Rosella Visintin

The Phosphatase Cdc14 Triggers Mitotic Exit by Reversal of Cdk-Dependent Phosphorylation

Exit from mitosis requires the inactivation of mitotic cyclin-dependent kinases (CDKs) by an unknown mechanism. We show that the Cdc14 phosphatase triggers mitotic exit by three parallel mechanisms, each of which inhibits Cdk activity. Cdc14 dephosphorylates Sic1, a Cdk inhibitor, and Swi5, a transcription factor for SIC1, and induces degradation of mitotic cyclins, likely by dephosphorylating the activator of mitotic cyclin degradation, Cdh1/Hct1. Feedback between these pathways may lead to precipitous collapse of mitotic CDK activity and help coordinate exit from mitosis.

Cfi1 prevents premature exit from mitosis by anchoring Cdc14 phosphatase in the nucleolus.

In eukaryotes, the activation of mitotic cyclin-dependent kinases (CDKs) induces mitosis, and their inactivation causes cells to leave mitosis. In budding yeast, two redundant mechanisms induce the inactivation of mitotic CDKs. In one mechanism, a specialized ubiquitin-dependent proteolytic system (called the APC-dependent proteolysis machinery) degrades the mitotic (Clb) cyclin subunit. In the other, the kinase-inhibitor Sic1 binds to mitotic CDKs and inhibits their kinase activity,. The highly conserved protein phosphatase Cdc14 promotes both Clb degradation and Sic1 accumulation. Cdc14 promotes SIC1 transcription and the stabilization of Sic1 protein by dephosphorylating Sic1 and its transcription factor Swi5. Cdc14 activates the degradation of Clb cyclins by dephosphorylating the APC-specificity factor Cdh1 (refs 3, 4). So how is Cdc14 regulated? Here we show that Cdc14 is sequestered in the nucleolus for most of the cell cycle. During nuclear division, Cdc14 is released from the nucleolus, allowing it to reach its targets. A highly conserved signalling cascade, critical for the exit from mitosis, is required for this movement of Cdc14 during anaphase. Furthermore, we have identified a negative regulator of Cdc14, Cfi1, that anchors Cdc14 in the nucleolus.

Separase, polo kinase, the kinetochore protein Slk19, and Spo12 function in a network that controls Cdc14 localization during early anaphase

In budding yeast, the phosphatase Cdc14, a key regulator of exit from mitosis, is released from its inhibitor Cfi1/Net1 in the nucleolus during anaphase. A signaling cascade, known as the mitotic exit network (MEN), controls this release. We have identified a regulatory network, the FEAR (Cdc fourteen early anaphase release) network that promotes Cdc14 release from the nucleolus during early anaphase. The FEAR network is comprised of the polo kinase Cdc5, the separase Esp1, the kinetochore-associated protein Slk19, and Spo12. We also show that the FEAR network initiates Cdc14 release from Cfi1/Net1 during early anaphase, and MEN maintains Cdc14 in the released state during late anaphase. We propose that one function of Cdc14 released by the FEAR network is to stimulate MEN activity.

A Mechanism for Coupling Exit from Mitosis to Partitioning of the Nucleus

Exit from mitosis must not occur prior to partitioning of chromosomes between daughter cells. We find that the GTP binding protein Tem1, a regulator of mitotic exit, is present on the spindle pole body that migrates into the bud during S phase and mitosis. Tem1s exchange factor, Lte1, localizes to the bud. Thus, Tem1 and Lte1 are present in the same cellular compartment (the bud) only after the nucleus enters the bud during nuclear division. We also find that the presence of Tem1 and Lte1 in the bud is required for mitotic exit. Our results suggest that the spatial segregation of Tem1 and Lte1 ensures that exit from mitosis only occurs after the genetic material is partitioned between mother and daughter cell.

THE REGULATION OF CDC20 PROTEOLYSIS REVEALS A ROLE FOR THE APC COMPONENTS CDC23 AND CDC27 DURING S PHASE AND EARLY MITOSIS

BACKGROUND In eukaryotic cells, a specialized proteolysis machinery that targets proteins containing destruction-box sequences for degradation and that uses a ubiquitin ligase known as the anaphase-promoting complex/cyclosome (APC) plays a key role in the regulation of mitosis. APC-dependent proteolysis triggers the separation of sister chromatids at the metaphase-anaphase transition and the destruction of mitotic cyclins at the end of mitosis. Recently, two highly conserved WD40-repeat proteins, Cdc20 and Cdh1/Hct1, have been identified as substrate-specific regulators for APC-dependent proteolysis in the budding yeast Saccharomyces cerevisiae. Here, we have investigated the cell cycle regulation of Cdc20 and Cdh1/Hct1. RESULTS Whereas the levels CDH1/HCT1 RNA and Cdh1/Hct1 protein are constant throughout the cell cycle, CDC20 RNA and Cdc20 protein are present only during late S phase and mitosis and Cdc20 protein is unstable throughout the entire cell cycle. The instability of Cdc20 depends on CDC23 and CDC27, which encode components of the APC. During the G1 phase, a destruction box within Cdc20 mediates its instability, but during S phase and mitosis, although Cdc20 destruction is still dependent on CDC23 and CDC27, it does not depend on the Cdc20 destruction box. CONCLUSIONS There are remarkable differences in the regulation of Cdc20 and Cdh1/Hct1. Furthermore, the APC activator Cdc20 is itself a substrate of the Cdc27 have a role in the degradation of Cdc20 during S Phase and early mitosis that is not mediated by its destruction box.

The nucleolus: the magician's hat for cell cycle tricks

The nucleolus, for decades considered a ribosome factory and site for ribosomal RNA synthesis and processing, has recently acquired new fame. Analyses of proteins important for cell-cycle regulation have shown that this organelle is used to sequester proteins, thereby inhibiting their activity.