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Conservation and function of a potential substrate-binding domain in the yeast Clb5 B-type cyclin
1The Rockefeller University, New York, NY 10021, USA. fcross@rockvax.rockefeller.edu
Molecular and Cellular Biology
|June 10, 2000
Summary
The cyclin A-binding region is conserved across species, suggesting a shared role in substrate targeting. Mutations disrupting this conserved region impact cyclin function, supporting its modular nature.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Cyclin A possesses a key region for binding the p27 inhibitor and substrates.
- This region exhibits significant evolutionary conservation in cyclins, including yeast B-type cyclins, despite lacking a direct p27 counterpart.
- The yeast S-phase cyclin Clb5p demonstrates interaction with mammalian p27.
Purpose of the Study:
- To investigate the functional significance of the conserved cyclin A-binding region.
- To determine if mutations affecting cyclin A-p27 interactions impact yeast cyclin function.
- To explore the role of this conserved domain in substrate targeting and biological activity.
Main Methods:
- Utilized yeast two-hybrid assays to assess interactions between yeast and mammalian proteins.
- Introduced specific mutations (hpm, Q241A) based on cyclin A-p27 crystal structure to disrupt binding.
- Assessed the impact of mutations on cyclin biological activity and kinase activity in various assays.
- Examined the effects of analogous mutations in mitotic cyclins (Clb2p).
Main Results:
- Yeast Clb5p interacted with mammalian p27, and this interaction was sensitive to mutations disrupting hydrophobic or hydrogen bonding interactions.
- Mutations in the Clb5p p27-binding domain had minimal effect on Sic1p inhibition but significantly reduced Clb5p biological activity.
- Clb5p-associated kinase activity remained largely unaffected by these mutations.
- An analogous mutation in Clb2p reduced its mitotic function but enhanced its ability to perform Clb5p-specific functions in some contexts.
Conclusions:
- The study supports a model of a modular and structurally conserved cyclin domain crucial for substrate targeting.
- This conserved domain plays a significant role in regulating cyclin biological activity, independent of kinase activity.
- Findings suggest evolutionary conservation of a functional module within cyclins for substrate interaction.