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Regulation of the mitotic exit protein kinases Cdc15 and Dbf2
1Center for Cancer Research, Howard Hughes Medical Institute, Massachusetts Institute of Technology, E17-233, 40 Ames Street, Cambridge MA 02139, USA.
Abstract:
In budding yeast, the release of the protein phosphatase Cdc14 from its inhibitor Cfi1/Net1 in the nucleolus during anaphase triggers the inactivation of Clb CDKs that leads to exit from mitosis. The mitotic exit pathway controls the association between Cdc14 and Cfi1/Net1. It is comprised of the RAS-like GTP binding protein Tem1, the exchange factor Lte1, the GTPase activating protein complex Bub2-Bfa1/Byr4, and several protein kinases including Cdc15 and Dbf2. Here we investigate the regulation of the protein kinases Dbf2 and Cdc15. We find that Cdc15 is recruited to both spindle pole bodies (SPBs) during anaphase. This recruitment depends on TEM1 but not DBF2 or CDC14 and is inhibited by BUB2. Dbf2 also localizes to SPBs during anaphase, which coincides with activation of Dbf2 kinase activity. Both events depend on the mitotic exit pathway components TEM1 and CDC15. In cells lacking BUB2, Dbf2 localized to SPBs in cell cycle stages other than anaphase and telophase and Dbf2 kinase was prematurely active during metaphase. Our results suggest an order of function of mitotic exit pathway components with respect to SPB localization of Cdc15 and Dbf2 and activation of Dbf2 kinase. BUB2 negatively regulates all 3 events. Loading of Cdc15 on SPBs depends on TEM1, whereas loading of Dbf2 on SPBs and activation of Dbf2 kinase depend on TEM1 and CDC15.
Insights
The mitotic exit pathway in budding yeast involves protein kinases Cdc15 and Dbf2. Bub2 negatively regulates their spindle pole body localization and Dbf2 kinase activity, suggesting a specific order of function for these key cell cycle regulators.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- Mitotic exit is crucial for cell division, regulated by the protein phosphatase Cdc14 release.
- The mitotic exit pathway involves key proteins like Tem1, Lte1, Bub2-Bfa1/Byr4, Cdc15, and Dbf2.
- Understanding the precise roles and regulation of these components is essential for comprehending cell cycle control.
Purpose of the Study:
- To investigate the regulation of protein kinases Cdc15 and Dbf2 within the budding yeast mitotic exit pathway.
- To determine the order of function of mitotic exit pathway components regarding spindle pole body (SPB) localization and kinase activation.
- To elucidate the inhibitory role of Bub2 in these processes.
Main Methods:
- Utilized budding yeast as a model organism.
- Employed immunofluorescence microscopy to track protein localization (Cdc15, Dbf2) to spindle pole bodies (SPBs).
- Performed genetic analysis using deletion mutants (e.g., bub2, tem1, dbf2, cdc14) to assess pathway component dependencies and kinase activity.
Main Results:
- Cdc15 is recruited to SPBs during anaphase, dependent on TEM1 but inhibited by BUB2.
- Dbf2 also localizes to SPBs during anaphase, coinciding with its kinase activation; both depend on TEM1 and CDC15.
- Loss of BUB2 leads to ectopic Dbf2 SPB localization and premature kinase activation during metaphase.
Conclusions:
- Established a functional order for mitotic exit pathway components: TEM1 is required for Cdc15 SPB loading.
- TEM1 and CDC15 are necessary for Dbf2 SPB loading and kinase activation.
- BUB2 acts as a negative regulator, inhibiting SPB localization of Cdc15 and Dbf2, and Dbf2 kinase activation, ensuring timely mitotic exit.