BINDING OF CELLULAR PROTEINS TO A CONFORMATIONAL DOMAIN OF TUMOR-SUPPRESSOR PROTEIN P53

The genes regulated by p53, as well as the factors modulating its func tion, need to be identified before the mechanism of action of p53 in c ontrol of cell growth can be adequately understood, Binding of the SV4 0 large T-antigen protein to an evolutionally conserved (conformationa l) domain of p53 inhibits p53's DNA-binding and transcription activati on activities. Cellular proteins might also bind to this same region o f p53 to regulate its function. A hybrid protein composed of protein A fused to the conformational domain (amino acids 115-295) of p53 was e xpressed in Escherichia coli and used as an affinity probe for binding proteins in detergent lysates of non-small cell lung carcinoma (NSCLC ) cells. The wildtype p53 hybrid protein associated with several major proteins of molecular weights 45 K, 56 K, and 70 K, as web as other m inor species ranging in molecular weight from 30 K to 90 K. These prot eins bound specifically to the p53 sequence of the hybrid protein. Pro tein A did not associate with these proteins and the two p53 hybrid pr oteins containing missense mutations at codons 273 and 175 exhibited a 40-80% weaker association. In addition, T antigen competed with the c ellular proteins for binding to the conformational domain. The conditi ons of cell growth had a profound effect on the expression of the p53 binding proteins. Considerably more p53 binding proteins were expresse d in actively growing cells than in culture's maintained under conditi ons for slow growth. Quantitative differences in expression of p53-bin ding proteins were observed among different NSCLC cell lines. The expr ession of p53-binding proteins was not restricted to NSCLC cell lines; detergent extracts of an osteosarcoma cell line yielded similar p53-b inding proteins.