p53CP is p51/p63, the third member of the p53 gene family: partial purification and characterization

The p53 tumor suppressor is a transcription factor that upon activation by DNA-damaging agents induces growth arrest or apoptosis mainly through trans activation and transrepression of its downstream target genes. Two addition al; p53 family members, p73 and p51/p63, were recently identified and chara cterized. Although the three family members share some similarities in tran scription activation and apoptosis induction, each of them appears to play a distinct role in development and tumor suppression, We have previously id entified a nuclear protein, p53CP (p53 competing protein), that is not p53 but binds to the p53 consensus sequence. Here we report the partial purific ation of p53CP from HeLa cells by ammonium sulfate precipitation, followed by a series of chromatography steps through heparin-agarose, Mono S ion exc hange and DNA affinity columns, coupled with a gel shift assay. Although p5 3CP activity is readily detectable in HeLa cells by gel shift assay, only a trace amount of p53CP protein was partially purified, which was not suffic ient for direct protein sequencing. Using a monoclonal antibody (4A4) speci fic for ail p51/p63 isoforms or a polyclonal antibody (N-18) recognizing th e N-terminus-containing p51/p63 isoforms we detected a significant enrichme nt of p51/p63 protein in p53CP containing fractions following each step of purification. Significantly, p51/p63 was detected only in the DNA affinity column fractions that contain p53CP activity. Thus, p53CP appears to be p51 /p63, the third member of the p53 gene family.