Augmented DNA-binding activity of p53 protein encoded by a carboxyl-terminal alternatively spliced mRNA is blocked by p53 protein encoded by the regularly spliced form.

DNA-binding activity of the wild-type p53 is central to its function in vivo. However, recombinant or in vitro translated wild-type p53 proteins, unless modified, are poor DNA binders. The fact that the in vitro produced protein gains DNA-binding activity upon modification at the C terminus raises the possibility that similar mechanisms may exist in the cell. Data presented here show that a C-terminal alternatively spliced wild-type p53 (ASp53) mRNA expressed by bacteria or transcribed in vitro codes for a p53 protein that efficiently binds DNA. Our results support the conclusion that the augmented DNA binding activity of an ASp53 protein is probably due to attenuation of the negative effect residing at the C terminus of the wild-type p53 protein encoded by the regularly spliced mRNA (RSp53) rather than acquisition of additional functionality by the alternatively spliced C' terminus. In addition, we found that ASp53 forms a complex with the non-DNA-binding RSp53, which in turn blocks the DNA-binding activity of ASp53. Interaction between these two wild-type p53 proteins may underline a mechanism that controls the activity of the wild-type p53 protein in the cell.

The carboxyl-terminal domain of the p53 protein regulates sequence-specific DNA binding through its nonspecific nucleic acid-binding activity.

The murine p53 protein contains two nucleic acid-binding sites, a sequence-specific DNA-binding region localized between amino acid residues 102-290 and a nucleic acid-binding site without sequence specificity that has been localized to residues 364-390. Alternative splicing of mRNA generates two forms of this p53 protein. The normal, or majority, splice form (NSp53) retains its carboxyl-terminal sequence-nonspecific nucleic acid-binding site, which can negatively regulate the sequence-specific DNA-binding site. The alternative splice form of p53 (ASp53) replaces amino acid residues 364-390 with 17 different amino acids. This protein fails to bind nucleic acids nonspecifically and is constitutive for sequence-specific DNA binding. Thus, the binding of nucleic acids at the carboxyl terminus regulates sequence-specific DNA binding by p53. The implications of these findings for the activation of p53 transcriptional activity following DNA damage are discussed.

Cyclosporin A potentiates the dexamethasone-induced mouse mammary tumor virus-chloramphenicol acetyltransferase activity in LMCAT cells: a possible role for different heat shock protein-binding immunophilins in glucocorticosteroid receptor-mediated gene expression.

As previously observed for FK506, we report here that cyclosporin A (CsA) treatment of mouse fibroblast cells stably transfected with the mouse mammary tumor virus-chloramphenicol acetyltransferase (MMTV-CAT) reporter plasmid (LMCAT cells) results in potentiation of dexamethasone (Dex)-induced CAT gene expression. Potentiation by CsA is observed in cells treated with 10-100 nM Dex but not in cells treated with 1 microM Dex, a concentration of hormone which results in maximum CAT activity. At 10 nM Dex, 1-5 microM CsA provokes an approximately 50-fold increase in CAT gene transcription, compared with transcription induced by Dex alone. No induction of CAT gene expression is observed in cells treated with CsA or FK506 in the absence of Dex. The antisteroid RU 486 abolishes effects obtained in the presence of Dex. Using a series of CsA, as well as FK506, analogs, including some devoid of calcineurin phosphatase inhibition activity, we conclude that the potentiation effects of these drugs on Dex-induced CAT gene expression in LMCAT cells do not occur through a calcineurin-mediated pathway. Western-blotting experiments following immunoprecipitation of glucocorticosteroid receptor (GR) complexes resulted in coprecipitation of GR, heat shock protein hsp90 and two immunophilins: the FK506-binding protein FKBP59 and the CsA-binding protein cyclophilin 40 (CYP40). Two separate immunophilin-hsp90 complexes are present in LMCAT cells: one containing CYP40-hsp90, the other FKBP59-hsp90. Thus, both FKBP59 and CYP40 can be classified as hsp-binding immunophilins, and their possible involvement as targets of immunosuppressants potentiating the GR-mediated transcriptional activity is discussed.

Human TAFII31 protein is a transcriptional coactivator of the p53 protein.

The p53 protein activates transcription of a target gene by binding to a specific DNA response element and interacting with the transcriptional apparatus of RNA polymerase II. The amino-terminal domain of p53 interacts with a component of the TFIID basal transcription complex. The human TATA-binding-protein-associated factor TAFII31, a component of TFIID, has been identified as a critical protein required for p53-mediated transcriptional activation. TAFII31 and p53 proteins bind to each other via amino acid residues in the amino-terminal domain of p53 that are essential for transcription. Antibodies directed against TAFII31 protein inhibit p53-activated but not basal transcription in vitro. These results demonstrate that TAFII31 is a coactivator for the p53 protein.