Endotoxin induces rapid protein tyrosine phosphorylation in 70Z/3 cells expressing CD14.
CD14, a glycosylphosphatidylinositol-anchored glycoprotein of leukocytes, binds endotoxin (lipopolysaccharide (LPS)) with high affinity. After the murine pre-B cell line 70Z/3 is transfected with DNA encoding human CD14 (hCD14), the resultant stably transfected cell line, 70Z/3-hCD14, responds to 1000-fold lower LPS concentrations than the parental CD14-negative line. We have used 70Z/3-hCD14 cells, RAW264.7 cells, and elicited murine peritoneal exudate macrophages (PEM) to study LPS-induced protein tyrosine phosphorylation. LPS induces the rapid tyrosine phosphorylation of a 38-kDa protein (p38) in 70Z/3-hCD14 cells, PEM, and RAW264.7 cells and of two isoforms of mitogen-activated protein kinases (MAPK) in only RAW264.7 cells and PEM. p38 can be distinguished from the MAPK isoforms based on differences in mobilities on SDS-polyacrylamide gel electrophoresis and the lack of reactivity of p38 with anti-MAPK antibody even after dephosphorylation with potato acid phosphatase. Synthetic lipid A induces p38 phosphorylation in 70Z/3-hCD14 cells, whereas phorbol 12-myristate 13-acetate and interferon-gamma fail to induce tyrosine phosphorylation of p38. Pretreatment of 70Z/3-hCD14 cells with anti-hCD14 monoclonal antibody or the tyrosine kinase inhibitor herbimycin A inhibits LPS-induced tyrosine phosphorylation of p38. These results suggest that increased protein tyrosine phosphorylation occurs rapidly after LPS binds to CD14 and is likely to be an important event in mediating LPS-induced cell activation.
Han
J
J
Department of Immunology, Scripps Research Institute, La Jolla, California 92037.
Lee
J D
JD
Tobias
P S
PS
Ulevitch
R J
RJ
D000818:Animals; D015703:Antigens, CD / Q000096:biosynthesis / Q000378:metabolism*; D015214:Antigens, Differentiation, Myelomonocytic / Q000096:biosynthesis / Q000378:metabolism*; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides / Q000494:pharmacology*; D051379:Mice; D008809:Mice, Inbred C3H; D019950:Mitogen-Activated Protein Kinase 1; D016328:NF-kappa B / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism*
D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D016328:NF-kappa B; D011506:Proteins; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D019950:Mitogen-Activated Protein Kinase 1