The chemotactic factor N-formylmethionyl-leucyl-phenylalanine activates microtubule-associated protein 2 (MAP) kinase and a MAP kinase kinase in polymorphonuclear leucocytes.
Incubation of human polymorphonuclear leucocytes (PMN) with either the chemotactic factor N-formylmethionyl-leucylphenylalanine (FMLP) or phorbol 12-myristate 13-acetate (PMA) activates a kinase with phosphorylating activity towards a known microtubule-associated protein-2 (MAP) kinase substrate, the epidermal growth factor receptor peptide (T669). Activation of this enzyme by FMLP was maximal at 1 min, decreasing by 10 min. Activation by PMA was slightly slower than that by FMLP, but more prolonged (maximal at 5 min, with no significant decrease by 20 min). The enzyme induced by either stimulant bound strongly to phenyl-Sepharose, had a molecular mass of 40 kDa on gel filtration and phosphorylated three MAP kinase substrates, i.e. MAP, myelin basic protein and the T669 peptide. By use of antibodies to MAP kinases and phosphotyrosine, the enzyme was identified as the 42 kDa MAP kinase (also known as extracellular-signal-regulated kinase 2, ERK2). Stimulation of PMN with FMLP or PMA was also found to induce a kinase kinase which phosphorylated human recombinant MAP kinase on threonine and tyrosine, with concomitant activation. These results suggest that MAP kinase and the kinase kinase are involved in the activation of PMN by chemotactic factors such as FMLP.
Cytokine Biochemistry Department, Strangeways Research Laboratory, Worts Causeway, Cambridge, U.K.
H L
HL
Thompson
M
M
Shiroo
J
J
Saklatvala
D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002478:Cells, Cultured; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D066246:ErbB Receptors / Q000378:metabolism; D006801:Humans; D020929:Mitogen-Activated Protein Kinase Kinases; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D009504:Neutrophils / Q000201:enzymology*; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011817:Rabbits; D013379:Substrate Specificity; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D009240:N-Formylmethionine Leucyl-Phenylalanine; D011494:Protein Kinases; D066246:ErbB Receptors; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D013755:Tetradecanoylphorbol Acetate