Isolation of Temperature-sensitive p53 Mutations from a Comprehensive Missense Mutation Library

Kazuko Shiraishi, Shunsuke Kato, Shuang Yin Han, Wen Liu, Kazunori Otsuka, Masato Sakayori, Takanori Ishida, Motohiro Takeda, Ryunosuke Kanamaru, Noriaki Ohuchi, Chikashi Ishioka

Research output: Contribution to journalArticlepeer-review

72 Citations (Scopus)

Abstract

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. These mutants clustered in β-strands in the DNA-binding domain, particularly in one of the two β-sheets of the protein, and 15 residues (Thr155, Arg158, Met160, Ala161, Val172, His214, Ser215, Pro223, Thr231, Thr253, Ile254, Thr256, Ser269, Glu271, and Glu285) 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 β-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.

Original languageEnglish
Pages (from-to)348-355
Number of pages8
JournalJournal of Biological Chemistry
Volume279
Issue number1
DOIs
Publication statusPublished - 2004 Jan 2

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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