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Binding of cellular proteins to a conformational domain of tumor suppressor protein p53
1Departments of Thoracic and Cardiovascular Surgery, University of Texas M.D. Anderson Cancer Center, Houston 77030.
Abstract:
The genes regulated by p53, as well as the factors modulating its function, need to be identified before the mechanism of action of p53 in control of cell growth can be adequately understood. Binding of the SV40 large T-antigen protein to an evolutionally conserved (conformational) domain of p53 inhibits p53's DNA-binding and transcription activation activities. Cellular proteins might also bind to this same region of p53 to regulate its function. A hybrid protein composed of protein A fused to the conformational domain (amino acids 115-295) of p53 was expressed in Escherichia coli and used as an affinity probe for binding proteins in detergent lysates of non-small cell lung carcinoma (NSCLC) cells. The wild-type p53 hybrid protein associated with several major proteins of molecular weights 45 K, 56 K, and 70 K, as well as other minor species ranging in molecular weight from 30 K to 90 K. These proteins bound specifically to the p53 sequence of the hybrid protein. Protein A did not associate with these proteins and the two p53 hybrid proteins containing missense mutations at codons 273 and 175 exhibited a 40-80% weaker association. In addition, T antigen competed with the cellular proteins for binding to the conformational domain. The conditions of cell growth had a profound effect on the expression of the p53 binding proteins. Considerably more p53 binding proteins were expressed in actively growing cells than in cultures maintained under conditions for slow growth. Quantitative differences in expression of p53-binding proteins were observed among different NSCLC cell lines. The expression of p53-binding proteins was not restricted to NSCLC cell lines; detergent extracts of an osteosarcoma cell line yielded similar p53-binding proteins.
Insights
Researchers identified cellular proteins that bind to the p53 tumor suppressor protein, revealing new insights into cell growth regulation. These p53-binding proteins
Area of Science:
- Molecular Biology
- Cancer Research
- Cell Biology
Background:
- Understanding the tumor suppressor protein p53's mechanism in cell growth control requires identifying its regulated genes and modulating factors.
- The SV40 large T-antigen inhibits p53's DNA-binding and transcription activities by binding to a conserved conformational domain.
- Cellular proteins may also regulate p53 function by binding to this same domain.
Purpose of the Study:
- To identify cellular proteins that bind to the conformational domain of p53.
- To investigate the regulation of p53-binding proteins under different cellular conditions.
Main Methods:
- A hybrid protein of Protein A fused to the p53 conformational domain (amino acids 115-295) was expressed in E. coli.
- This hybrid protein was used as an affinity probe to detect binding proteins in non-small cell lung carcinoma (NSCLC) cell lysates.
- Binding specificity was assessed using mutated p53 hybrid proteins and competition assays with SV40 T-antigen.
Main Results:
- Wild-type p53 hybrid protein specifically associated with cellular proteins of 45 K, 56 K, and 70 K, plus other minor species.
- Mutations in the p53 hybrid protein (codons 273 and 175) significantly reduced binding affinity (40-80%).
- SV40 T-antigen competed with cellular proteins for binding to the p53 conformational domain.
- Expression of p53-binding proteins was higher in actively growing cells and varied among NSCLC cell lines and other cancer types.
Conclusions:
- Specific cellular proteins bind to the conformational domain of p53, potentially regulating its function.
- Cellular growth conditions and cell type influence the expression levels of these p53-binding proteins.
- These findings contribute to understanding p53's role in cell growth and cancer.