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Cyclin D1/cdk2 kinase is present in a G1 phase-specific protein complex Yi1 that binds to the mouse thymidine kinase
Q P Dou1, G Molnar, A B Pardee
1Department of Pharmacology, University of Pittsburgh School of Medicine, PA.
Abstract:
Our studies provide evidence for the presence of cyclin D1 in an early G1 cycle-specific DNA binding complex Yi1. Previously we identified several complexes including Yi and E2F that at different times during G0 to S transition bind to three distinct DNA sequences (MT1, MT2, MT3) located in the mouse thymidine kinase upstream promoter. These various complexes contain DNA binding proteins (Sp1, E2F, p110, p60), cyclins A and E, cyclin-dependent kinase 2 (cdk2), and retinoblastoma-related proteins (pRB, p107). Here we report that Yi1 is different from the E2F complexes. Yi1 contains cyclin D1/cdk2 kinase as shown by using specific antibodies to cyclins, cdks and the Yi1 DNA-binding protein in gel retardation, western blotting, and immunoprecipitation assays. Yi1 binding is specific to a consensus sequence different from that of E2F.
Insights
Researchers found cyclin D1 in a novel DNA-binding complex called Yi1 during early G1 phase. This complex differs from previously identified E2F complexes and binds to specific DNA sequences, advancing our understanding of cell cycle regulation.
Area of Science:
- Molecular Biology
- Cell Cycle Regulation
- Biochemistry
Background:
- Several DNA-binding complexes (Yi, E2F) involved in cell cycle progression from G0 to S phase have been identified.
- These complexes bind to specific DNA sequences in the mouse thymidine kinase promoter.
- Known components include Sp1, E2F, p110, p60, cyclins A and E, cyclin-dependent kinase 2 (cdk2), and retinoblastoma-related proteins (pRB, p107).
Purpose of the Study:
- To characterize a novel DNA-binding complex, Yi1, identified during early G1 phase.
- To determine the composition and DNA-binding specificity of the Yi1 complex.
- To differentiate Yi1 from previously identified E2F complexes.
Main Methods:
- Gel retardation assays using specific antibodies against cyclins, cdks, and the Yi1 DNA-binding protein.
- Western blotting to confirm protein presence and interactions.
- Immunoprecipitation assays to isolate and identify complex components.
Main Results:
- Evidence for the presence of cyclin D1 in the early G1 cycle-specific DNA-binding complex Yi1.
- Demonstration that Yi1 contains the cyclin D1/cdk2 kinase.
- Confirmation that Yi1 binding is specific to a consensus DNA sequence distinct from that of E2F complexes.
Conclusions:
- The Yi1 complex represents a distinct DNA-binding entity active in early G1 phase.
- Cyclin D1, in complex with cdk2, is a component of the Yi1 complex.
- Yi1 exhibits unique DNA-binding specificity compared to E2F complexes, contributing to cell cycle regulation insights.