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.
Powell
David W
DW
Departments of Biochemistry and Molecular Biology, University of Louisville, Louisville, KY 40202, USA.
Rane
Madhavi J
MJ
Joughin
Brian A
BA
Kalmukova
Ralitsa
R
Hong
Jeong-Ho
JH
Tidor
Bruce
B
Dean
William L
WL
Pierce
William M
WM
Klein
Jon B
JB
Yaffe
Michael B
MB
McLeish
Kenneth R
KR
D048948:14-3-3 Proteins; D000595:Amino Acid Sequence; D002460:Cell Line; D004252:DNA Mutational Analysis; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D020935:MAP Kinase Signaling System; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000201:enzymology / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D020543:Proteome; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D012984:Software; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D014357:Trypsin / Q000494:pharmacology; D014446:Tyrosine 3-Monooxygenase / Q000737:chemistry / Q000502:physiology*; D048051:p38 Mitogen-Activated Protein Kinases; D000099190:MAP-Kinase-Activated Kinase 2
D048948:14-3-3 Proteins; D005982:Glutathione Transferase; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D020928:Mitogen-Activated Protein Kinases; D009240:N-Formylmethionine Leucyl-Phenylalanine; D017346:Protein Serine-Threonine Kinases; D020543:Proteome; D019908:Proto-Oncogene Proteins c-raf; D011994:Recombinant Proteins; D014357:Trypsin; D014446:Tyrosine 3-Monooxygenase; D048051:p38 Mitogen-Activated Protein Kinases; D000099190:MAP-Kinase-Activated Kinase 2