Phosphatidylinositol 3-kinase-dependent activation of protein kinase C-zeta in bacterial lipopolysaccharide-treated human monocytes.
The isoform identity of activated protein kinase C (PKC) and its regulation were investigated in bacterial lipopolysaccharide (LPS)-treated human monocytes. Resolution of detergent-soluble lysates prepared from LPS-treated, peripheral blood monocytes using Mono Q anion-exchange chromatography revealed two principal peaks of myelin basic protein kinase activity. Immunoblotting and immunoprecipitation with isoform-specific anti-PKC antibodies showed that the major and latest eluting peak is accounted for by PKC-zeta. In addition to primary monocytes, activation of PKC-zeta in response to LPS was also observed in the human promonocytic cell lines, U937 and THP-1. Consistent with its identity as PKC-zeta, the kinase did not depend upon the presence of lipids, Ca2+, or diacylglycerol for activity. In addition, the kinase phosphorylates peptide epsilon and myelin basic protein with equal efficiency but phosphorylates Kemptide and protamine sulfate poorly. Translocation of PKC-zeta from the cytosolic to the particulate membrane fraction upon exposure of monocytes to LPS provided further evidence for activation of the kinase. Preincubation of monocytes with the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitors, wortmannin or LY294002, abrogated LPS-induced activation of PKC-zeta. Furthermore, activation of PKC-zeta failed to occur in U937 cells transfected with a dominant negative mutant of the p85 subunit of PI 3-kinase. PKC-zeta activity was also observed to be enhanced in vitro by the addition of phosphatidylinositol 3,4,5P3. These findings are consistent with a model in which PKC-zeta is activated downstream of PI 3-kinase in monocytes in response to LPS.
Herrera-Velit
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Department of Microbiology and Immunology, The University of British Columbia Faculties of Medicine and Science and The Research Institute of the Vancouver Hospital and Health Sciences Center, Vancouver, British Columbia, Canada V5Z 3J5.
Knutson
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Reiner
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D000595:Amino Acid Sequence; D002460:Cell Line; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007527:Isoenzymes / Q000187:drug effects* / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009000:Monocytes / Q000187:drug effects / Q000201:enzymology*; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000502:physiology*; D011493:Protein Kinase C / Q000187:drug effects* / Q000378:metabolism
D007527:Isoenzymes; D008070:Lipopolysaccharides; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D011493:Protein Kinase C