Effect of serum starvation on expression and phosphorylation of PKC-alpha and p53 in V79 cells: implications for cell death.

Abstract

The effect of serum starvation on the expression and phosphorylation of PKC-alpha and p53 in Chinese hamster V79 cells was investigated. Serum starvation led to growth arrest, rounding up of cells and the appearance of new PKC-alpha and p53 bands on Western blots. Prolonged incubation (> or = 48 hr) in serum-deprived medium led to cell detachment and death. Moving cells to fresh medium containing 10% serum before, but not after, cell detachment reversed the changes observed in PKC-alpha and p53, and also prevented later cell detachment. Radiolabelling studies showed that the higher-molecular-weight PKC-alpha and p53 bands result from increased phosphorylation, while a lower-molecular-weight PKC-alpha band reflects newly synthesized protein. Immunocomplex kinase assays have shown that the increased phosphorylation of PKC-alpha is associated with its increased activity. To study the relationship between PKC-alpha, p53 and cell death, cells were treated either with TPA, to down-regulate PKC or with staurosporine, to inhibit PKC activity. Staurosporine, a potent PKC inhibitor and inducer of programmed cell death, caused the appearance of new PKC-alpha and p53 bands similar to those induced by serum starvation. If serum starvation was preceded by prolonged (48 hr) TPA treatment to down-regulate PKC-alpha, cell detachment and death did not take place within the same time frame. Intracellular fractionation of cells demonstrated that increased expression of PKC-alpha and the appearance of the associated higher and lower molecular-weight bands occurred in the nucleus. These data highlight the association of PKC-alpha and p53 with cellular events leading to cell death.