Characterization of two different forms of mitogen-activated protein kinase kinase induced in polymorphonuclear leukocytes following stimulation by N-formylmethionyl-leucyl-phenylalanine or granulocyte-macrophage colony-stimulating factor.
Incubation of polymorphonuclear leukocytes with chemoattractants, granulocyte-macrophage colony-stimulating factor (GM-CSF), or phorbol 12-myristate 13-acetate (PMA) activated both mitogen-activated protein kinase kinase (MAPKK) and mitogen-activated protein kinase (MAPK). Activation by chemoattractants was rapid and transient, being maximal by 1 min and decreasing by 10 min. The order of efficacy was formyl-met-leu-phe > C5a > > LTB4 > interleukin 8 > platelet-activating factor. In contrast, activation by GM-CSF or PMA was slow and sustained being maximal at 5 min and with little decrease by 30 min. Sustained MAPK activation required continuous activation of the MAPKK. The MAPKK induced by N-formylmethionyl-leucyl-phenylalanine, GM-CSF, or PMA was resolved into two forms by anion exchange chromatography (Mono Q). Both corresponded to a 45-kDa MAPKK antigen by Western blotting and were inactivated by serine/threonine protein phosphatase 2A. Rechromatography of both forms after dephosphorylation resulted in the antigen's eluting slightly earlier on the Mono Q gradient than when in the active state. However, the two peaks remained separate, suggesting that they are not merely different phosphoforms of the same enzyme. The MAPK cascade is a signaling pathway common to many polymorphonuclear leukocyte stimulants, which may be activated transiently or in a sustained manner.
Thompson
H L
HL
Department of Development and Signalling, Babraham Institute, Cambridge, United Kingdom.
Marshall
C J
CJ
Saklatvala
J
J
D015153:Blotting, Western; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000097:blood* / Q000378:metabolism; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D015936:Complement C5a / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology*; D006801:Humans; D015151:Immunoblotting; D016209:Interleukin-8 / Q000494:pharmacology; D007527:Isoenzymes / Q000096:biosynthesis / Q000097:blood / Q000302:isolation & purification; D007975:Leukotriene B4 / Q000494:pharmacology; D020929:Mitogen-Activated Protein Kinase Kinases; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology*; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000032:analysis; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010972:Platelet Activating Factor / Q000494:pharmacology; D017354:Point Mutation; D011494:Protein Kinases / Q000096:biosynthesis* / Q000097:blood / Q000302:isolation & purification; D054648:Protein Phosphatase 2; D011994:Recombinant Proteins / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D016209:Interleukin-8; D007527:Isoenzymes; D010748:Phosphopeptides; D010972:Platelet Activating Factor; D011994:Recombinant Proteins; D007975:Leukotriene B4; D009240:N-Formylmethionine Leucyl-Phenylalanine; D015936:Complement C5a; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; D013755:Tetradecanoylphorbol Acetate