Isolation of temperature-sensitive p53 mutations from a comprehensive missense mutation library.
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 beta-strands in the DNA-binding domain, particularly in one of the two beta-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 beta-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.
Shiraishi
Kazuko
K
Department of Clinical Oncology, Institute of Development, Aging and Cancer, Tohoku University, Sendai 980-8575, Japan.
Kato
Shunsuke
S
Han
Shuang-Yin
SY
Liu
Wen
W
Otsuka
Kazunori
K
Sakayori
Masato
M
Ishida
Takanori
T
Takeda
Motohiro
M
Kanamaru
Ryunosuke
R
Ohuchi
Noriaki
N
Ishioka
Chikashi
C
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D015723:Gene Library; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D011994:Recombinant Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D013050:Spectrometry, Fluorescence; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry / Q000235:genetics*
D011994:Recombinant Proteins; D016159:Tumor Suppressor Protein p53; D004247:DNA