title: Activation of MAP kinase-activated protein kinase 2 in human neutrophils after phorbol ester or fMLP peptide stimulation.
abstract: In response to extracellular stimulation, one of the earliest events in human neutrophils is protein phosphorylation, which mediates signal transduction and leads to the regulation of cellular functions. Mitogen-activated protein (MAP) kinases are rapidly activated by a variety of mitogens, cytokines, and stresses. The activated MAP kinases in turn regulate their substrate molecules by phosphorylation. MAP kinase-activated protein (MAPKAP) kinase 2, a Ser/Thr kinase, has been shown to be phosphorylated by p38 MAP kinase both in vivo and in vitro. Phosphorylation of the Thr-334 site of MAPKAP kinase 2 results in a conformational change with subsequent activation of the enzyme. To better define the role of MAPKAP kinase 2 in the activation of human neutrophils, its enzymatic activity was measured after stimulation by either a phorbol ester (phorbol myristate acetate [PMA]), a potent protein kinase C activator, or the tripeptide fMLP, which is a chemotactic factor. The in vitro kinase assays indicate that both PMA and fMLP stimulated a transient increase in the enzymatic activity of cellular MAPKAP kinase 2. The induced kinase activation was concentration-dependent and reached a maximum at 5 minutes for PMA and 1 minute for fMLP. To identify potential substrate molecules for MAPKAP kinase 2, a highly active kinase mutant was generated by mutating the MAP kinase phosphorylation site in the C-terminal region. The replacement of threonine 334 with alanine resulted in a marked augmentation of catalytic activity. Analysis of in vitro protein phosphorylation in the presence of the active kinase indicates that a 60-kD cytosolic protein (p60) was markedly phosphorylated and served as the major substrate for MAPKAP kinase 2 in human neutrophils. Based on the MAPKAP kinase 2 phosphorylation site of Hsp27, a competitive inhibitory peptide was synthesized. This competitive inhibitory peptide specifically inhibited MAPKAP kinase 2 enzymatic activity, as well as the in vitro and in vivo kinase-induced p60 phosphorylation. To assess the contribution of MAPKAP kinase 2 in neutrophil function, the oxidative burst response after manipulation of endogenous kinase activity was measured. Intracellular delivery of the competitive inhibitory peptide into human neutrophils reduced both PMA- and fMLP-stimulated superoxide anion production. Thus, the results strongly suggest that MAPKAP kinase 2 is involved in the activation of human neutrophils.
authors.affiliation: Department of Physiology, University of Connecticut Health Center, Farmington, CT, USA.
authors.forename: Y L
authors.initials: YL
authors.lastname: Zu
authors.forename: Y
authors.initials: Y
authors.lastname: Ai
authors.forename: A
authors.initials: A
authors.lastname: Gilchrist
authors.forename: M E
authors.initials: ME
authors.lastname: Labadia
authors.forename: R I
authors.initials: RI
authors.lastname: Sha'afi
authors.forename: C K
authors.initials: CK
authors.lastname: Huang
mesh_terms: D000595:Amino Acid Sequence; D001667:Binding, Competitive; D002454:Cell Differentiation / Q000187:drug effects; D002630:Chemotactic Factors / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000494:pharmacology; D006360:Heat-Shock Proteins / Q000737:chemistry; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology*; D010446:Peptide Fragments / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D016897:Respiratory Burst / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects*; D013481:Superoxides / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D000099190:MAP-Kinase-Activated Kinase 2
chemical_list: D002630:Chemotactic Factors; D004791:Enzyme Inhibitors; D006360:Heat-Shock Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009240:N-Formylmethionine Leucyl-Phenylalanine; D010446:Peptide Fragments; D011493:Protein Kinase C; D017346:Protein Serine-Threonine Kinases; D013481:Superoxides; D013755:Tetradecanoylphorbol Acetate; D000099190:MAP-Kinase-Activated Kinase 2