Functional interactions of p53 with poly (ADP-ribose) polymerase (PARP) during apoptosis following DNA damage: covalent poly (ADP-ribosyl) ation of p53 by exogenous PARP and noncovalent binding of p53 to the M (r) 85,000 proteolytic fragment
Kumari, S.R.; Mendoza-Alvarez, H.; Alvarez-Gonzalez, R.
Cancer Research 58(22): 5075-5078
1998
ISSN/ISBN: 0008-5472 PMID: 9823314 Document Number: 490968
We have examined the domain-specific interactions between p53 and poly(ADP-ribose)polymerase (PARP) (E.C. 2.4.2.30) in apoptotic HeLa cells. Apoptosis was induced by exposing cells to 50 muM N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) for increasing lengths of time and was confirmed by: (a) oligonucleosomal fragmentation of chromatin; (b) increase in p53 levels; and (c) degradation of PARP into the characteristic Mr 85,000 (COOH-terminal catalytic domain) and Mr 29,000 (DNA-binding domain) peptide fragments. We also immunodetected p53 in immunoprecipitates obtained with a PARP-specific antibody. However, intact PARP coimmunoprecipitated with a p53-specific antibody during the initial 30 min of MNNG treatinenL After 60 min, only the COOH-terminal fragment coimmunoprecipitated with pS3, indicating that PARP noncovalently binds p53 via its Mr 85,000 catalytic domain. Therefore, we next examined p53 as a covalent target for poly(ADP-ribosyl)ation. Although p53 was not endogenously poly(ADP-ribosyl)ated in situ, incubation of cell extracts with full-length PARP from calf thymus and (32P)betaNAD+ resulted in its time-dependent poly(ADP-ribosyl)ation. In summary, our results are consistent with the conclusion that PARP and p53 are activated with nonoverlapping kinetics during apoptosis.