Cyclin E-mediated elimination of p27 requires its interaction with the nuclear pore-associated protein mNPAP60.
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.
Müller
D
D
Institute for Molecular Biology and Tumour Research, University of Marburg, Emil Mannkopff-Strasse 2, 35033 Marburg, Martinsried, Germany.
Thieke
K
K
Bürgin
A
A
Dickmanns
A
A
Eilers
M
M
D016475:3T3 Cells; D000818:Animals; D042846:CDC2-CDC28 Kinases; D002453:Cell Cycle; D018797:Cell Cycle Proteins; D019927:Cyclin E / Q000378:metabolism*; D051357:Cyclin-Dependent Kinase 2; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation; D017354:Point Mutation; D018272:Porins / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D015398:Signal Transduction; D025521:Tumor Suppressor Proteins
C493961:Cdkn1b protein, mouse; D018797:Cell Cycle Proteins; D019927:Cyclin E; D008869:Microtubule-Associated Proteins; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; C105412:Nup50 protein, mouse; D018272:Porins; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; C495893:Cdk2 protein, mouse; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases