Cascades of multisite phosphorylation control Sic1 destruction at the onset of S phase

Kõivomägi, Mardo; Valk, Ervin; Venta, Rainis; Iofik, Anna; Lepiku, Martin; Balog, Eva Rose M.; Rubin, Seth M.; Morgan, David; Loog, Mart

doi:10.1038/nature10560

Cited by 200 publications

(301 citation statements)

References 25 publications

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“…To define which steps of mitotic exit are altered as a result, we monitored mitotic progression until septation in clp1 + cdc25-GFP, clp1 + cdc25-13A-GFP, clp1Δ cdc25-GFP, and clp1Δ cdc25-13A-GFP cells by measuring the distance between SPBs (marked by Sid4-RFP) as an indication of spindle length. S. pombe has three phases of spindle dynamics (23). In phase 1, a 2-to 2.5-μm spindle is formed.…”

Section: Multisite Phosphorylation Of Cdc25 By Cdk1 Contributes To Cdc25mentioning

confidence: 99%

“…Cdk1 phosphorylation could activate Cdc25 by a stepwise mechanism, in which a specific number of phosphorylations is necessary for full Cdc25 activation or a progressive mechanism, in which Cdc25 is gradually activated as it becomes more highly phosphorylated. Furthermore, phosphorylation could occur in an ordered or disordered manner (8,11,23). To differentiate between these possibilities, we created more Cdc25 mutants, decreasing two available Cdk1 phosphosites in each subsequent phosphomutant (cdc25-7A to cdc25-11A) (Fig.…”

Section: Multisite Phosphorylation Of Cdc25 By Cdk1 Contributes To Cdc25mentioning

confidence: 99%

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Multisite phosphoregulation of Cdc25 activity refines the mitotic entrance and exit switches

Lu¹,

Domingo-Sananes

Huzarska³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Cyclin-dependent kinase 1 (Cdk1) kinase dephosphorylation and activation by Cdc25 phosphatase are essential for mitotic entry. Activated Cdk1 phosphorylates Cdc25 and other substrates, further activating Cdc25 to form a positive feedback loop that drives the abrupt G2/mitosis switch. Conversely, mitotic exit requires Cdk1 inactivation and reversal of Cdk1 substrate phosphorylation. This dephosphorylation is mediated, in part, by Clp1/Cdc14, a Cdk1-antagonizing phosphatase, which reverses Cdk1 phosphorylation of itself, Cdc25, and other Cdk1 substrates. Thus, Cdc25 phosphoregulation is essential for proper G2-M transition, and its contributions to cell cycle control have been modeled based on studies using Xenopus and human cell extracts. Because cell extract systems only approximate in vivo conditions where proteins interact within dynamic cellular environments, here, we use Schizosaccharomyces pombe to characterize, both experimentally and mathematically, the in vivo contributions of Cdk1-mediated phosphorylation of Cdc25 to the mitotic transition. Through comprehensive mapping of Cdk1 phosphosites on Cdc25 and characterization of phosphomutants, we show that Cdc25 hyperphosphorylation by Cdk1 governs Cdc25 catalytic activation, the precision of mitotic entry, and unvarying cell length but not Cdc25 localization or abundance. We propose a mathematical model that explains Cdc25 regulation by Cdk1 through a distributive and disordered phosphorylation mechanism that ultrasensitively activates Cdc25. We also show that Clp1/Cdc14 dephosphorylation of Cdk1 sites on Cdc25 controls the proper timing of cell division, a mechanism that is likely due to the double negative feedback loop between Clp1/Cdc14 and Cdc25 that controls the abruptness of the mitotic exit switch. mitotic bistability | multisite phosphorylation

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Section: Multisite Phosphorylation Of Cdc25 By Cdk1 Contributes To Cdc25mentioning

confidence: 99%

Section: Multisite Phosphorylation Of Cdc25 By Cdk1 Contributes To Cdc25mentioning

confidence: 99%

Multisite phosphoregulation of Cdc25 activity refines the mitotic entrance and exit switches

Lu¹,

Domingo-Sananes

Huzarska³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…Recent work on the canonical Cdc4 target Sic1 shows that maximal degradation of Sic1 by SCF Cdc4 also requires multiple phospho-degrons (36,37). We find that in Hst3, two phosphorylation sites-S420 and S421-are required for maximal turnover even though the two sites alone are not sufficient for any turnover.…”

