Proteomic identification of 14-3-3ζ as a mitogen-activated protein kinase-activated protein kinase 2 substrate: Role in dimer formation and ligand binding

David W. Powell, Madhavi J. Rane, Brian A. Joughin, Ralitsa Kalmukova, Jeong Ho Hong, Bruce Tidor, William L. Dean, William M. Pierce, Jon B. Klein, Michael B. Yaffe, Kenneth R. McLeish

Research output: Contribution to journalArticlepeer-review

110 Citations (Scopus)

Abstract

Mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MAPKAPK2) mediates multiple p38 MAPK-dependent inflammatory responses. To define the signal transduction pathways activated by MAPKAPK2, we identified potential MAPKAPK2 substrates by using a functional proteomic approach consisting of in vitro phosphorylation of neutrophil lysate by active recombinant MAPKAPK2, protein separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and phosphoprotein identification by peptide mass fingerprinting with matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and protein database analysis. One of the eight candidate MAPKAPK2 substrates identified was the adaptor protein, 14-3-3ζ. We confirmed that MAPKAPK2 interacted with and phosphorylated 14-3-3ζ in vitro and in HEK293 cells. The chemoattractant formyl-methionyl-leucyl-phenylalanine (fMLP) stimulated p38-MAPK-dependent phosphorylation of 14-3-3 proteins in human neutrophils. Mutation analysis showed that MAPKAPK2 phosphorylated 14-3-3ζ at Ser-58. Computational modeling and calculation of theoretical binding energies predicted that both phosphorylation at Ser-58 and mutation of Ser-58 to Asp (S58D) compromised the ability of 14-3-3ζ to dimerize. Experimentally, S58D mutation significantly impaired both 14-3-3ζ dimerization and binding to Raf-1. These data suggest that MAPKAPK2-mediated phosphorylation regulates 14-3-3ζ functions, and this MAPKAPK2 activity may represent a novel pathway mediating p38 MAPK-dependent inflammation.

Original languageEnglish
Pages (from-to)5376-5387
Number of pages12
JournalMolecular and cellular biology
Volume23
Issue number15
DOIs
Publication statusPublished - 2003 Aug
Externally publishedYes

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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