A Yeast GSK-3 Kinase Mck1 Promotes Cdc6 Degradation to Inhibit DNA Re-Replication

Abstract

Cdc6p is an essential component of the pre-replicative complex (pre-RC), which binds to DNA replication origins to promote initiation of DNA replication. Only once per cell cycle does DNA replication take place. After initiation, the pre-RC components are disassembled in order to prevent re-replication. It has been shown that the N-terminal region of Cdc6p is targeted for degradation after phosphorylation by Cyclin Dependent Kinase (CDK). Here we show that Mck1p, a yeast homologue of GSK-3 kinase, is also required for Cdc6 degradation through a distinct mechanism. Cdc6 is an unstable protein and is accumulated in the nucleus only during G1 and early S-phase in wild-type cells. In mck1 deletion cells, CDC6p is stabilized and accumulates in the nucleus even in late S phase and mitosis. Overexpression of Mck1p induces rapid Cdc6p degradation in a manner dependent on Threonine-368, a GSK-3 phosphorylation consensus site, and SCFCDC4. We show evidence that Mck1p-dependent degradation of Cdc6 is required for prevention of DNA re-replication. Loss of Mck1 activity results in synthetic lethality with other pre-RC mutants previously implicated in re-replication control, and these double mutant strains over-replicate DNA within a single cell cycle. These results suggest that a GSK3 family protein plays an unexpected role in preventing DNA over-replication through Cdc6 degradation in Saccharomyces cerevisiae. We propose that both CDK and Mck1 kinases are required for Cdc6 degradation to ensure a tight control of DNA replication.

Plasma membrane/cell wall perturbation activates a novel cell cycle checkpoint during G1 in Saccharomyces cerevisiae

“…Thus, Cdc6 is selectively degraded in a Mck1-dependent manner after membrane damage. Previously, we showed that Mck1 phosphorylates Cdc6 at T39 and T368 (30,31). Motivated by these findings, we tested if the Cdc6 phosphorylation mutant, Cdc6-T39A-T368A, is stabilized upon SDS stress.…”

“…S3B). Previously, we showed that Cdc6 degradation is mediated by Mck1 during an unperturbed cell cycle or during a DNA damage response (30,31). Δmck1 deletion cells are sensitive to various stress stimuli, including plasma membrane stress (Fig.…”

“…consensus site, at Thr-39 and Thr-368, promoting degradation of Cdc6 to guarantee genome integrity (30). Cdc6-Thr368 phosphorylation requires a priming phosphorylation at Ser372 by Cyclin/CDK (31).…”

Hst3 is turned over by a replication stress-responsive SCF Cdc4 phospho-degron

“…Mck1 phosphorylates its substrates at a consensus site that contains a priming phosphorylated residue in the +4 position (26). Subsequent phosphorylation by Mck1 generates a di-phosphorylation matching the Cdc4 phospho-degron spacing, and Mck1 has been implicated previously in controlling turnover of several Cdc4 substrates (23,(27)(28)(29). Two of the phosphorylation sites we identified by mass spectrometry, T380 and S416, exactly match the Mck1 consensus (with a phosphorylated residue in the +4 position).…”

Regulation of the Histone Deacetylase Hst3 by Cyclin-dependent Kinases and the Ubiquitin Ligase SCFCdc4

“…6A) strongly suggest that residues other than Hst3 Thr-380/ Thr-384 can be phosphorylated by CDKs. It is also possible that phosphorylation of non-CDK sites may contribute to Hst3 deg- radation, as has been observed for some substrates of SCF Cdc4 and SCF Fbw7 (29,49,51). Hst4 is redundant with Hst3 for H3K56 deacetylation, and interestingly, Hst4 was previously identified through a screen for proteins that directly bind to Cdc4 (25).…”