The requirement for the p53 proline-rich functional domain for mediation of apoptosis is correlated with specific PIG3 gene transactivation and with transcriptional repression

Abstract

Wild-type p53 is a tumor suppressor gene which can activate or repress transcription, as well as induce apoptosis. The human p53 proline-rich domain localized between amino acids 64 and 92 has been reported to be necessary for efficient growth suppression. This study shows that this property mainly results from impaired apoptotic activity. Although deletion of the proline-rich domain does not affect transactivation of several promoters, such as WAF1, MDM2 and BAX, it does alter transcriptional repression, reactive oxygen species production and sequence-specific transactivation of the PIG3 gene, and these are activities which affect apoptosis. Whereas gel retardation assays revealed that this domain did not alter in vitro the specific binding to the p53-responsive element of PIG3, this domain plays a critical role in transactivation from a synthetic promoter containing this element. To explain this discrepancy, evidence is given for a prolinerich domain-mediated cellular activation of p53 DNA binding.

Tumor-derived mutations within the DNA-binding domain of p53 that phenotypically resemble the deletion of the proline-rich domain

“…Twenty-four hours after transfection, luciferase activity was determined, re¯ecting promoter activation by the di erent p53 mutants. As shown in Figure 1, p53 activated both promoters whereas p53D62-91 induced the mdm2 promoter but not the PIG3 promoter, con®rming previous results (Venot et al, 1998). Further, the mutations S315A, S376A, the combination of two mutations S376A/S378A, and S392A, were all without signi®cant in¯uence on the ability of p53 to activate both promoters in this assay.…”

“…In a previous report, p53 and p53D62-91 were shown to bind PIG3 promoter DNA with di erent e ciencies only when produced in mammalian cells but not when puri®ed from baculovirus-infected insect cells. Further, no signi®cant di erence was reported in binding e ciency of p53 and p53D62-91 to the p21 promoter DNA (Venot et al, 1998). In contrast, our results reveal that p53D62-91 and p53M246I bind to PIG3 promoter DNA and mdm2 promoter DNA, as well as p21 promoter DNA, with lower e ciencies than wild type p53.…”

“…On the other hand, p53D62-91 reportedly fails to do so (Venot et al, 1998). To test the ability of tumorderived p53-mutants to confer RSV-LTR promoter repression, a reporter construct with the RSV-3'LTR regulating luciferase expression was cotransfected along with p53 expression constructs, followed by a luciferase assay.…”

“…This inability of p53D62-91 to induce apoptosis correlated with a selective defect in transcriptional activation. While p53D62-91 activates the p21 promoter and many other p53-responsive promoters, its ability to activate a transiently transfected PIG3 promoter (Venot et al, 1998) and to activate the expression of endogenous PIG3, PIG6 and PIG11 (Zhu et al, 1999) turned out to be severely reduced. This correlation further supports the previously proposed model that PIGs are mediators of p53-induced apoptosis.…”

Twenty years of p53 research: structural and functional aspects of the p53 protein

“…The fact that this region is proline-rich and contains ®ve repeats of the SH3 binding motif PXXP suggests that it may be involved in physical interaction with elements of signal transduction pathways, for example c-abl. Although deletion of the proline-rich domain does not a ect transactivation of several promoters, such as WAF1, mdm2 and BAX, it does alter transcriptional repression (Venot et al, 1998). A chief point of interest is the fact that p53 gene is polymorphic at amino acid 72 which results in either a proline (p53 Pro) or a arginine (p53 Arg) at this position (Storey et al, 1998).…”