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Protein kinase A represses skeletal myogenesis by targeting myocyte enhancer factor 2D

Min Du1, Robert L S Perry, Nathaniel B Nowacki

  • 1Department of Biology, York University, 4700 Keele Street, Toronto M3J 1P3, Ontario, Canada.

Molecular and Cellular Biology
|February 27, 2008
PubMed

Insights

Protein kinase A (PKA) activation inhibits skeletal muscle development by directly phosphorylating myocyte enhancer factor 2D (MEF2D). This phosphorylation represses MEF2D activity, hindering muscle differentiation.

Area of Science:

  • Molecular Biology
  • Cellular Biology
  • Biochemistry

Background:

  • Skeletal myogenesis is inhibited by protein kinase A (PKA) activation.
  • Myogenic regulatory factors (MRFs) are indirectly modulated during PKA-mediated repression.
  • Myocyte enhancer factor 2 (MEF2) proteins are crucial for myogenesis and partner with MRFs.

Purpose of the Study:

  • To investigate the role of MEF2 proteins in PKA-mediated inhibition of skeletal myogenesis.
  • To determine if PKA directly phosphorylates MEF2 proteins.

Main Methods:

  • In silico analysis to identify PKA phosphoacceptor sites on MEF2.
  • In vitro kinase assays to assess PKA phosphorylation of MEF2D.
  • Mass spectrometry to identify specific phosphorylation sites on MEF2D.
  • Transcriptional reporter gene assays to evaluate MEF2D function.
  • Site-directed mutagenesis to create PKA-resistant MEF2D.
  • Analysis of histone deacetylase 4 (HDAC4) nuclear accumulation.

Main Results:

  • PKA directly and efficiently phosphorylates MEF2D at serine 121 and serine 190.
  • PKA activation represses MEF2D's transactivation activity.
  • Mutating MEF2D phosphorylation sites (S121A, S190A) confers resistance to PKA-mediated repression.
  • PKA activation increases nuclear accumulation of HDAC4, enhancing the MEF2D-HDAC4 repressor complex.

Conclusions:

  • MEF2D is a direct target of PKA signaling in myoblasts.
  • PKA-mediated phosphorylation of MEF2D inhibits skeletal muscle differentiation.
  • HDAC4 involvement in the MEF2D repressor complex is regulated by PKA signaling.

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