Regulation of the mitotic exit protein kinases Cdc15 and Dbf2

In budding yeast, the release of the protein phosphatase Cdc14 from its inh ibitor Cfi1/Net1 in the nucleolus during anaphase triggers the inactivation of Clb CDKs that leads to exit from mitosis. The mitotic exit pathway cont rols the association between Cdc14 and Cfi1/Net1. It is comprised of the RA S-like GTP binding protein Tem1, the exchange factor Lte1, the GTPase activ ating protein complex Bub2-Bfa1 / Byr4, and several protein kinases includi ng Cdc15 and Dbf2. Here we investigate the regulation of the protein kinase s. Dbf2 and Cdc15. We find that Cdc15 is recruited to both spindle pole bod ies (SPBs) during anaphase. This recruitment depends on TEM1 but not DBF2 o r CDC14 and is inhibited by BUB2. Dbf2 also localizes to SPBs during anapha se, which coincides with activation of Dbf2 kinase activity. Both events de pend on the mitotic exit pathway components TEM1 and CDC15. In cells lackin g BUB2, Dbf2 localized to SPBs. in cell cycle stages other than anaphase an d telophase and Dbf2 kinase was prematurely active during metaphase. Our re sults 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 dep end on TEM1 and CDC15.