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Molecular and Cellular Biology, August 2003, p. 5376-5387, Vol. 23, No. 15
0270-7306/03/$08.00+0     DOI: 10.1128/MCB.23.15.5376-5387.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Proteomic Identification of 14-3-3 as a Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 Substrate: Role in Dimer Formation and Ligand Binding

Departments of Biochemistry and Molecular Biology,¹ Medicine,² Pharmacology, University of Louisville,⁶ The Veterans Affairs Medical Center, Louisville, Kentucky 40202,⁷ Center for Cancer Research, Department of Biology,³ Division of Biological Engineering,⁴ Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139-4307⁵

Received 12 November 2002/ Returned for modification 12 March 2003/ Accepted 14 May 2003

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, 14-3-3. We confirmed that MAPKAPK2 interacted with and phosphorylated 14-3-3 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 14-3-3 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 14-3-3 to dimerize. Experimentally, S58D mutation significantly impaired both 14-3-3 dimerization and binding to Raf-1. These data suggest that MAPKAPK2-mediated phosphorylation regulates 14-3-3 functions, and this MAPKAPK2 activity may represent a novel pathway mediating p38 MAPK-dependent inflammation.

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