A cyclin D-CDK6 dimer helps to reshuffle cyclin-dependent kinase inhibitors (CKI) to overcome TGF-beta-mediated
Sarah E Nataraj1, Stacy W Blain1,2
1Program in Molecular and Cellular Biology, School of Graduate Studies, SUNY Downstate Medical Center, Brooklyn, New York.
Abstract:
The cyclin D-CDK4/6 complex has two distinct functions. Its kinase-dependent role involves its ability to act as serine/threonine kinase, responsible for phosphorylation of substrates required for cell cycle transitions, while its kinase-independent function involves its ability to act as a reservoir for p27Kip1. This association sequesters p27 from cyclin E-CDK2 complexes, allowing them to remain active. The aim of this current study is two-fold: to understand the contribution of the kinase-dependent and kinase-independent functions of CDK4 and CDK6 in epithelial cells and to directly compare CDK4 and CDK6 in a simple model system, TGF-β treatment, where arrest is initiated by the expression of p15Ink4b. Cells that overexpressed a catalytically inactive, p15-insensitive CDK6 variant (p27 sequestration only mutant) were able to overcome TGF-β-mediated arrest by maintaining CDK2 activity, while cells expressing the identical mutations in CDK4 were not. This result can be partially explained by the presence of a previously unidentified cyclin D-CDK6 dimer, which serves as a sink for free p27 during TGF-β treatment, enabling CDK2 to remain inhibitor free. The use of the TGF-β model system and the characterization of CDK pool dynamics and p27 switching is relevant to the CDK4/6 specific inhibitors, such as palbociclib, whose mechanism of action may resemble that of p15.
Insights
Cyclin D-CDK4/6 complexes have kinase-dependent and independent roles. CDK6
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Cyclin D-CDK4/6 complexes regulate cell cycle transitions.
- These complexes possess both kinase-dependent and kinase-independent functions.
- Kinase-independent function involves sequestering p27 Kip1, maintaining cyclin E-CDK2 activity.
Purpose of the Study:
- To elucidate the roles of kinase-dependent and -independent functions of CDK4 and CDK6 in epithelial cells.
- To compare CDK4 and CDK6 functions using a TGF-β treatment model.
- To investigate the mechanism of TGF-β-induced cell cycle arrest mediated by p15 Ink4b.
Main Methods:
- Overexpression of catalytically inactive CDK6 and CDK4 variants in epithelial cells.
- Utilizing TGF-β treatment to induce cell cycle arrest.
- Assessing CDK2 activity and p27 Kip1 levels in response to TGF-β.
- Characterizing cyclin D-CDK6 dimer formation.
Main Results:
- Cells overexpressing a p15-insensitive CDK6 variant overcame TGF-β arrest by maintaining CDK2 activity.
- Cells expressing the equivalent CDK4 mutation did not overcome arrest.
- A novel cyclin D-CDK6 dimer was identified as a p27 Kip1 sink during TGF-β treatment.
- This dimer facilitated sustained CDK2 activity by keeping it inhibitor-free.
Conclusions:
- CDK6's kinase-independent function, via p27 sequestration, is crucial for overcoming TGF-β-induced arrest.
- A previously unrecognized cyclin D-CDK6 dimer plays a key role in regulating p27 Kip1 availability.
- Findings offer insights into CDK4/6 inhibitors like palbociclib, suggesting similar mechanisms involving p15 Ink4b.
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