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

The effect of serum starvation on the expression and phosphorylation of PKC -alpha and p53 in Chinese hamster V79 cells was investigated, Serum starvat ion led to growth arrest, rounding up of cells and the appearance of new PK C-alpha and p53 hands on Western blots, Prolonged incubation (greater than or equal to 48 hr) in serum-deprived medium led to cell detachment and deat h, Moving cells to fresh medium containing 10% serum before, but not after, cell detachment reversed the changes observed in PKC-alpha and p53, and al so prevented later cell detachment. Radioiabelling studies showed that the higher-molecular-weight PKC-alpha and p53 bands result from increased phosp horylation, while a lower-molecular-weight PKC-alpha band reflects newly sy nthesized protein. Immunocomplex kinase assays have shown that the increase d phosphorylation of PKC-alpha is associated with its increased activity. T o study the relationship between PKC-alpha, p53 and cell death, cells were treated either with TPA, to down-regulate PKC or with staurosporine, to inh ibit PKC activity. Staurosporine, a potent PKC inhibitor and inducer of pro grammed cell death, caused the appearance of new PKC-alpha and p53 bands si milar to those induced by serum starvation, If serum starvation was precede d by prolonged (48 hr) TPA treatment to down-regulate PKC-alpha, cell detac hment and death did not take place within the same time frame, Intracellula r fractionation of cells demonstrated that increased expression of PKC-alph a and the appearance of the associated higher and lower molecular-weight ba nds occurred in the nucleus, These data highlight the association of PKC-al pha and p53 with cellular events leading to cell death. (C) 1999 Wiley-Liss , Inc.