Functions and regulation of the Polo-like kinase Cdc5 in the absence and presence of DNA damage

Abstract

Polo-like kinases are essential cell cycle regulators that are conserved from yeast to humans. Unlike higher eukaryotes, who express multiple Polo-like kinase family members that perform many important functions, budding yeast express only a single Polo-like kinase, Cdc5, which is the homolog of mammalian cell cycle master regulator Polo-like kinase 1. Cdc5 is a fascinating multifaceted protein that is programmed to target its many substrates in a timely, sequential manner to ensure proper cell cycle progression. Over the years, many lessons about Polo-like kinase 1 have been learned by studying Cdc5 in budding yeast. Cdc5 has been well documented in regulating mitotic entry, chromosome segregation, mitotic exit, and cytokinesis. Cdc5 also plays important roles during cell division after DNA damage. Here, we briefly review the many functions of Cdc5 and its regulation in the absence and presence of DNA damage.

Keywords: Cdc5; Cell cycle; DNA damage; Mitosis; Plk1; Polo-like kinase 1.

Figure 1.. Key motifs found inside Cdc5.

Cdc5 consists of a kinase domain in its N-terminal half, and a polo-box domain (PBD) towards its C-terminus. The destruction sequences (two Destruction Boxes and a KEN motif), which are recognized by APC^Cdh1 for proteasome-mediated degradation in G1 phase, lie in the N-terminus. The Cdc5 kinase domain, through which Cdc5 phosphorylates its substrates, contains a nuclear localization signal. Residue T242 is phosphorylated by Cdk1, which is required for the kinase activity of Cdc5. Phosphorylation of T238 is required for full kinase activity and for recruitment of Cdc5 to SPBs after DNA-damage. The L251W mutation is found in the adaptation-defective cdc5-ad allele. The Polo Box Domain (PBD), made up of two polo boxes, is required for Cdc5 binding to its substrates. Mutating W517/V518/L530 to F/A/A (cdc5-FAA) prevents Cdc5 from localizing to its substrates and is lethal (Song et al. 2000). The cdc5–16 (W517F H641A K643M) PBD mutant cannot bind to substrates that have undergone a priming phosphorylation event; however, this mutant still allows for cell viability.

Figure 2.. Overview of Cdc5 functions in key cell cycle pathways.

A) To initiate mitotic entry, Cdc5 promotes expression of mitotic cyclin Clb2 and inactivates Swe1 kinase. Swe1 phosphorylates Cdk1 and prevents Cdk1 from interacting with Clb2. At G2/M, Swe1 gets phosphorylated by Cdc5. Hsl1/7 are required for efficient phosphorylation of Swe1 by Cdc5. This phosphorylation of Swe1 targets it for degradation. Mih1 dephosphorylates Cdk1 and allows Cdk1 to bind Clb2 and trigger entry into mitosis. B) In metaphase, Cdc5 phosphorylates cohesin kleisin subunit Scc1. This phosphorylation is antagonized by PP2A^Cdc55, which is down-regulated by Esp1 once the spindle assembly checkpoint (SAC) (Wang et al. 2014) has been satisfied. Esp1 cleaves phosphorylated Scc1, allowing sister chromatid separation. C) Mitotic exit in budding yeast is driven by Cdc14 phosphatase, which antagonizes Cdk1 activity. Prior to anaphase, Cdc14 is sequestered in the nucleolus, spatially separated from its nuclear and cytoplasmic substrates, by anchoring protein Net1/Cfi1. In early anaphase, phosphorylation of Net1/Cfi1 by Cdk1 and Cdc5 triggers the release of Cdc14 to the nucleus (FEAR). FEAR is also controlled by other components including Esp1, Slk19, and Spo12. The second mitotic exit pathway, the MEN, is triggered in the cytoplasm by the Tem1 GTPase, which is negatively regulated by the Bfa1/Bub2 GAP and positively regulated by the Lte1 GEF. When Cdc14 is released from the nucleus by FEAR, it triggers the release of Cdc5 from the nucleus to the cytoplasm. In the cytoplasm, Cdc5 activates the MEN by phosphorylating the Bfa1/Bub2 and Cdc15. MEN promotes the release of Cdc14 from the nucleus to the cytoplasm, further driving mitotic exit. D) Cdc5 controls multiple aspects of cytokinesis. Cdc5 phosphorylates Rho1 GEFs Tus1 and Rom2, which promotes contractile actin ring (CAR) assembly. Cdc5 promotes the recruitment of F-BAR protein Hof1 to the bud neck to trigger CAR contraction. Cdc5 also promotes cell separation by inhibiting Cdc42 and regulates the cell size at the bud neck by preventing Sec4 from interacting with its effector protein Sec15.

Figure 3.. Cdc5 localization during the cell cycle.

Cdc5 starts to accumulate at the SPB in S-phase. Following SPB duplication, in G2, Cdc5 accumulates in the nucleus and decorates the nuclear surface of SPBs. Cdc5 remains in the nucleus and at both SPBs in early anaphase. Later in anaphase, Cdc5 is released from the nucleus to the cytoplasm. In late anaphase, Cdc5 localizes strongly at the cytoplasmic side of the daughter SPB, and weakly at the mother SPB, as well as at the bud neck. After cytokinesis is complete, Cdc5 gets targeted by APC^Cdh1 for degradation in G1 of the following cell cycle.