Temporal control of the dephosphorylation of Cdk substrates by mitotic exit pathways in budding yeast

Abstract

The temporal phosphorylation of cell cycle-related proteins by cyclin-dependent kinases (Cdks) is critical for the correct order of cell cycle events. In budding yeast, CDC28 encodes the only Cdk and its association with various cyclins governs the temporal phosphorylation of Cdk substrates. S-phase Cdk substrates are phosphorylated earlier than mitotic Cdk substrates, which ensures the sequential order of DNA synthesis and mitosis. However, it remains unclear whether Cdk substrates are dephosphorylated in temporally distinct windows. Cdc14 is a conserved protein phosphatase responsible for the dephosphorylation of Cdk substrates. In budding yeast, FEAR (Cdc14 early anaphase release) and MEN (mitotic exit network) activate phosphatase Cdc14 by promoting its release from the nucleolus in early and late anaphase, respectively. Here, we show that the sequential Cdc14 release and the distinct degradation timing of different cyclins provides the molecular basis for the differential dephosphorylation windows of S-phase and mitotic cyclin substrates. Our data also indicate that FEAR-induced dephosphorylation of S-phase Cdk substrates facilitates anaphase progression, revealing an extra layer of mitotic regulation.Cdc14 phosphatase ͉ spindle ͉ Clb5 ͉ Clb2 ͉ anaphase

Cdc14-Dependent Dephosphorylation of a Kinetochore Protein Prior to Anaphase in Saccharomyces cerevisiae

“…Unexpectedly, the Cdc14-dependent dephosphorylation of Dsn1 occurs prior to anaphase when Cdc14 is sequestered in nucleolus and the bulk of known Cdc14 targets are not dephosphorylated, including the Fin1 kinetochore protein that is readily dephosphorylated by Cdc14 at the onset of anaphase (e.g., Shou et al 1999;Visintin et al 1999;Jaspersen and Morgan 2000;Pereira and Schiebel 2003;Jin et al 2008;Akiyoshi et al 2009;Konig et al 2010). To our knowledge, this is the first observation of a nonnucleolar protein that is dephosphorylated in a Cdc14-dependent manner prior to anaphase onset, suggesting that there is a pool of active Cdc14 outside of nucleolus.…”

The multilayer regulation of the metaphase-to-anaphase transition

“…In support of the negative role of Clb5 in anaphase, we have shown that overexpression of CLB5 results in the accumulation of large budded cells with an undivided nucleus. 16 In summary, Clb5-Cdk1 promotes the initiation of DNA synthesis by phosphorylating DNA synthesisrelated proteins. However, on the other hand, Clb5-Cdk1 plays a negative role in anaphase by phosphorylating microtubule-associated proteins before anaphase onset.…”

“…The loss of function of the phosphatase Cdc14 abolishes the dephosphorylation of both Sld2 and Pol12, suggesting that either FEAR or MEN-dependent Cdc14 release is required for the dephosphorylation of both Clb5 and Clb2 substrates. 4,16 The FEAR-induced Cdc14 release during early anaphase raises the possibility that the FEAR pathway contributes to the dephosphorylation of Clb5 substrates immediately after anaphase onset. We found that the inactivation of the MEN pathway prevents the dephosphorylation of Clb2 substrate, but the Clb5 substrate is still able to be dephosphorylated.…”

“…Strikingly, the inactivation of both MEN and FEAR blocks the dephosphorylation of Clb5 substrates, indicating that FEAR-induced Cdc14 release is sufficient for the dephosphorylation of Clb5 substrates, but not for Clb2 substrates. 16 However, we need to keep in mind that not all the Clb5 substrates are subjected to Cdc14-dependent dephosphorylation. For example, the phosphorylation status of Orc6, another Clb5-specific substrate, is insensitive to Cdc14 release.…”

“…We conclude that the balance between the phosphatase Cdc14 and the cyclin protein levels determines dephosphorylation window of a given Cdk substrate. 16 …”