Independent modulation of the kinase and polo-box activities of Cdc5 protein unravels unique roles in the maintenance of genome stability

Abstract

Polo-like kinases (PLKs) are evolutionarily conserved kinases essential for cell cycle regulation. These kinases are characterized by the presence of a C-terminal phosphopeptide-interaction domain, the polo-box domain (PBD). How the functional domains of PLKs work together to promote cell division is not understood. To address this, we performed a genetic screen to identify mutations that independently modulate the kinase and PBD activities of yeast PLK/Cdc5. This screen identified a mutagenic hotspot in the F-helix region of Cdc5 kinase domain that allows one to control kinase activity in vivo. These mutations can be systematically engineered into other major eukaryotic cell cycle kinases to similarly regulate their activity in live cells. Here, using this approach, we show that the kinase activity of Cdc5 can promote the execution of several stages of mitosis independently of PBD activity. In particular, we observe that the activation of Cdc14 and execution of mitotic exit are uniquely sensitive to the modulation of Cdc5 kinase activity. In contrast, PBD-defective mutants are capable of completing mitosis but are unable to maintain spindle pole body integrity. Consistent with this defect, PBD-deficient cells progressively double the size of their genome and ultimately lose genome integrity. Collectively, these results highlight the specific contributions of Cdc5 functional domains to cell division and reveal unexpected mechanisms controlling spindle pole body behavior and genome stability.

Mitotic Exit and Separation of Mother and Daughter Cells

“…This is clearly the case for some Cdc5 substrates (Yoshida et al 2006). Intriguingly, however, recent analyses indicate that phosphopeptide binding by the Cdc5 PBD is not truly essential, and instead is required for only a subset of Cdc5-dependent processes (Chen and Weinreich 2010;Ratsima et al 2011).…”

“…As discussed in detail below, in essence Cdc5 turns on both pathways and thereby increases the activity of Cdc14 (Figure 8). This is probably triggered by changes in Cdc5 kinase activity rather than in PBD-mediated docking to substrates (Ratsima et al 2011). Intriguingly, since Cdc5 is a robust target of APC Cdh1 in G1 phase, the kinase effectively plants the seeds of its own destruction by upregulating Cdc14 (Visintin et al 2008).…”

Phosphorylation of Chs2p regulates interaction with COPII

“…In light of the role of Cdc14p in Chs2p export from the ER (Chin et al, 2012), we hypothesized that the Chs2p trafficking defects observed by mutation of CDC5 are caused by defects in release of Cdc14p from the nucleolus. To explore this, we took advantage of a cdc5 mutant strain, cdc5-77 ts , which carries a single amino acid mutation D263Q in the kinase domain resulting in a diminished MEN-induced nucleolar Cdc14p release (Ratsima et al, 2011). Trafficking of Chs2-3xGFP to the bud-neck is also compromised in cdc5-77 ts (Fig.…”

Structural Maintenance of Chromosome (SMC) Proteins Link Microtubule Stability to Genome Integrity

“…Samples were taken at intervals and processed for microscopic characterization of yeast bud, spindle, and nuclear morphology, as described previously (23). Yeast images were acquired on a Leica DM5500B microscope equipped with an EM-CCD camera (Hamamatsu).…”

“…Brightness and contrast adjustments were applied to primary micrographs using Photoshop software (Adobe). During time course experiments, yeast culture samples were also processed for determination of DNA content by flow cytometry using published procedures (23).…”