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Cooperative signals governing ARF-mdm2 interaction and nucleolar localization of the complex
J D Weber1, M L Kuo, B Bothner
1Howard Hughes Medical Institute, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.
Abstract:
The ARF tumor suppressor protein stabilizes p53 by antagonizing its negative regulator, Mdm2 (Hdm2 in humans). Both mouse p19(ARF) and human p14(ARF) bind to the central region of Mdm2 (residues 210 to 304), a segment that does not overlap with its N-terminal p53-binding domain, nuclear import or export signals, or C-terminal RING domain required for Mdm2 E3 ubiquitin ligase activity. The N-terminal 37 amino acids of mouse p19(ARF) are necessary and sufficient for binding to Mdm2, localization of Mdm2 to nucleoli, and p53-dependent cell cycle arrest. Although a nucleolar localization signal (NrLS) maps within a different segment (residues 82 to 101) of the human p14(ARF) protein, binding to Mdm2 and nucleolar import of ARF-Mdm2 complexes are both required for cell cycle arrest induced by either the mouse or human ARF proteins. Because many codons of mouse ARF mRNA are not recognized by the most abundant bacterial tRNAs, we synthesized ARF minigenes containing preferred bacterial codons. Using bacterially produced ARF polypeptides and chemically synthesized peptides conjugated to Sepharose, residues 1 to 14 and 26 to 37 of mouse p19(ARF) were found to interact independently and cooperatively with Mdm2, while residues 15 to 25 were dispensable for binding. Paradoxically, residues 26 to 37 of mouse p19(ARF) are also essential for ARF nucleolar localization in the absence of Mdm2. However, the mobilization of the p19(ARF)-Mdm2 complex into nucleoli also requires a cryptic NrLS within the Mdm2 C-terminal RING domain. The Mdm2 NrLS is unmasked upon ARF binding, and its deletion prevents import of the ARF-Mdm2 complex into nucleoli. Collectively, the results suggest that ARF binding to Mdm2 induces a conformational change that facilitates nucleolar import of the ARF-Mdm2 complex and p53-dependent cell cycle arrest. Hence, the ARF-Mdm2 interaction can be viewed as bidirectional, with each protein being capable of regulating the subnuclear localization of the other.
Insights
The ARF tumor suppressor protein stabilizes p53 by binding Mdm2. This interaction triggers ARF-Mdm2 complex nucleolar import, leading to p53-dependent cell cycle arrest.
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Research
Background:
- ARF (alternative reading frame) is a tumor suppressor that stabilizes p53.
- Mdm2 is a negative regulator of p53 and an E3 ubiquitin ligase.
- ARF antagonizes Mdm2, preventing p53 degradation.
Purpose of the Study:
- To elucidate the molecular mechanisms of ARF-Mdm2 interaction.
- To determine the roles of specific ARF and Mdm2 domains in complex formation and nucleolar localization.
- To understand how this interaction leads to p53-dependent cell cycle arrest.
Main Methods:
- Bacterial expression and synthesis of ARF minigenes and polypeptides.
- Peptide conjugation to Sepharose for binding assays.
- Analysis of protein-protein interactions and subnuclear localization (nucleolar import).
Main Results:
- ARF binds to the central region of Mdm2 (residues 210-304).
- Specific ARF residues (1-14 and 26-37) are crucial for Mdm2 binding.
- ARF binding unmasks a cryptic nucleolar localization signal (NrLS) in Mdm2's RING domain, facilitating complex import.
- ARF also possesses an NrLS essential for its own nucleolar localization.
Conclusions:
- ARF-Mdm2 interaction is bidirectional, with each protein regulating the other's subnuclear localization.
- ARF binding induces a conformational change in Mdm2, promoting nucleolar import of the complex.
- This facilitated nucleolar import is critical for ARF-mediated p53 stabilization and cell cycle arrest.