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The subcellular locations of p15(Ink4b) and p27(Kip1) coordinate their inhibitory interactions with cdk4 and cdk2
1Cell Biology Program and Howard Hughes Medical Institute, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
Abstract:
In dividing cells, p27(Kip1) is predominantly bound to cyclin D-cdk4 without inhibiting this kinase. Upon being induced by TGF-beta or with a conditional expression system in lung epithelial cells, p15(Ink4b) binds to and inhibits the cyclin D-dependent kinases, prevents p27 binding to these cdk complexes, and promotes p27 binding and inhibition of cyclin-cdk2. In vitro, however, p15 prevents p27 binding only if it has access to cyclin D-cdk4 first. We present evidence that the different subcellular location of p15 and p27 ensures the prior access of p15 to cdk4. In the cell, p15 is localized mostly in the cytoplasm, whereas p27 is nuclear. p15 prevails over p27 or a p27 construct consisting of the cdk inhibitory domain tagged with a nuclear localization signal. However, when p15 and p27 are forced to reside in the same subcellular location, either the cytoplasm or the nucleus, p15 no longer prevents p27 from binding to cdk4. These properties allow p15 and p27 to coordinately inhibit cdk4 and cdk2.
Insights
Cellular localization dictates the interaction of p15(Ink4b) and p27(Kip1) with cyclin-dependent kinases (cdks). This spatial regulation ensures p15 inhibits cyclin D-cdk4, allowing p27 to inhibit cyclin-cdk2, coordinating cell cycle control.
Area of Science:
- Cell biology
- Molecular biology
- Biochemistry
Background:
- p27(Kip1) binds cyclin D-cdk4 in dividing cells but does not inhibit it.
- p15(Ink4b) is induced by TGF-beta and inhibits cyclin D-dependent kinases.
- The interplay between p15 and p27 in regulating cell cycle kinases is not fully understood.
Purpose of the Study:
- To investigate the role of subcellular localization in the interaction of p15(Ink4b) and p27(Kip1) with cyclin-dependent kinases.
- To elucidate how p15 and p27 coordinately regulate cdk4 and cdk2 activity.
Main Methods:
- Conditional expression system in lung epithelial cells.
- In vitro binding assays.
- Subcellular localization studies using wild-type and engineered p15 and p27 proteins.
Main Results:
- p15(Ink4b) predominantly localizes to the cytoplasm, while p27(Kip1) is primarily nuclear.
- Cytoplasmic p15(Ink4b) effectively prevents p27(Kip1) binding to cyclin D-cdk4.
- When co-localized, p15 no longer inhibits p27 binding to cdk4, allowing coordinated inhibition of cdk4 and cdk2.
Conclusions:
- Subcellular localization is critical for the distinct roles of p15 and p27 in cell cycle regulation.
- p15's cytoplasmic localization ensures its prior access to cyclin D-cdk4, enabling sequential inhibition.
- These findings reveal a mechanism for coordinate regulation of cdk4 and cdk2 by p15 and p27 through spatial control.