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ARF function does not require p53 stabilization or Mdm2 relocalization
Chandrashekhar Korgaonkar1, Lili Zhao, Modestos Modestou
1Department of Pharmacology. Molecular Biology Graduate Program, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA.
Abstract:
It is generally accepted that the ARF tumor suppressor induces p53-dependent growth arrest by sequestering the p53 antagonist Mdm2 in the nucleolus. Previous mutagenic studies of murine ARF suggested that residues 1 through 14 and 26 through 37 were critical for Mdm2 binding, while the latter domain also governed ARF nucleolar localization. We show that mouse ARF residues 6 to 10 and 21 to 25 are required for ARF-induced growth arrest whereas residues 1 to 5 and 29 to 34 are dispensable. Deletion of the putative nucleolar localization signal (31)RRPR(34) did not prevent nucleolar localization. Surprisingly, unlike wild-type ARF, growth-inhibitory mutants D1-5 and D29-34 failed to stabilize p53 yet induced its transcriptional activation in reporter assays. This suggests that p53 stabilization is not essential for ARF-mediated activation of p53. Like wild-type ARF, both mutants also exhibited p53-independent function since they were able to arrest p53/Mdm2-null cells. Notably, other mutants lacking conserved residues 6 to 10 or 21 to 25 were unable to suppress growth in p53-positive cells despite nucleolar localization and the ability to import Mdm2. Those observations stood in apparent contrast to the ability of wild-type ARF to block growth in some cells without relocalizing endogenous Mdm2 to nucleoli. Together, these data show a lack of correlation between ARF activity and Mdm2 relocalization, suggesting that additional events other than Mdm2 import are required for ARF function.
Insights
The ARF tumor suppressor
Area of Science:
- Oncology
- Molecular Biology
- Cellular Biology
Background:
- The ARF tumor suppressor is known to induce p53-dependent growth arrest by sequestering Mdm2 in the nucleolus.
- Previous studies identified residues 1-14 and 26-37 of murine ARF as critical for Mdm2 binding and nucleolar localization.
Purpose of the Study:
- To investigate the specific roles of ARF residues in Mdm2 binding, nucleolar localization, and p53-dependent and -independent growth arrest.
- To determine if p53 stabilization is essential for ARF-mediated p53 transcriptional activation.
- To explore the relationship between ARF's growth-suppressive activity and Mdm2 relocalization.
Main Methods:
- Site-directed mutagenesis of mouse ARF to create deletion mutants.
- Assays for ARF-induced growth arrest, p53 stabilization, and p53 transcriptional activation.
- Analysis of ARF nucleolar localization and Mdm2 import in p53-positive and p53/Mdm2-null cells.
Main Results:
- ARF residues 6-10 and 21-25 are crucial for ARF-induced growth arrest, while residues 1-5 and 29-34 are not.
- Mutants D1-5 and D29-34 induced p53 transcriptional activation without stabilizing p53, and retained p53-independent growth arrest.
- Mutants lacking residues 6-10 or 21-25 failed to suppress growth in p53-positive cells despite nucleolar localization and Mdm2 import.
Conclusions:
- p53 stabilization is not required for ARF-mediated p53 transcriptional activation.
- ARF exhibits p53-independent growth-suppressive functions.
- ARF activity is not directly correlated with Mdm2 relocalization, indicating other mechanisms are involved in ARF tumor suppression.