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

Fingerprint

Mitogen-Activated Protein Kinases
Proteomics
Protein Kinases
Ligands
p38 Mitogen-Activated Protein Kinases
Phosphorylation
14-3-3 Proteins
Mutation
Neutrophils
methionyl-leucyl-phenylalanine
Protein Databases
Peptide Mapping
HEK293 Cells
Phosphoproteins
Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
Chemotactic Factors
Dimerization
Sodium Dodecyl Sulfate
Polyacrylamide Gel Electrophoresis
Signal Transduction

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Cell Biology

Cite this

Proteomic identification of 14-3-3ζ as a mitogen-activated protein kinase-activated protein kinase 2 substrate : Role in dimer formation and ligand binding. / Powell, David W.; Rane, Madhavi J.; Joughin, Brian A.; Kalmukova, Ralitsa; Hong, Jeong-Ho; Tidor, Bruce; Dean, William L.; Pierce, William M.; Klein, Jon B.; Yaffe, Michael B.; McLeish, Kenneth R.

In: Molecular and Cellular Biology, Vol. 23, No. 15, 01.08.2003, p. 5376-5387.

Research output: Contribution to journalArticle

Powell, David W. ; Rane, Madhavi J. ; Joughin, Brian A. ; Kalmukova, Ralitsa ; Hong, Jeong-Ho ; Tidor, Bruce ; Dean, William L. ; Pierce, William M. ; Klein, Jon B. ; Yaffe, Michael B. ; McLeish, Kenneth R. / Proteomic identification of 14-3-3ζ as a mitogen-activated protein kinase-activated protein kinase 2 substrate : Role in dimer formation and ligand binding. In: Molecular and Cellular Biology. 2003 ; Vol. 23, No. 15. pp. 5376-5387.
@article{bafc752846bb4a1b96ea4fff0642d4c2,
title = "Proteomic identification of 14-3-3ζ as a mitogen-activated protein kinase-activated protein kinase 2 substrate: Role in dimer formation and ligand binding",
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.",
author = "Powell, {David W.} and Rane, {Madhavi J.} and Joughin, {Brian A.} and Ralitsa Kalmukova and Jeong-Ho Hong and Bruce Tidor and Dean, {William L.} and Pierce, {William M.} and Klein, {Jon B.} and Yaffe, {Michael B.} and McLeish, {Kenneth R.}",
year = "2003",
month = "8",
day = "1",
doi = "10.1128/MCB.23.15.5376-5387.2003",
language = "English",
volume = "23",
pages = "5376--5387",
journal = "Molecular and Cellular Biology",
issn = "0270-7306",
publisher = "American Society for Microbiology",
number = "15",

}

TY - JOUR

T1 - Proteomic identification of 14-3-3ζ as a mitogen-activated protein kinase-activated protein kinase 2 substrate

T2 - Role in dimer formation and ligand binding

AU - Powell, David W.

AU - Rane, Madhavi J.

AU - Joughin, Brian A.

AU - Kalmukova, Ralitsa

AU - Hong, Jeong-Ho

AU - Tidor, Bruce

AU - Dean, William L.

AU - Pierce, William M.

AU - Klein, Jon B.

AU - Yaffe, Michael B.

AU - McLeish, Kenneth R.

PY - 2003/8/1

Y1 - 2003/8/1

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0043092653&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0043092653&partnerID=8YFLogxK

U2 - 10.1128/MCB.23.15.5376-5387.2003

DO - 10.1128/MCB.23.15.5376-5387.2003

M3 - Article

VL - 23

SP - 5376

EP - 5387

JO - Molecular and Cellular Biology

JF - Molecular and Cellular Biology

SN - 0270-7306

IS - 15

ER -