aorta / metabolism
  • Apoptosis
  • Apoptosis Regulatory Proteins / metabolism
  • Apoptosis Regulatory Proteins / physiology*
  • Cell Proliferation
  • DNA / metabolism
  • Electrophoresis, Gel, Two-Dimensional
  • Enzyme Activation
  • Microscopy, Fluorescence / methods
  • Muscle, Smooth, Vascular / metabolism
  • Myosin-Light-Chain Phosphatase / metabolism*
  • Phosphorylation
  • RNA / chemistry
  • Rats

Substances

  • Apoptosis Regulatory Proteins
  • prostate apoptosis response-4 protein
  • RNA
  • DNA
  • Myosin-Light-Chain Phosphatase