1. Academic Validation
  2. Post-translational regulation of mitogen-activated protein kinase phosphatase (MKP)-1 and MKP-2 in macrophages following lipopolysaccharide stimulation: the role of the C termini of the phosphatases in determining their stability

Post-translational regulation of mitogen-activated protein kinase phosphatase (MKP)-1 and MKP-2 in macrophages following lipopolysaccharide stimulation: the role of the C termini of the phosphatases in determining their stability

  • J Biol Chem. 2014 Oct 17;289(42):28753-64. doi: 10.1074/jbc.M114.591925.
Sara Crowell 1 Lyn M Wancket 2 Yasmine Shakibi 1 Pingping Xu 1 Jianjing Xue 1 Lobelia Samavati 3 Leif D Nelin 4 Yusen Liu 5
Affiliations

Affiliations

  • 1 From the Center for Perinatal Research, The Research Institute at Nationwide Children's Hospital, Columbus, Ohio 43205.
  • 2 Department of Veterinary Bioscience, The Ohio State University College of Veterinary Medicine, Columbus, Ohio 43210, and.
  • 3 Department of Medicine, Division of Pulmonary, Critical Care and Sleep Medicine, Wayne State University School of Medicine and Detroit Medical Center, Detroit, Michigan 48201.
  • 4 From the Center for Perinatal Research, The Research Institute at Nationwide Children's Hospital, Columbus, Ohio 43205, Department of Pediatrics, The Ohio State University College of Medicine, Columbus, Ohio 43205.
  • 5 From the Center for Perinatal Research, The Research Institute at Nationwide Children's Hospital, Columbus, Ohio 43205, Department of Veterinary Bioscience, The Ohio State University College of Veterinary Medicine, Columbus, Ohio 43210, and Department of Pediatrics, The Ohio State University College of Medicine, Columbus, Ohio 43205, [email protected].
Abstract

MAPK phosphatases (MKPs) are critical modulators of the innate immune response, and yet the mechanisms regulating their accumulation remain poorly understood. In the present studies, we investigated the role of post-translational modification in the accumulation of MKP-1 and MKP-2 in macrophages following LPS stimulation. We found that upon LPS stimulation, MKP-1 and MKP-2 accumulated with different kinetics: MKP-1 level peaked at ∼1 h, while MKP-2 levels continued to rise for at least 6 h. Accumulation of both MKP-1 and MKP-2 were attenuated by inhibition of the ERK cascade. Interestingly, p38 inhibition prior to LPS stimulation had little effect on MKP-1 and MKP-2 protein levels, but hindered their detection by an M-18 MKP-1 antibody. Studies of the epitope sequence recognized by the M-18 MKP-1 antibody revealed extensive phosphorylation of two serine residues in the C terminus of both MKP-1 and MKP-2 by the ERK pathway. Remarkably, the stability of both MKP-1 and MKP-2 was markedly decreased in macrophages in the presence of an ERK pathway inhibitor. Mutation of the two C-terminal serine residues in MKP-1 and MKP-2 to alanine decreased their half-lives, while mutating these residues to aspartate dramatically increased their half-lives. Deletion of the C terminus from MKP-1 and MKP-2 also considerably increased their stabilities. Surprisingly, enhanced stabilities of the MKP-1 and MKP-2 mutants were not associated with decreased ubiquitination. Degradation of both MKP-1 and MKP-2 was attenuated by proteasomal inhibitors. Our studies suggest that MKP-1 and MKP-2 stability is regulated by ERK-mediated phosphorylation through a degradation pathway independent of polyubiquitination.

Keywords

Lipopolysaccharide (LPS); MAP Kinase Phosphatase; Macrophage; Phosphorylation; Post-translational Modification (PTM); Protein Stability; Ubiquitylation (Ubiquitination).

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