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 transformed and nontransformed rat cell lines constitutively expressing the temperature-sensitive p53135val. It was found that in both cell types at 37.5 degrees C, where overexpressed p53 exhibits mutant conformation and cytoplasmic localization, a considerable part of the protein was poly(ADP-ribosyl)ated. Using densitometric scanning, the molecular mass of the modified protein was estimated as 64 kD. Immunofluorescence 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 depleted of pADPRT. A distinct intracellular localization and action of pADPRT was found in the cell lines cultivated at 32.5 degrees C, where p53 adopts wild-type form. Despite nuclear coexistence of both proteins no significant modification of p53 was found. Since the strikingly shared compartmentalization of p53 and pADPRT was indicative of possible complex formation between the two proteins, reciprocal immunoprecipitation and immunoblotting were performed with anti-p53 and anti-pADPRT antibodies. A poly(ADP-ribosyl)ated protein of 116 kD constantly precipitated at stringent conditions was identified as the automodified enzyme. It is concluded that mutant cytoplasmic p53 is tightly complexed to pADPRT and becomes modified. At 32.5 degrees C binding to DNA of p53 or its temperature-dependent conformational alteration might prevent an analogous modification of the tumor suppressor protein.
Wesierska-Gadek
J
J
Institute of Tumorbiology-Cancer Research, University of Vienna, Austria.
Bugajska-Schretter
A
A
Cerni
C
C
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000818:Animals; D002455:Cell Division / Q000235:genetics; D002460:Cell Line; D002471:Cell Transformation, Neoplastic / Q000235:genetics; D006358:Hot Temperature; D009154:Mutation; D010641:Phenotype; D051381:Rats; D011916:Rats, Inbred F344; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*
D016159:Tumor Suppressor Protein p53; D000246:Adenosine Diphosphate Ribose