Identification of the p16-Arc Subunit of the Arp 2/3 Complex as a Substrate of MAPK-activated Protein Kinase 2 by Proteomic Analysis

Abstract

The p38 MAPK pathway regulates multiple neutrophil functional responses via activation of the serine-threonine kinase MAPK-activated protein kinase 2 (MAP-KAPK2). 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.

Recent advances in blood‐related proteomics

“…The conclusion was that lipopolysaccharides induce an up-regulation of inflammatory mediators and signaling molecules, as well as the remodeling of the cytoskeleton, which may explain the release of secretory granules and the migration of neutrophils towards the site of infection [138]. The central pathway in the regulation of neutrophil function is the p38 mitogen-activated protein kinase signal transduction as shown by Singh et al [139]. Activation of neutrophil phorbol 12-myristate, tumor necrosis factor a or interferon g has been shown to induce tyrosylation of a number of endogenous proteins such as lactoferrin, catalase, vimentin, filamin A, myeloperoxidase, ATP synthetase b, annexin 1, cytokeratin 10, or glyceraldehyde 3-phosphate dehydrogenase after 2-DE and MS [140].…”

Myeloid-Related Protein-14 Is a p38 MAPK Substrate in Human Neutrophils

“…Neutrophil lysate was prepared as previously described (22,23). In brief, 10 8 cells were lysed in 500 l of lysis buffer containing 2 M thiourea, 7 M urea, 65 mM CHAPS, 58 mM DTT, and 4.5% ampholytes (pH 3-10).…”

“…Lysates were cleared by centrifugation at 12,000 ϫ g for 20 min at 15°C. Before addition of exogenous p38 MAPK, lysate urea concentration was reduced to 1 M by size exclusion chromatography as previously described (22,23).…”

“…Neutrophil lysates (400 g of total protein) were incubated with 10 Ci of [␥-32 P]ATP (ICN Biomedicals) in the presence and absence of 400 ng of active recombinant p38 MAPK␣ (Upstate Biotechnology) at 30°C for 1 h. Kinase reactions were stopped by adding rehydration buffer (8 M urea, 2% CHAPS, 0.01 M DTT, 2% ampholytes pH 3-10, and 0.01% bromphenol blue) to give a total volume of 450 l. Proteins were separated by isoelectric focusing with 18-cm, pH 3-10 immobilized pH gradient (IPG) strips, followed by size separation with 10% Duracyl (Genomic Solutions), as previously described (23). Proteins were stained with SYPRO Ruby fluorescent stain (Molecular Probes), and phosphorylation was visualized by autoradiography.…”

“…The only p38 MAPK substrates that have been identified in human neutrophils, however, are MAP-KAPK-2 and p47 phox (1,9,10,21). The goal of the present study was to identify p38 MAPK substrates in human neutrophils using a functional proteomic approach developed in our laboratory that combines in vitro phosphorylation of neutrophil lysate by recombinant kinase, separation of proteins by two-dimensional gel electrophoresis, and phosphoprotein identification by MALDI-TOF mass spectrometry (MALDI-TOF MS) (22,23). One of the proteins identified was myeloid-related protein-14 (MRP-14), a member of the S100 protein family of calcium binding proteins (24,25).…”