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C-terminus of p53 is required for G(2) arrest
Seiichi Nakamura1, Yoshihito Gomyo, Jack A Roth
1Section of Thoracic Molecular Oncology, Department of Thoracic and Cardiovascular Surgery, Box 109, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, Texas, TX 77030, USA.
Abstract:
Mutation of four lysine residues in the p53 C-terminal domain inhibits MDM2-dependent ubiquitination of p53 and alters its subcellular distribution. This implies that modification (such as acetylation and phosphorylation) of amino acid residues in p53 C-terminal domain, regulate the biological functions of p53. In this study, we demonstrated that p53 with lysine residues 372, 373, 381, and 382 mutated to alanine (the A4 mutant) retained the transactivation activity of wild-type p53, although the transactivation activity of p21 promoter by the A4 mutant was slightly reduced. The inducible expression of wild-type p53 and the A4 mutant in H1299 cells caused growth inhibition due to cell-cycle arrest. Consistent with previous studies, the expression of wild-type p53 elicited G(1) and G(2) arrests. However, the cells expressing the A4 mutant underwent G(1) arrest but not G(2) arrest. Cyclin B1-associated kinase activity was reduced in cells expressing wild-type p53 but not A4, when the cells underwent G(2) arrest. This suggests that modification of the p53 C-terminal domain might inhibit p53-mediated G(2) arrest. In other words, p53 requires an intact C-terminus to induce G(2) arrest.
Insights
The p53 C-terminal domain is crucial for inducing G(2) cell-cycle arrest. Mutations in this region impair p53
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Post-translational modifications of the p53 C-terminal domain regulate its biological functions.
- Lysine residue mutations in the p53 C-terminal domain affect MDM2-dependent ubiquitination and subcellular localization.
- The p53 protein plays a critical role in cell-cycle regulation and tumor suppression.
Purpose of the Study:
- To investigate the role of specific lysine residues in the p53 C-terminal domain on its function.
- To determine the impact of mutations in the p53 C-terminal domain on transactivation activity and cell-cycle arrest.
- To elucidate the mechanism by which p53 induces G(2) arrest.
Main Methods:
- Site-directed mutagenesis to create the A4 mutant (lysines 372, 373, 381, 382 mutated to alanine).
- Inducible expression of wild-type p53 and the A4 mutant in H1299 cells.
- Cell-cycle analysis (flow cytometry) to assess G(1) and G(2) arrest.
- Western blotting to detect protein expression and cell-cycle regulators.
- Assay of cyclin B1-associated kinase activity.
Main Results:
- The A4 mutant retained transactivation activity, though slightly reduced for the p21 promoter.
- Both wild-type p53 and the A4 mutant induced growth inhibition and cell-cycle arrest.
- Wild-type p53 induced both G(1) and G(2) arrest, while the A4 mutant induced only G(1) arrest.
- Cyclin B1-associated kinase activity was reduced in wild-type p53 expressing cells during G(2) arrest, but not in A4 mutant expressing cells.
Conclusions:
- The p53 C-terminal domain is essential for mediating G(2) cell-cycle arrest.
- Modifications of the p53 C-terminal domain may inhibit p53-mediated G(2) arrest.
- An intact p53 C-terminus is required for the induction of G(2) arrest.