Abstract
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.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, Non-P.H.S.
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Actin-Related Protein 2
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Actin-Related Protein 3
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Actins / metabolism
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Amino Acid Sequence
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Cytoskeletal Proteins / chemistry*
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Cytoskeleton / metabolism
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Electrophoresis, Gel, Two-Dimensional
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Glutathione Transferase / metabolism
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Humans
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Intracellular Signaling Peptides and Proteins
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Mass Spectrometry
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Microfilament Proteins / chemistry*
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Microfilament Proteins / metabolism*
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Mitogen-Activated Protein Kinases / metabolism
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Models, Molecular
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Molecular Sequence Data
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Mutation
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Neutrophils / chemistry
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Neutrophils / enzymology*
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Neutrophils / metabolism
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Phosphorylation
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Protein Binding
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Protein Conformation
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Protein Isoforms
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Protein Serine-Threonine Kinases / chemistry*
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Protein Serine-Threonine Kinases / metabolism
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Protein Structure, Tertiary
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Serine / chemistry
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Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
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Substrate Specificity
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Trypsin / pharmacology
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p38 Mitogen-Activated Protein Kinases
Substances
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ACTR2 protein, human
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ACTR3 protein, human
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Actin-Related Protein 2
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Actin-Related Protein 3
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Actins
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Cytoskeletal Proteins
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Intracellular Signaling Peptides and Proteins
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Microfilament Proteins
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Protein Isoforms
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Serine
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Glutathione Transferase
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MAP-kinase-activated kinase 2
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Protein Serine-Threonine Kinases
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Mitogen-Activated Protein Kinases
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p38 Mitogen-Activated Protein Kinases
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Trypsin