Temperature-sensitive (ts) mutations have been used as a genetic and molecular tool to study the functions of many gene products. Each ts mutant protein may contain a temperature-dependent intramolecular mechanism such as ts conformational change. To identify key ts structural elements controlling the protein function, we screened ts p53 mutants from a comprehensive mutation library consisting of 2,314 p53 missense mutations for their sequence-specific transactivity through p53-binding sequences in Saccharomyces cerevisiae. We isolated 142 ts p53 mutants, including 131 unreported ts mutants. were ts hot spots. Among the 142 mutants, 54 were examined further in human osteosarcoma Saos-2 cells, and it was confirmed that 89% of the mutants were also ts in mammalian cells. The ts mutants represented distinct ts transactivities for the p53 binding sequences and a distinct epitope expression pattern for conformation-specific anti-p53 antibodies. These results indicated that the intramolecular ␤-sheet in the core DNA-binding domain of p53 was a key structural element controlling the protein function and provided a clue for finding a molecular mechanism that enables the rescue of the mutant p53 function.p53 tumor suppressor is a 393-amino acid transcription factor that activates the transcription of a number of downstream genes through p53 binding to two copies of the specific consensus DNA sequence 5Ј-RRRC(A/T)(T/A)GYYY-3Ј (in which R is a purine nucleoside and Y is a pyrimidine nucleoside) in their regulatory regions (1). These molecular switches are activated by post-translational modifications, including phosphorylation, acetylation, and prolyl isomerization (2-5) of p53 in response to genotoxic or non-genotoxic stresses. The resulting biological effects are cell cycle arrest, apoptosis, DNA repair, and angiogenesis (6 -10). A growing number of p53 downstream genes have been isolated, and p53 has been structurally and functionally divided into three portions, namely the NH 2 -terminal portion containing the transactivation domain, the central core portion corresponding to the DNA-binding domain, and the COOH-terminal portion containing the oligomerization domain. The evolution of the DNA-binding domain is highly conserved in p53 orthologues (11) and also in the conserved human homologues p63 and p73 (12, 13).The structure of the DNA-binding domain (residues 94 -312) was resolved by x-ray crystallography (14). The domain consists of two ␣-helixes (H1 and H2) and 11 ␤-strands (S1, S2, S2Ј, and S3-S10) that were interconnected by loops (long L1-L3 loops and other short loops). Two anti-parallel ␤-sheets containing four (S1, S3, S5, and S8) and five (S4, S6, S7, S9, and S10) ␤-strands make up a large ␤-sandwich that serves as a scaffold for a loop-sheet-helix (LSH) motif (L1, S2, S2Ј, S10, and H2) and two large loops (L2 and L3). The loop-sheet-helix consists of two separate regions as follows: (i) the L1 loop (residues 113-123) and the S2-S2Ј ␤-hairpin (residues 124 -135) that correspond to evolutionary conserved regio...

Somatic mutations in the gene encoding the catalytic subunit of protein phosphatase 6 (Ppp6c) have been identified in malignant melanoma and are thought to function as a driver in B-raf- or N-ras-driven tumorigenesis. To assess the role of Ppp6c in carcinogenesis, we generated skin keratinocyte-specific Ppp6c conditional knockout mice and performed two-stage skin carcinogenesis analysis. Ppp6c deficiency induced papilloma formation with 7,12-dimethylbenz (a) anthracene (DMBA) only, and development of those papillomas was significantly accelerated compared with that seen following DMBA/TPA (12-O-tetradecanoylphorbol 13-acetate) treatment of wild-type mice. NF-κB activation either by tumor necrosis factor (TNF)-α or interleukin (IL)-1β was enhanced in Ppp6c-deficient keratinocytes. Overall, we conclude that Ppp6c deficiency predisposes mice to skin carcinogenesis initiated by DMBA. This is the first report showing that such deficiency promotes tumor formation in mice.

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