Global Analysis of Cdc14 Phosphatase Reveals Diverse Roles in Mitotic Processes

Abstract

Cdc14 phosphatase regulates multiple events during anaphase and is essential for mitotic exit in budding yeast. Cdc14 is regulated in both a spatial and temporal manner. It is sequestered in the nucleolus for most of the cell cycle by the nucleolar protein Net1 and is released into the nucleus and cytoplasm during anaphase. To identify novel binding partners of Cdc14, we used affinity purification of Cdc14 and mass spectrometric analysis of interacting proteins from strains in which Cdc14 localization or catalytic activity was altered. To alter Cdc14 localization, we used a strain deleted for NET1, which causes full release of Cdc14 from the nucleolus. To alter Cdc14 activity, we generated mutations in the active site of Cdc14 (C283S or D253A), which allow binding of substrates, but not dephosphorylation, by Cdc14. Using this strategy, we identified new interactors of Cdc14, including multiple proteins involved in mitotic events. A subset of these proteins displayed increased affinity for catalytically inactive mutants of Cdc14 compared with the wild-type version, suggesting they are likely substrates of Cdc14. We have also shown that several of the novel Cdc14-interacting proteins, including Kar9 (a protein that orients the mitotic spindle) and Bni1 and Bnr1 (formins that nucleate actin cables and may be important for actomyosin ring contraction) are specifically dephosphorylated by Cdc14 in vitro and in vivo. Our findings suggest the dephosphorylation of the formins may be important for their observed localization change during exit from mitosis and indicate that Cdc14 targets proteins involved in wide-ranging mitotic events.

Mechanisms of cytokinesis in budding yeast

“…Bnr1 targets to the septin hourglass via distinct domains during different phases of the cell cycle [Gao et al, 2010] but details surrounding its interactions with the septins remain to be determined. The mitotic exit phosphatase Cdc14 is not only required for the timed disappearance of Bnr1 from the bud neck but also required for the timed appearance of Bni1 at the division site during cytokinesis [Bloom et al, 2011]. Consistently, Bni1 fails to localize in the MEN mutants such as cdc15-2 cells [Bloom et al, 2011].…”

“…The mitotic exit phosphatase Cdc14 is not only required for the timed disappearance of Bnr1 from the bud neck but also required for the timed appearance of Bni1 at the division site during cytokinesis [Bloom et al, 2011]. Consistently, Bni1 fails to localize in the MEN mutants such as cdc15-2 cells [Bloom et al, 2011]. However, another study indicates that Bni1 is able to localize to the division site in the cdc15-2 cells [Yoshida et al, 2006].…”

Hippo signalling in the G2/M cell cycle phase: Lessons learned from the yeast MEN and SIN pathways

“…By changing the phosphorylation status of the formins Bnr1 and Bni1, Cdc14p can affect the repolarisation of the cytoskeleton at the end of mitosis [42]. Cdc14p may also directly regulate Boi1, Boi2, Chs1, Shs1 and other mitotic factors to facilitate cytokinesis [33], however, the direct regulation of these proteins by Cdc14p is yet to be established.…”

Cdk1-dependent phosphorylation of Iqg1 governs actomyosin ring assembly prior to cytokinesis

“…Dephosphorylation of Cdk1 substrates underlies many events of late mitosis and cytokinesis (Bloom et al, 2011;Chin et al, 2012;Khmelinskii et al, 2009;Mishima et al, 2004;Sullivan et al, 2008;Teh et al, 2009). Cdc14 localizes to the bud neck prior to the onset of cytokinesis and is known to promote primary septum deposition (Palani et al, 2012).…”