CD14-dependent activation of protein kinase C and mitogen-activated protein kinases (p42 and p44) in human monocytes treated with bacterial lipopolysaccharide.
To investigate mechanisms of mononuclear phagocyte cell signaling, the effects of bacterial LPS on protein kinase activities in normal human peripheral blood monocytes were examined. Incubation of intact monocytes with LPS brought about time- and concentration-dependent increases in myelin basic protein (MBP) phosphotransferase activity in high speed supernatants of cell lysates. Anion-exchange chromatography on Mono Q demonstrated that LPS treatment resulted in two principal peaks of stimulated MBP kinase activity. Evidence was obtained to indicate that the first eluted peak of MBP kinase activity is accounted for by p42 and p44 mitogen-activated protein (MAP) kinases. Thus, 1) MBP kinase activity within peak 1 was quantitatively precipitated by anti-MAP kinase Abs, 2) the enzyme effectively phosphorylated a specific peptide substrate, 3) peak 1 contained proteins of subunit size M(r) 42,000 and M(r) 44,000 that reacted specifically with anti-MAP kinase Abs, and that 4) were recognized by anti-phosphotyrosine Abs only after stimulation of cells with LPS. Studies of the second peak of LPS-stimulated MBP kinase activity indicate that it is an isoform of protein kinase C (PKC) because: 1) enzyme activity was quantitatively immunoprecipitated by anti-PKC Abs, 2) the activity of the enzyme was potently and selectively inhibited by a specific peptide modeled on the autoinhibitory domain of PKC, and 3) the presence of a protein of subunit size M(r) 80,000 recognized by anti-PKC Abs. Because the second peak of MBP kinase activity (like the first) was active in the absence of added calcium and in the presence of 2 mM EGTA, it appears to be a type II, calcium-independent isoform of PKC. Abs to CD14 completely abrogated LPS-induced activation of both Mono Q peaks of MBP phosphotransferase activity. These results indicate that LPS coordinately activates both an apparently calcium-independent PKC and MAP kinase in mononuclear phagocytes and these responses appear to be initiated by signaling through the cell surface receptor, CD14.
Liu
M K
MK
Department of Medicine, University of British Columbia Faculties of Medicine and Science, Vancouver, Canada.
Herrera-Velit
P
P
Brownsey
R W
RW
Reiner
N E
NE
D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000502:physiology*; D015214:Antigens, Differentiation, Myelomonocytic / Q000502:physiology*; D015153:Blotting, Western; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000502:physiology*; D002478:Cells, Cultured; D002852:Chromatography, Ion Exchange; D004789:Enzyme Activation / Q000502:physiology; D006801:Humans; D007375:Interleukin-1 / Q000096:biosynthesis; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides / Q000494:pharmacology; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D008969:Molecular Sequence Data; D009000:Monocytes / Q000201:enzymology* / Q000276:immunology; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000502:physiology*; D011505:Protein-Tyrosine Kinases / Q000502:physiology*; D012333:RNA, Messenger / Q000096:biosynthesis; D015398:Signal Transduction / Q000502:physiology*
D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D007375:Interleukin-1; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D011505:Protein-Tyrosine Kinases; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases