Central and carboxy‐terminal regions of human p53 protein are essential for interaction and complex formation with PARP‐1

Abstract

It has been previously described by different groups that poly(ADP‐ribose) polymerase‐1 (PARP‐1) and the product of the tumor suppressor gene p53 form tight complexes. We investigated which domains of human PARP‐1 and of human wild‐type p53 were involved in this protein–protein interaction. We generated baculoviral constructs encoding full length protein or distinct functional domains of both proteins. Baculovirally expressed wild‐type p53 was posttranslationally modified. Full length PARP‐1 was simultaneously coexpressed in insect cells with full length wt p53 protein or its distinct truncated fragments and vice versa. Reciprocal immunoprecipitation of Sf9 cell lysates revealed that the central and carboxy‐terminal fragments of p53 were sufficient to confer binding to PARP‐1. The amino‐terminal part harboring the transactivation functional domain of p53 was dispensable. On the other hand, the amino‐terminal and central fragments of PARP‐1 were necessary for complex formation with p53 protein. Finally, we explored the functional significance of the interaction between both proteins. Inactivation of PARP‐1 resulted in the reduction of p53 steady‐state levels. Inhibition of nuclear export by leptomycin B prevented accelerated degradation of p53 in PARP‐1 KO cells and led to accumulation of p53 protein. Considering the fact that the accelerated p53 nuclear export in the absence of PARP‐1 contributes to enhanced p53 degradation, we conclude that PARP‐1 may mask the NES of p53 through complex formation with its carboxy‐terminal part, thereby preventing the export. J. Cell. Biochem. 89: 220–232, 2003.