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Cyclin E-mediated elimination of p27 requires its interaction with the nuclear pore-associated protein mNPAP60
1Institute for Molecular Biology and Tumour Research, University of Marburg, Emil Mannkopff-Strasse 2, 35033 Marburg, Martinsried, Germany.
Abstract:
The Cdk2 inhibitor, p27(Kip1), is degraded in a phosphorylation- and ubiquitylation-dependent manner at the G(1)-S transition of the cell cycle. Degradation of p27(Kip1) requires import into the nucleus for phosphorylation by Cdk2. Phosphorylated p27(Kip1) is thought to be subsequently re-exported and degraded in the cytosol. Using two-hybrid screens, we now show that p27(Kip1) interacts with a nuclear pore-associated protein, mNPAP60, map the interaction to the 3(10) helix of p27 and identify a point mutant in p27(Kip1) that is deficient for interaction (R90G). In vivo and in vitro, the loss-of-interaction mutant is poorly transported into the nucleus, while ubiquitylation of p27R90G occurs normally. In vivo, co-expression of cyclin E and Cdk2 rescues the import defect. However, mutant p27(Kip1) accumulates in a phosphorylated form in the nucleus and is not efficiently degraded, arguing that at least one step in the degradation of phosphorylated p27(Kip1) requires an interaction with the nuclear pore. Our results identify a novel component involved in p27(Kip1) degradation and suggest that degradation of p27(Kip1) is tightly linked to its intracellular transport.
Insights
The cell cycle protein p27(Kip1) degradation requires nuclear import for phosphorylation. A novel interaction with nuclear pore protein mNPAP60 is crucial for this process and subsequent degradation.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- p27(Kip1) is a Cdk2 inhibitor degraded during the G1-S cell cycle transition.
- Degradation is phosphorylation- and ubiquitylation-dependent, requiring nuclear import for Cdk2-mediated phosphorylation.
- Phosphorylated p27(Kip1) is hypothesized to be re-exported for cytosolic degradation.
Purpose of the Study:
- To identify novel proteins involved in p27(Kip1) degradation.
- To elucidate the role of intracellular transport in p27(Kip1) regulation.
Main Methods:
- Yeast two-hybrid screens to identify interacting proteins.
- Site-directed mutagenesis to create interaction-deficient p27(Kip1) mutants (R90G).
- In vivo and in vitro assays to assess nuclear transport, phosphorylation, ubiquitylation, and degradation.
Main Results:
- Identified mNPAP60, a nuclear pore-associated protein, as an interactor of p27(Kip1).
- Mapped the interaction to the 3(10) helix of p27(Kip1); the R90G mutant shows impaired nuclear import.
- Loss of mNPAP60 interaction leads to nuclear accumulation of phosphorylated p27(Kip1), hindering its degradation.
Conclusions:
- mNPAP60 is a novel component critical for p27(Kip1) degradation.
- p27(Kip1) degradation is intrinsically linked to its nuclear transport, with nuclear pore interaction being essential for efficient clearance of phosphorylated p27(Kip1).