P53CP, A PUTATIVE P53 COMPETING PROTEIN THAT SPECIFICALLY BINDS TO THE CONSENSUS P53 DNA-BINDING SITES - A 3RD MEMBER OF THE P53 FAMILY

p53 tumor suppressor protein negatively regulates cell growth, mainly through the transactivation of its downstream target genes. As a seque nce-specific DNA binding transcription factor, p53 specifically binds to a 20-bp consensus motif 5'-PuPuPuC(A/T) (T/A)GPyPyPyPuPuPuC(A/T) (T / A)GPyPyPy-3'. We have now identified, partially purified, and charac terized an additional approximate to 40-kDa nuclear protein, p53CP (p5 3 competing protein), that specifically binds to the consensus p53 bin ding sites found in several p53 downstream target genes, including Waf -1, Gadd45, Mdm2, Bax, and RGC. The minimal sequence requirement for b inding is a 14-bp motif, 5'-CTTGCTTGAACAGG-3' [5'-C(A/T) (T/A)GPyPyPyP uPuPuC(A/T)(T/A)G-3'], which includes the central nucleotides of the t ypical p53 binding site with one mismatch. p53CP and p53 (complexed wi th antibody) showed a similar binding specificity to Waf-1 site but di fferences in Gadd45 and T3SF binding. Like p53, p53CP also binds both double-and single-stranded DNA oligonucleotides. Important to note, ce ll cycle blockers and DNA damaging reagents, which induce p53 binding activity, were found to inhibit p53CP binding in p53-positive, but not in p53-negative, cells. This finding suggested a p53-dependent coordi nate regulation of p53 and p53CP in response to external stimuli. p53C P therefore could be a third member of the p53 family, in addition to p53 and p73, a newly identified p53 homolog. p53CP, if sequestering p5 3 from its DNA binding sites through competitive binding, may provide a novel mechanism of p53 inactivation. Alternatively, p53CP may have p 53-like functions by binding and transactivating p53 downstream target genes. Cloning of the p53CP gene ultimately will resolve this issue.