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.
Singh
Saurabh
S
Department of Biochemistry and Molecular Biology, Medicine, and Pharmacology, University of Louisville, KY 40202, USA.
Powell
David W
DW
Rane
Madhavi J
MJ
Millard
Tom H
TH
Trent
John O
JO
Pierce
William M
WM
Klein
Jon B
JB
Machesky
Laura M
LM
McLeish
Kenneth R
KR
D051377:Actin-Related Protein 2; D051378:Actin-Related Protein 3; D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D003598:Cytoskeletal Proteins / Q000737:chemistry*; D003599:Cytoskeleton / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D013058:Mass Spectrometry; D008840:Microfilament Proteins / Q000737:chemistry* / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009504:Neutrophils / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D020033:Protein Isoforms; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012694:Serine / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D014357:Trypsin / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases; D000099190:MAP-Kinase-Activated Kinase 2
D051377:Actin-Related Protein 2; D051378:Actin-Related Protein 3; D000199:Actins; D003598:Cytoskeletal Proteins; D005982:Glutathione Transferase; D047908:Intracellular Signaling Peptides and Proteins; D008840:Microfilament Proteins; D020928:Mitogen-Activated Protein Kinases; D020033:Protein Isoforms; D017346:Protein Serine-Threonine Kinases; D012694:Serine; D014357:Trypsin; D048051:p38 Mitogen-Activated Protein Kinases; D000099190:MAP-Kinase-Activated Kinase 2; C495926:ACTR2 protein, human; C495928:ACTR3 protein, human