A Phosphoproteomic Screen Identifies a Guanine Nucleotide Exchange Factor for Rab3A Protein as a Mitogen-activated Protein (MAP) Kinase Phosphatase-5-regulated MAP Kinase Target in Interleukin 6 (IL-6) Secretion and Myogenesis

J Biol Chem. 2017 Mar 3;292(9):3581-3590. doi: 10.1074/jbc.M116.769208. Epub 2017 Jan 17.

Abstract

The mitogen-activated protein kinases (MAPKs) have been shown to regulate skeletal muscle function. Previously, we showed that MAPK phosphatase-5 (MKP-5) negatively regulates myogenesis and regeneration of skeletal muscle through inhibition of p38 MAPK and c-Jun N-terminal kinase (JNK). However, the identity and contribution of MKP-5-regulated MAPK targets in the control of skeletal muscle function and regenerative myogenesis have not been established. To identify MKP-5-regulated MAPK substrates in skeletal muscle, we performed a global differential phospho-MAPK substrate screen in regenerating skeletal muscles of wild type and MKP-5-deficient mice. We discovered a novel MKP-5-regulated MAPK substrate called guanine nucleotide exchange factor for Rab3A (GRAB) that was hyperphosphorylated on a phospho-MAPK motif in skeletal muscle of MKP-5-deficient mice. GRAB was found to be phosphorylated by JNK on serine 169. Myoblasts overexpressing a phosphorylation-defective mutant of GRAB containing a mutation at Ser-169 to Ala-169 (GRAB-S169A) inhibited the ability of C2C12 myoblasts to differentiate. We found that GRAB phosphorylation at Ser-169 was required for the secretion of the promyogenic cytokine interleukin 6 (IL-6). Consistent with this observation, MKP-5-deficient mice exhibited increased circulating IL-6 expression as compared with wild type mice. Collectively, these data demonstrate a novel mechanism whereby MKP-5-mediated regulation of JNK negatively regulates phosphorylation of GRAB, which subsequently controls secretion of IL-6. These data support the notion that MKP-5 serves as a negative regulator of MAPK-dependent signaling of critical skeletal muscle signaling pathways.

Keywords: cytokine; dual specificity phosphoprotein phosphatase; mitogen-activated protein kinase (MAPK); myogenesis; phosphoproteomics; skeletal muscle.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Motifs
  • Animals
  • Cell Movement
  • Cell Proliferation
  • Dual-Specificity Phosphatases / metabolism*
  • Gene Expression Regulation, Enzymologic*
  • Guanine Nucleotide Exchange Factors / metabolism*
  • Interleukin-6 / metabolism*
  • MAP Kinase Signaling System
  • Mice
  • Mice, Knockout
  • Muscle Development*
  • Muscle, Skeletal / metabolism
  • Mutation
  • Myoblasts / metabolism
  • Phosphorylation
  • Proteomics
  • Regeneration
  • Serine / chemistry
  • rab3A GTP-Binding Protein / metabolism*

Substances

  • GRAB protein, mouse
  • Guanine Nucleotide Exchange Factors
  • Interleukin-6
  • interleukin-6, mouse
  • Serine
  • Dusp10 protein, mouse
  • Dual-Specificity Phosphatases
  • rab3A GTP-Binding Protein