Activation of the cryptic DNA binding function of mutant forms of p53

Abstract

Wild type p53 assembles into a latent multiprotein complex which can be activated for sequence-specific DNA binding in vitro by proteins targeting the carboxy-terminal domain. Using an optimized system coupling the post-translational modification of wild type p53 to activation of sequence specific DNA binding, we examined the affects of common mutations on the cryptic DNA binding function of p53. Two mutant forms of p53 were shown to be efficiently converted from the latent state by PAb421 and DnaK, but were defective in activation by casein kinase II, indicating that mutant p53 may not be receptive to allosteric regulation by casein kinase II phosphorylation. A reactive sulfhydryl group is absolutely required for DNA binding by wild type and mutant forms of p53 once converted to the activated state. Together, these data show that some mutant forms of p53 harbour the wild-type machinery required to engage in sequence-specific DNA binding and define a signalling pathway whose inactivation may directly result in a loss of p53 function.

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

“…Phosphorylation by PKC and casein kinase2 (CK2) in vitro stimulate p53 to bind to DNA (Hupp et al, 1993) probably through a conformational change of the protein. However, the activation of PKC by phorbol ester does not cause a change in the phosphorylation of the C-terminal of mouse (Milne et al, 1996), suggesting that the PKC site may be phosphorylated constitutively.…”

“…Redox modulation The DNA-binding activity and conformation of p53 are also regulated by the redox state of the protein such that oxidation inhibits DNA binding, whereas reduction favors it (Hainaut et al, 1993a,b;Hupp et al, 1993;Rainwater et al, 1995). Several cysteine residues in the core DNA-binding domain have been involved in zinc coordination, and mutational analysis has allowed to identify cysteines at positions 173, 235 and 239 which participate in DNA binding and are also critical for transcriptional activation and suppression of transformation (Rainwater et al, 1995).…”

DNA-conformation is an important determinant of sequence-specific DNA binding by tumor suppressor p53

“…This sequence is particularly interesting insofar, as p53 binds this DNA, in contrast to RGC-DNA, only after activation, e.g. by addition of PAb421 (Hupp et al, 1992(Hupp et al, , 1993(Hupp et al, , 1994. Using the approach outlined in Figure 4, three di erent isoforms were prepared in which this site was present in di erent conformations, as shown in Figure 6a (referred to as Hu/La-du/ds, Hu/ Figure 5 Binding of p53 to single-stranded oligonucleotides capable of forming stem-loop structures.…”

Restoration of transcriptional activity of p53 mutants in human tumour cells by intracellular expression of anti-p53 single chain Fv fragments

“…Coexpression of either scFvs or scFv421-NLS with the p53 mutant His273 led to an increase in the His273-associated transcriptional activity. This e ect was not observed with the His175 mutant which had previously been shown to be unsensitive to Pab421 activation (Hupp et al, 1993). This also demonstrated that the in vivo restoration of p53 transactivation function by these scFvs was not due to a non p53-mediated e ect.…”

“…This inhibition can be relieved by partial proteolysis, DnaK association and by the action of some anti-p53 monoclonal antibodies (Hupp et al, 1992(Hupp et al, , 1993Halazonetis and Kandil, 1993;Hupp and Lane, 1994;Bayle et al, 1995;Waterman et al, 1995). Thus, the monoclonal antibody (mAb) PAb421 (Harlow et al, 1981) is able to increase the sequencespeci®c DNA binding of wt p53 and to restore the sequence-speci®c DNA binding of some p53 mutants in vitro (Hupp et al, 1992(Hupp et al, , 1995Halazonetis and Kandil, 1993;Niewolik et al, 1995, Abarzua et al, 1996.…”