title: Activation of multiple proline-directed kinases by bacterial lipopolysaccharide in murine macrophages.
abstract: Bacterial LPS stimulation of murine macrophages leads to increased tyrosine phosphorylation and activation of the 42- and 44-kDa mitogen-activated protein kinases (MAPK) and the activation of stress-activated protein kinases (SAPK)/c-Jun N-terminal kinase (JNK) and p38, related to the high osmolarity glycerol protein kinase in Saccharomyces cerevisiae (HOG1). LPS caused a rapid increase (10 min) in phosphotransferase activity toward myelin basic protein (MBP), a polypeptide that encompassed the first 169 residues of c-Jun fused to gluthathione S-transferase (GST-c-Jun (1-169)) and 27-kDa heat shock protein (hsp27). MonoQ fractionation of cell extracts resolved phosphotransferase activity peaks toward MBP, GST-c-Jun (1-169), and hsp27, which contained MAPK, SAPK/JNK, and MAPKAPK2, respectively, as indicated by immunoblotting data. In RAW 264.7 macrophages, LPS stimulation of MAPKAPK2, a substrate of p38 HOG1 and MAPK, appeared to occur predominantly via p38 HOG1 and not the MAPK. PMA, which activated the MAPK as potently as LPS, did not strongly activate MAPKAPK2, as assessed by hsp27 phosphorylation. Consistent with p38 HOG1-mediating LPS activation of MAPKAPK2, treatment with LPS, but not PMA, increased the tyrosine phosphorylation of p38 HOG1, a modification known to elevate the enzymatic capacity of this kinase. In LPS-treated cells, the activity of SAPK/JNK was increased 5- to 10-fold, as measured by precipitating SAPK/JNK with Abs or immobilized GST-c-Jun and performing an in vitro kinase assay. In addition, the kinases thought to be upstream of SAPK/JNK, SAPK/ERK kinase 1 (SEK1), and MAPK/ERK kinase kinase 1 (MEKK1), were activated following LPS, but not PMA, exposure (5-fold and 2.5-fold, respectively.
authors.affiliation: Biomedical Research Centre and Department of Medicine, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: J S
authors.initials: JS
authors.lastname: Sanghera
authors.forename: S L
authors.initials: SL
authors.lastname: Weinstein
authors.forename: M
authors.initials: M
authors.lastname: Aluwalia
authors.forename: J
authors.initials: J
authors.lastname: Girn
authors.forename: S L
authors.initials: SL
authors.lastname: Pelech
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D048031:JNK Mitogen-Activated Protein Kinases; D007700:Kinetics; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008262:Macrophage Activation / Q000187:drug effects; D008264:Macrophages / Q000187:drug effects* / Q000201:enzymology*; D051379:Mice; D020928:Mitogen-Activated Protein Kinases; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics; D038461:Proline-Directed Protein Kinases; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases; D000099190:MAP-Kinase-Activated Kinase 2
chemical_list: D017871:Calcium-Calmodulin-Dependent Protein Kinases; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D048031:JNK Mitogen-Activated Protein Kinases; D008070:Lipopolysaccharides; D020928:Mitogen-Activated Protein Kinases; D010455:Peptides; D038461:Proline-Directed Protein Kinases; D017346:Protein Serine-Threonine Kinases; D013755:Tetradecanoylphorbol Acetate; D048051:p38 Mitogen-Activated Protein Kinases; D000099190:MAP-Kinase-Activated Kinase 2