Activation of protein kinase C and specific phosphorylation of a Mr 90,000 membrane protein of promotable BALB/3T3 and C3H/10T1/2 cells by tumor promoters

Abstract

The activation of protein kinase C and protein phosphorylation by tumor promoters were examined using quiescent cultures of BALB/3T3 and C3H/10T1/2 cells, because in these cells tumor promoters enhance chemically induced transformation and also induce DNA synthesis and ornithine decarboxylase. The cytosol and membrane fractions were partially purified, and the activity of protein kinase C was assayed. In quiescent cells, protein kinase C activity was found only in the cytosol fraction. Treatment with 100 ng of 12-O-tetradecanoylphorbol-13-acetate or teleocidin B per ml caused rapid translocation of protein kinase C from the cytosol to the membrane fraction. The activity in the cytosol disappeared almost completely after 15 min when the activity in the membrane reached a peak. The membrane activity gradually decreased to the control level after 6 h, while no activity reappeared in the cytosol within 6 h. Under these circumstances, a membrane protein with a molecular weight of 90,000 and pl of 4.0-4.4 (termed p90) was specifically phosphorylated, possibly by the activated protein kinase C, in both cell-free and intact-cell systems. On treatment of quiescent BALB/3T3 cells with 100 ng of 12-O-tetradecanoylphorbol-13-acetate, p90 phosphorylation increased 2-fold in 1 min, reaching a peak in 15 min of 3.4-fold the initial value. The phosphorylation of p90 increased with increase in the concentrations of 12-O-tetradecanoylphorbol-13-acetate between 0.1 and 10 ng/ml and reached a plateau at 10 ng/ml. p90 phosphorylation also occurred on exposure of the cells to non-phorbol ester tumor promoters (mezerein and teleocidin B) and growth factors, such as platelet-derived growth factor and fibroblast growth factor. p90 was not immunoprecipitated by antibody against the insulin receptor. Phosphorylation of p90 occurred at a serine residue. The present study suggests that activation of protein kinase C and phosphorylation of p90 by it are early events leading to tumor promotion.