Identification of the p16-Arc subunit of the Arp 2/3 complex as a substrate of MAPK-activated protein kinase 2 by proteomic analysis.
The p38 MAPK pathway regulates multiple neutrophil functional responses via activation of the serine-threonine kinase MAPK-activated protein kinase 2 (MAPKAPK2). To identify substrates of MAPKAPK2 that mediate these responses, a proteomic approach was used in which in vitro phosphorylation of neutrophil lysates by exogenously added active recombinant MAPKAPK2 was followed ... by protein separation using two-dimensional electrophoresis. Peptide mass fingerprinting of peptides defined by MALDI-MS was then utilized to identify phosphorylated proteins detected by autoradiography. Six candidate substrates were identified, including the p16 subunit of the seven-member Arp2/3 complex (p16-Arc). In vitro studies confirmed that MAPKAPK2 interacts with and phosphorylates the A isoform, but not the B isoform, of p16-Arc with a stoichiometry of 0.6 to 0.7. MAPKAPK2 also phosphorylated p16-Arc in intact Arp2/3 complexes precipitated from neutrophil lysates. Mutation of serine-77 to alanine on the A isoform prevented phosphorylation by MAPKAPK2. The ability of MAPKAPK2 to phosphorylate one isoform of p16-Arc suggests a possible mechanism by which the p38 MAPK cascade regulates remodeling of the actin cytoskeleton.
Mesh Terms:
Actin-Related Protein 2, Actin-Related Protein 3, Actins, Amino Acid Sequence, Cytoskeletal Proteins, Cytoskeleton, Electrophoresis, Gel, Two-Dimensional, Glutathione Transferase, Humans, Intracellular Signaling Peptides and Proteins, Mass Spectrometry, Microfilament Proteins, Mitogen-Activated Protein Kinases, Models, Molecular, Molecular Sequence Data, Mutation, Neutrophils, Phosphorylation, Protein Binding, Protein Conformation, Protein Isoforms, Protein Structure, Tertiary, Protein-Serine-Threonine Kinases, Serine, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Substrate Specificity, Trypsin, p38 Mitogen-Activated Protein Kinases
Actin-Related Protein 2, Actin-Related Protein 3, Actins, Amino Acid Sequence, Cytoskeletal Proteins, Cytoskeleton, Electrophoresis, Gel, Two-Dimensional, Glutathione Transferase, Humans, Intracellular Signaling Peptides and Proteins, Mass Spectrometry, Microfilament Proteins, Mitogen-Activated Protein Kinases, Models, Molecular, Molecular Sequence Data, Mutation, Neutrophils, Phosphorylation, Protein Binding, Protein Conformation, Protein Isoforms, Protein Structure, Tertiary, Protein-Serine-Threonine Kinases, Serine, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Substrate Specificity, Trypsin, p38 Mitogen-Activated Protein Kinases
J. Biol. Chem.
Date: Sep. 19, 2003
PubMed ID: 12829704
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