Proteomic identification of 14-3-3zeta as a mitogen-activated protein kinase-activated protein kinase 2 substrate: role in dimer formation and ligand binding.
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, 14-3-3zeta. We confirmed that MAPKAPK2 interacted with and phosphorylated 14-3-3zeta 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 14-3-3zeta 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 14-3-3zeta to dimerize. Experimentally, S58D mutation significantly impaired both 14-3-3zeta dimerization and binding to Raf-1. These data suggest that MAPKAPK2-mediated phosphorylation regulates 14-3-3zeta functions, and this MAPKAPK2 activity may represent a novel pathway mediating p38 MAPK-dependent inflammation.
Mesh Terms:
14-3-3 Proteins, Amino Acid Sequence, Cell Line, DNA Mutational Analysis, Dimerization, Electrophoresis, Polyacrylamide Gel, Genetic Vectors, Glutathione Transferase, Humans, Intracellular Signaling Peptides and Proteins, Ligands, MAP Kinase Signaling System, Mitogen-Activated Protein Kinases, Models, Molecular, Molecular Sequence Data, Mutation, N-Formylmethionine Leucyl-Phenylalanine, Neutrophils, Phosphorylation, Precipitin Tests, Protein Binding, Protein-Serine-Threonine Kinases, Proteome, Proto-Oncogene Proteins c-raf, Recombinant Proteins, Signal Transduction, Software, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Substrate Specificity, Trypsin, Tyrosine 3-Monooxygenase, p38 Mitogen-Activated Protein Kinases
14-3-3 Proteins, Amino Acid Sequence, Cell Line, DNA Mutational Analysis, Dimerization, Electrophoresis, Polyacrylamide Gel, Genetic Vectors, Glutathione Transferase, Humans, Intracellular Signaling Peptides and Proteins, Ligands, MAP Kinase Signaling System, Mitogen-Activated Protein Kinases, Models, Molecular, Molecular Sequence Data, Mutation, N-Formylmethionine Leucyl-Phenylalanine, Neutrophils, Phosphorylation, Precipitin Tests, Protein Binding, Protein-Serine-Threonine Kinases, Proteome, Proto-Oncogene Proteins c-raf, Recombinant Proteins, Signal Transduction, Software, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Substrate Specificity, Trypsin, Tyrosine 3-Monooxygenase, p38 Mitogen-Activated Protein Kinases
Mol. Cell. Biol.
Date: Aug. 01, 2003
PubMed ID: 12861023
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