Inhibition of the transformation-specific kinase in ASV-transformed cells by N-α-tosyl-L-lysyl chloromethyl ketone
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We find that the protease inhibitor N-α-tosyl-L-lysyl chloromethyl ketone (TLCK) inhibits the transformation-specific kinase activity (Collett and Erikson, 1978) associated with p60src, the avian sarcoma virus (ASV) gene product responsible for the transformation of fibroblasts. TLCK has been shown to induce the phenotypic reversion of ASV-transformed cells to normal (Weber, 1975). Kinase activity was measured in extracts of chick embryo fibroblasts (CEF) transformed by the Schmidt-Ruppin strain of ASV (SR-ASV) with antiserum from rabbits bearing ASV-induced tumors. The immunoprecipitates were incubated with γ-32P-ATP under conditions in which the phosphorylation of the IgG heavy chain in the immunoprecipitate was directly proportional to the concentration of cell extract. When ASV-transformed CEF were treated with 0.1 mM TLCK, the kinase activity was reduced by 60% after 2 hr and by 80% after 6 hr, and continued to remain low for up to 40 hr when TLCK was present. When TLCK was removed, the kinase activity rose slowly over a period of many hours, suggesting that the enzyme is irreversibly inactivated by TLCK and new enzyme must be synthesized. The effect of TLCK in vivo is concentration-dependent and specific. Other serine protease inhibitors had no effect on kinase activity. At low concentrations (0.03 mM), TPCK produced partial inhibition (≤20%), but at higher concentrations TPCK was extremely toxic to the cells and therefore could not be tested. The inhibition by TLCK was not due to its ability to inhibit protein synthesis since cycloheximide treatment (1 μg/ml) did not significantly reduce kinase activity. TLCK also inhibited kinase activity when added directly to cell extracts, but about 5 times higher concentrations of TLCK were required to produce 50% inhibition. Under these conditions both TLCK and TPCK were comparable inhibitors, whereas PMSF had no effect. Our finding that the inhibition of the kinase by TLCK in vivo parallels the reversion of cell morphology to normal suggests that the kinase has an important role in transformation and offers a biochemical rationale for treatment of tumors with this agent. © 1979.
author list (cited authors)
Richert, N., Davies, P., Jay, G., & Pastan, I.