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.
Laboratory of Cell Biology, National Institutes of Health, Bethesda, Maryland 20892.
M
M
Fiscella
S J
SJ
Ullrich
N
N
Zambrano
M T
MT
Shields
D
D
Lin
S P
SP
Lees-Miller
C W
CW
Anderson
W E
WE
Mercer
E
E
Appella
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D015152:Blotting, Northern; D002453:Cell Cycle / Q000502:physiology*; D002460:Cell Line; D016158:Genes, p53; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D011494:Protein Kinases / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012694:Serine; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D009838:Oligodeoxyribonucleotides; D010455:Peptides; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53; D012694:Serine; D011494:Protein Kinases