Phosphorylation sites in the amino-terminal region of mouse p53.

Phosphorylation is an attractive mechanism for regulating the functions of p53. The p34cdc2 kinase, which is involved in regulation of the cell cycle, phosphorylates serine-315 of human p53 in vitro. Casein kinase II phosphorylates serine-389 of mouse p53 in vitro. The amino-terminal region of mouse p53 contains a cluster of potential serine phosphorylation sites. Those sites have been proposed to be sites for phosphorylation by a double-stranded DNA-dependent kinase (DNA-PK) from HeLa cells and can be dephosphorylated by protein phosphatase 2A. To identify in vivo phosphorylation sites in the amino-terminal region of mouse p53, we mutated potential phosphorylation sites and analyzed the mutant proteins by tryptic phosphopeptide mapping. We identified serine-7, -9, -18, and -37 as in vivo phosphorylation sites. We further showed that mouse p53 expressed in bacteria is phosphorylated by DNA-PK on amino-terminal serine residues in vitro.