Section: Hst3 Turnover Is Involved In Proper Regulation Of Acetylatedmentioning

confidence: 72%

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

Edenberg

Vashisht

Topacio

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Hst3 is the histone deacetylase that removes histone H3K56 acetylation. H3K56 acetylation is a cell-cycle-and damage-regulated chromatin marker, and proper regulation of H3K56 acetylation is important for replication, genomic stability, chromatin assembly, and the response to and recovery from DNA damage. Understanding the regulation of enzymes that regulate H3K56 acetylation is of great interest, because the loss of H3K56 acetylation leads to genomic instability. HST3 is controlled at both the transcriptional and posttranscriptional level. Here, we show that Hst3 is targeted for turnover by the ubiquitin ligase SCF Cdc4 after phosphorylation of a multisite degron. In addition, we find that Hst3 turnover increases in response to replication stress in a Rad53-dependent way. Turnover of Hst3 is promoted by Mck1 activity in both conditions. The Hst3 degron contains two canonical Cdc4 phospho-degrons, and the phosphorylation of each of these is required for efficient turnover both in an unperturbed cell cycle and in response to replication stress.H istone H3K56 acetylation is a cell-cycle-and damageregulated histone modification (1). In Saccharomyces cerevisiae, K56 acetylation is important for replication, genomic stability, chromatin assembly, and the response and recovery from DNA damage (1-6). Because of the critical nature of this chromatin mark, the enzymes that control it are themselves very important.In the budding yeast S. cerevisiae, Hst3 is a sirtuin histone deacetylase that, along with its close homolog Hst4, removes K56 acetylation (5, 7, 8). Overexpression of Hst3 or deletion of Hst3, along with related deacetylase Hst4, sensitizes cells to replication stress, suggesting that cells must control Hst3 levels precisely (5, 7). Hst3 is regulated both transcriptionally and by protein turnover. During an unperturbed cell cycle, HST3 transcript levels cycle, and Hst3 protein turns over very quickly (5,9). In response to DNA damage, transcription of HST3 is turned down, and Hst3 protein turnover is even faster (5,8,(10)(11)(12).Many cell-cycle-regulated proteins are targeted for proteasomal degradation after ubiquitination by a member of the SCF E3 ubiquitin ligase family. SCF ligases are multisubunit ubiquitin ligases composed of Skp1, a cullin (Cdc53), a RING-finger protein (Rbx1), and an F-box protein (13). The F-box protein is the substrate recognition module that confers substrate specificity to SCF ligases, with different F-box proteins recognizing different sets of substrates. The essential F-box protein Cdc4 regulates many cell-cycle-regulated proteins after their phosphorylation, including Sic1, Eco1, Cln3, and many others (14-19).Here, we have characterized the mechanism of Hst3 protein turnover. We find that Hst3 is targeted for degradation by SCF Cdc4 through a multisite phospho-degron. We find that Hst3 turnover is increased in response to replication stress in a Rad53-dependent way. Finally, we show that stabilizing Hst3 leads to misregulation of K56 acetylation in response to DNA damage and that...

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“…26 Interestingly, it was shown that Cdc28 is also regulated by a priming mechanism; in particular, Cks1 serves as a phospho-acceptor binding site that recruits the Cdc28 holoenzyme to at least some of its substrates. 27 Interestingly, these 200 ORFs belong to most highly transcribed genes in yeast, e.g., PMA1. To dissect the involvement of Cdc28 in basal transcription, we then used a mutant strain harboring the cdc28-as1 allele, which encodes a form of Cdc28 that is highly sensitive to the inhibitor 1-NM-PP1, 24 and found that PMA1 transcription depends on Cdc28 kinase activity.…”

Section: Mutual Regulation Of Cdc28 and Kin28mentioning

confidence: 99%

The cell cycle rallies the transcription cycle

Chymkowitch

Enserink

2013

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Cascades of multisite phosphorylation control Sic1 destruction at the onset of S phase

Cited by 200 publications

References 25 publications

Multisite phosphoregulation of Cdc25 activity refines the mitotic entrance and exit switches

Multisite phosphoregulation of Cdc25 activity refines the mitotic entrance and exit switches

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

The cell cycle rallies the transcription cycle

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Cascades of multisite phosphorylation control Sic1 destruction at the onset of S phase

Cited by 200 publications

References 25 publications

Multisite phosphoregulation of Cdc25 activity refines the mitotic entrance and exit switches

Multisite phosphoregulation of Cdc25 activity refines the mitotic entrance and exit switches

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

The cell cycle rallies the transcription cycle

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