Role of cysteine residues in regulation of p53 function.
Previous studies of p53 have implicated cysteine residues in site-specific DNA binding via zinc coordination and redox regulation (P. Hainaut and J. Milner, Cancer Res. 53:4469-4473, 1993; T. R. Hupp, D. W. Meek, C. A. Midgley, and D. P. Lane, Nucleic Acids Res. 21:3167-3174, 1993). We show here that zinc binding and redox regulation are, at least in part, distinct determinants of the binding of p53 to DNA. Moreover, by substituting serine for each cysteine in murine p53, we have investigated the roles of individual cysteines in the regulation of p53 function. Substitution of serine for cysteine at position 40, 179, 274, 293, or 308 had little or no effect on p53 function. In contrast, replacement of cysteine at position 173, 235, or 239 markedly reduced in vitro DNA binding, completely blocked transcriptional activation, and led to a striking enhancement rather than a suppression of transformation by p53. These three cysteines have been implicated in zinc binding by X-ray diffraction studies (Y. Cho, S. Gorina, P.D. Jeffrey, and N.P. Pavletich, Science 265:346-355, 1994); our studies demonstrate the functional consequences of the inability of the central DNA-binding domain of p53 to studies demonstrate the functional consequences of the inability of the central DNA-binding domain of p53 to bind zinc. Lastly, substitutions for cysteines at position 121, 132, 138, or 272 partially blocked both transactivation and the suppression of transformation by p53. These four cysteines are located in the loop-sheet-helix region of the site-specific DNA-binding domain of p53. Like the cysteines in the zinc-binding region, therefore, these cysteines may cooperate to modulate the structure of the DNA-binding domain. Our findings argue that p53 is subject to more than one level of conformational modulation through oxidation-reduction of cysteines at or near the p53-DNA interface.
Rainwater
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Biology Department, University of Alaska, Anchorage 99508, USA.
Parks
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Anderson
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Tegtmeyer
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Mann
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D017500:Adenovirus E1A Proteins / Q000378:metabolism; D000478:Alkylation; D000818:Animals; D001483:Base Sequence; D002471:Cell Transformation, Neoplastic; D003545:Cysteine; D004252:DNA Mutational Analysis; D051379:Mice; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011485:Protein Binding; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013489:Suppression, Genetic; D015533:Transcriptional Activation; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism; D018631:ras Proteins / Q000378:metabolism
D017500:Adenovirus E1A Proteins; D013438:Sulfhydryl Compounds; D016159:Tumor Suppressor Protein p53; D018631:ras Proteins; D015032:Zinc; D003545:Cysteine