ADP-RIBOSYLATION OF P53 TUMOR-SUPPRESSOR PROTEIN - MUTANT BUT NOT WILD-TYPE P53 IS MODIFIED

Poly(ADP-ribosyl)ation of mutant and wild-type p53 was studied in tran sformed and nontransformed rat cell lines constitutively expressing th e temperature-sensitive p53(135val). It was found that in both cell ty pes at 37.5 degrees C, where overexpressed p53 exhibits mutant conform ation and cytoplasmic localization, a considerable part of the protein was poly(ADP-ribosyl)ated. Using densitometric scanning, the molecula r mass of the modified protein was estimated as 64 kD. Immunofluoresce nce studies with affinity purified anti-poly(ADP-ribose) transferase ( pADPRT) antibodies revealed that, contrary to predictions, the active enzyme was located in the cytoplasm, while in nuclei chromatin was dep leted of pADPRT. A distinct intracellular localization and action of p ADPRT was found in the cell lines cultivated at 32.5 degrees C, where p53 adopts wild-type form. Despite nuclear coexistence of both protein s no significant modification of p53 was found. Since the strikingly s hared compartmentalization of p53 and pADPRT was indicative of possibl e complex Formation between the two proteins, reciprocal immunoprecipi tation and immunoblotting were performed with anti-p53 and anti-pADPRT antibodies. A poly(ADP-ribosyl)ated protein of 116 kD constantly prec ipitated at stringent conditions was identified as the automodified en zyme. It is concluded that mutant cytoplasmic p53 is tighly complexed to pADPRT and becomes modified. At 32.5 degrees C binding to DNA of p5 3 or its temperature-dependent conformational alteration might prevent an analogous modification of the tumor suppressor protein. (C) 1996 W iley-Liss, Inc.