Phosphorylation of a C-SRC-related protein in macrophages activated in vitro with lymphokine.
Treatment of proteose peptone elicited peritoneal macrophages from C3H/HeN mice or the macrophage cell line B6MP102 with a T-cell lymphokine preparation induces cytotoxicity for SV3T3 tumor cells. The Triton X-100 (TX-100) insoluble fractions from activated macrophages possessed kinase activity for an endogenous 53 kDa phosphoprotein (pp53) which was markedly greater than extracts from untreated macrophages. Addition of the tyrosine phosphatase inhibitor, Na3 VO4 to the cytotoxicity assay also enhanced tumor cell lysis and Na3 VO4 treated macrophages showed increased phosphorylation of pp53. Moreover, addition of Na3 VO4 to the cytoskeleton kinase assay enhanced the phosphorylation of pp53 in a dose dependent manner. Pp53 was immunoprecipitated from the in vitro phosphorylated TX-100 insoluble fraction with monoclonal antibody to pp60v-src. Anti-pp60v-src also precipitated a 53 and a 60 kDa phosphoprotein from whole cell extracts and from TX-100 cytoskeleton extracts of macrophages phosphorylated as viable intact cells. Addition of a known tyrosine kinase inhibitor, quercetin, to the macrophage cytoskeleton kinase assay inhibited phosphorylation of pp53, and the in vitro phosphorylated pp53 was resistant to 1 N NaOH hydrolysis, indicating phosphorylation of tyrosine residues. Immune complex kinase assays of anti-pp60v-src precipitated TX-100 insoluble macrophage fractions revealed strong phosphorylation for alpha-casein which was inhibited by quercetin. These data suggest that macrophage pp53 is a c-src-related gene product that is inducible by stimuli that activate macrophages to cytotoxicity.
Soong
M M
MM
Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Illinois, Urbana 61801.
Tompkins
W A
WA
D000818:Animals; D002478:Cells, Cultured; D003602:Cytotoxicity, Immunologic; D004591:Electrophoresis, Polyacrylamide Gel; D008222:Lymphokines / Q000494:pharmacology*; D008262:Macrophage Activation; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008809:Mice, Inbred C3H; D009363:Neoplasm Proteins / Q000378:metabolism*; D009857:Oncogenes; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism; D016159:Tumor Suppressor Protein p53
D008222:Lymphokines; D009363:Neoplasm Proteins; D010750:Phosphoproteins; D016159:Tumor Suppressor Protein p53; D010770:Phosphotransferases