MUTATION OF THE SERINE 15 PHOSPHORYLATION SITE OF HUMAN P53 REDUCES THE ABILITY OF P53 TO INHIBIT CELL-CYCLE PROGRESSION

Overexpression of wild-type p53 prevents cells from entering the S phase of the cell cycle. The amino-terminal transactivation region of p53 is phosphorylated by several protein kinases, including DNA-PK, a nuclear serine/threonine protein kinase that in vitro requires DNA for activity. DNA-PK was recently shown to phosphorylate serines 15 and 37 of human p53 (Lees-Miller et al, 1992. Mol. Cell. Biol., 12, 5041-5049). To prevent phosphorylation at these sites, mutants were constructed that changed the codons for serine 15 or serine 37 to alanine codons. Expression of p53-Ala-37 in stably transformed T98G cells blocked progression of the cells into S phase as well as did the expression of wild-type p53. In contrast, p53-Ala-15 was partially defective in blocking cell cycle progression. Several cell clones transformed with the mutant p53-Ala-15 gene expressed normal levels of p53 mRNA but accumulated little or no detectable p53 protein. However, by using a transient expression system driven by a strong cytomegalovirus promoter, we showed that the inability of p53-Ala-15 to fully block cell cycle progression was not due to inadequate levels of expression or to a failure of the mutant protein to accumulate in the nucleus. These results suggest that phosphorylation of Ser-15 may affect p53 function.