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.