Bacterial lipopolysaccharide induces the association and coordinate activation of p53/56lyn and phosphatidylinositol 3-kinase in human monocytes.
p53/56lyn and other src family tyrosine kinases become activated in monocytes treated with LPS. In a variety of systems, phosphatidylinositol 3-kinase (PI 3-kinase) is believed to be a downstream effector of tyrosine kinases, and activation of PI 3-kinase results in increased levels of D3-phosphorylated metabolites of phosphatidylinositol (PtdIns). To examine whether LPS activates PI 3-kinase, freshly isolated human, peripheral blood monocytes were labeled in vitro with [32P]orthophosphate, and inositol phospholipids were detected after extraction and separation of lipids by TLC. Levels of PtdIns 3,4,5-trisphosphate (PtdIns 3,4,5-P3) were elevated within minutes of exposure of cells to LPS. Analysis of 32P-labeled lipid extracts of U937 cells by HPLC confirmed that levels of PtdIns 3,4,5-P3 increased rapidly following LPS treatment. Increased levels of PtdIns 3,4,5-P3 in LPS-treated cells resulted from an increase in the specific activity of PI 3-kinase. Thus, anti-PI 3-kinase immunoprecipitates prepared from unlabeled monocytes and assayed in an in vitro phosphorylation assay, using PtdIns as substrate, showed higher enzymatic activity when these were prepared from lysates of LPS-treated cells as compared with control cells. PI 3-kinase activity in immunoprecipitates was elevated as early as 2 min after LPS exposure and was dose dependent, with increased activity being observed at LPS concentrations as low as 10 pg/ml. Activation of PI 3-kinase involved signaling through the monocyte cell surface molecule CD14, since pretreatment of cells with Abs to CD14 abrogated LPS-induced increases in PtdIns 3,4,5-P3. Immunoprecipitates of p53/56lyn from LPS-treated cells showed a time-dependent and transient increase in PI 3-kinase activity assayed in vitro, coordinate with activation of p53/56lyn, indicating that LPS induces the association and simultaneous activation of these two enzymes in vivo. These findings indicate that monocytes respond to LPS with the rapid activation of PI 3-kinase, resulting in transient increases in levels of PtdIns 3,4,5-P3. This process is CD14 dependent and involves the physical association of PI 3-kinase with activated p53/56lyn.
Herrera-Velit
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Department of Medicine (Division of Infectious Diseases), University of British Columbia Faculties of Medicine and Science, Canada.
Reiner
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D000730:Androstadienes / Q000633:toxicity; D002851:Chromatography, High Pressure Liquid; D004306:Dose-Response Relationship, Immunologic; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007700:Kinetics; D018950:Lipopolysaccharide Receptors / Q000502:physiology; D008070:Lipopolysaccharides / Q000494:pharmacology*; D016403:Lymphoma, Large B-Cell, Diffuse; D008262:Macrophage Activation / Q000187:drug effects; D009000:Monocytes / Q000201:enzymology*; D019869:Phosphatidylinositol 3-Kinases; D018129:Phosphatidylinositol Phosphates / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors / Q000187:drug effects* / Q000378:metabolism; D014407:Tumor Cells, Cultured; D000077191:Wortmannin; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000187:drug effects*
D000730:Androstadienes; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D018129:Phosphatidylinositol Phosphates; C060974:phosphatidylinositol 3,4,5-triphosphate; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases; D000077191:Wortmannin