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 journalArticle

109 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 1
Externally publishedYes

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Cell Biology

Fingerprint Dive into the research topics of 'Proteomic identification of 14-3-3ζ as a mitogen-activated protein kinase-activated protein kinase 2 substrate: Role in dimer formation and ligand binding'. Together they form a unique fingerprint.

  • Cite this

    Powell, D. W., Rane, M. J., Joughin, B. A., Kalmukova, R., Hong, J-H., Tidor, B., Dean, W. L., Pierce, W. M., Klein, J. B., Yaffe, M. B., & McLeish, K. R. (2003). Proteomic identification of 14-3-3ζ as a mitogen-activated protein kinase-activated protein kinase 2 substrate: Role in dimer formation and ligand binding. Molecular and Cellular Biology, 23(15), 5376-5387. https://doi.org/10.1128/MCB.23.15.5376-5387.2003