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.
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 microM 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 M(r) 85,000 (COOH-terminal catalytic domain) and M(r) 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 treatment. After 60 min, only the COOH-terminal fragment coimmunoprecipitated with p53, indicating that PARP noncovalently binds p53 via its M(r) 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]beta NAD+ 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.
Kumari
S R
SR
Department of Molecular Biology and Immunology, University of North Texas Health Science Center, Fort Worth 76107, USA.
Mendoza-Alvarez
H
H
Alvarez-Gonzalez
R
R
D017209:Apoptosis; D002273:Carcinogens; D004249:DNA Damage; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D008769:Methylnitronitrosoguanidine; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000737:chemistry / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
D002273:Carcinogens; D010446:Peptide Fragments; D016159:Tumor Suppressor Protein p53; D008769:Methylnitronitrosoguanidine; D011065:Poly(ADP-ribose) Polymerases