Identification of a minimal transforming domain of p53: negative dominance through abrogation of sequence-specific DNA binding.

Abstract

Mutations in the p53 gene are most frequent in cancer. Many p53 mutants possess transforming activity in vitro. In cells transformed by such mutants, the mutant protein is oligomerized with endogenous cell p53. To determine the relevance of oligomerization for transformation, miniproteins containing C-terminal portions of p53 were generated. These miniproteins, although carrying no point mutation, transformed at least as efficiently as full-length mutant p53. Transforming activity was coupled with the ability to oligomerize with wild-type p53, as well as with the ability to abrogate sequence-specific DNA binding by coexpressed wild-type p53. These findings suggest that p53-mediated transformation may operate through a dominant negative mechanism, involving the generation of DNA binding-incompetent oligomers.

p53 can inhibit cell proliferation through caspase-mediated cleavage of ERK2/MAPK

“…Immortal C2C12 myoblasts and NIH3T3 fibroblasts were transformed with the v-Ha-Ras oncogene by analyses of p53 and ERK2 proteins on total cell extracts (TCEs) from cells treated as in (a). Expression of dnp53 protein (i.e., the p53 oligomerization domain DD 43 ) stabilized the endogenous p53 protein as expected. ERK2 expression and phosphorylation were detected by C-14 Ab that recognizes both the unphosphorylated and phosphorylated forms of ERK2.…”

p53 protein stability in tumour cells is not determined by mutation but is dependent on Mdm2 binding

“…The plasmids are referred to throughout as pCMVVal135, pCMVKH215, pCMVHis169, pCMVGln242 pCMVND37 etc. The p53DSS deletion was recreated by deleting a SacII-StuI fragment (Shaulian et al, 1992) which e ectively substitutes a single proline for residues 327 ± 341. The deletion of all 5' untranslated sequences (5'UTRD) was created by transferring an end®lled StyI restriction fragment including the 5' 251 base pairs of mouse p53 to EcoRV cut pBluescript.…”

p53 protein is activated during muscle differentiation and participates with MyoD in the transcription of muscle creatine kinase gene

“…To further evaluate the possible role of p53 in the expression of MCK, we used dominant negative mini protein of p53 (Shaulian et al, 1992). The mini protein is a short C-terminal fragment of p53 that can complex with wild type p53 and inhibit its activities (p53-DD) (Shaulian et al, 1992).…”

“…The mini protein is a short C-terminal fragment of p53 that can complex with wild type p53 and inhibit its activities (p53-DD) (Shaulian et al, 1992). A second peptide that was used as a control for the dominant negative peptide is a derivative of p53-DD containing an in-frame deletion of the`core oligomerization domain' (p53-DDSS) (deletion of amino acids 330-344).…”

“…Double-stranded oligonucleotides of the MyoD right site or the MEF2 site both of the MCK enhancer, were inserted to the HindIII site of pBS-p53-80 MCK-CAT or pBS-80 MCK-CAT. The expression plasmids of p53-pCMV-p53, pCMV-DD and pCMV-DDSS, are described elsewhere and were kindly provided by M Oren from the Weizmann Institute (Shaulian et al, 1992). Expression plasmids of MyoD;tm167,tm270 are described elsewhere and were kindly provided by S Tapscott, Fred Hutchinson Center, Seattle, WA.…”