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 transactivation and transrepression of its downstream target genes. Two additional; p53 family members, p73 and p51/p63, were recently identified and characterized. Although the three family members share some similarities in transcription activation and apoptosis induction, each of them appears to play a distinct role in development and tumor suppression, We have previously identified a nuclear protein, p53CP (p53 competing protein), that is not p53 but binds to the p53 consensus sequence. Here we report the partial purification of p53CP from HeLa cells by ammonium sulfate precipitation, followed by a series of chromatography steps through heparin-agarose, Mono S ion exchange and DNA affinity columns, coupled with a gel shift assay. Although p53CP activity is readily detectable in HeLa cells by gel shift assay, only a trace amount of p53CP protein was partially purified, which was not sufficient for direct protein sequencing. Using a monoclonal antibody (4A4) specific for ail p51/p63 isoforms or a polyclonal antibody (N-18) recognizing the N-terminus-containing p51/p63 isoforms we detected a significant enrichment 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.