Structural basis of human separase regulation by securin and CDK1–cyclin B1

Yu, Jun; Raia, Pierre; Ghent, Chloe M.; Raisch, Tobias; Sadian, Yashar; Cavadini, Simone; Sabale, Pramod M.; Barford, David; Raunser, Stefan; Morgan, David; Boland, Andreas

doi:10.1038/s41586-021-03764-0

Cited by 65 publications

(79 citation statements)

References 55 publications

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“…Our work provides evidence that a recently described phosphate-binding pocket on B-type cyclins 21 contributes to the regulation of Cdk1 substrate phosphorylation.…”

Section: Discussionsupporting

confidence: 55%

“…Recent structural studies led to the discovery of a previously uncharacterized phosphate-binding pocket on the surface of human cyclin B1 21 . The three basic residues that form this pocket are highly conserved throughout the eukaryotic B-type cyclins and are found in all six of the budding yeast Clb proteins.…”

Section: Phosphorylation Of N-terminal Sites Depends On Priming By Phosphate-binding Sites In Cks1 and Clb2mentioning

confidence: 99%

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Multisite phosphorylation by Cdk1 initiates delayed negative feedback to control mitotic transcription

Asfaha¹,

Örd

Loog

et al. 2021

Preprint

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Ordered phosphorylation of cyclin-dependent kinase (CDK) substrates leads to the sequential transcriptional activation and inhibition of hundreds of cell cycle-regulated genes. We find that Ndd1, an activator of genes required for mitotic progression, is both positively and negatively regulated by CDK activity. CDK activity initially stimulates Ndd1-dependent transcription as cells enter mitosis, but prolonged high CDK activity in a mitotic arrest inhibits transcription. The result is a time-delayed negative feedback circuit that generates a pulse of mitotic gene expression. Our results suggest that high CDK activity catalyzes the formation of multiple weak phosphodegrons on Ndd1, leading to its destabilization. Cyclin specificity and phosphorylation kinetics contribute to the timing of Ndd1 destruction. Failure to degrade Ndd1 in a mitotic arrest leads to elevated mitotic gene expression. We conclude that a combination of positive and negative Ndd1 regulation by CDKs governs the timing and magnitude of the mitotic transcriptional program.

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“…Our work provides evidence that a recently described phosphate-binding pocket on B-type cyclins 21 contributes to the regulation of Cdk1 substrate phosphorylation.…”

Section: Discussionsupporting

confidence: 55%

Section: Phosphorylation Of N-terminal Sites Depends On Priming By Phosphate-binding Sites In Cks1 and Clb2mentioning

confidence: 99%

Multisite phosphorylation by Cdk1 initiates delayed negative feedback to control mitotic transcription

Asfaha¹,

Örd

Loog

et al. 2021

Preprint

Self Cite

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show abstract

“…Cyclin B1–Cdk1 itself binds to an accessory Cks protein (Cyclin-dependent kinase regulatory subunit Cks1 or Cks2) that recognizes and binds to phospho-threonine ([ 3 – 8 ]; reviewed in [ 9 ]). In mitosis, Cyclin B1 binds strongly to the MAD-1 checkpoint protein [ 10 – 12 ], and later to the separase enzyme in a phospho-dependent manner [ 13 – 15 ]. Cyclin B1 levels start to increase late in S phase and continue to accumulate in the cytoplasm of G2 cells [ 16 , 17 ].…”

Section: Introductionmentioning

confidence: 99%

Cell cycle-dependent binding between Cyclin B1 and Cdk1 revealed by time-resolved fluorescence correlation spectroscopy

et al. 2022

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Measuring the dynamics with which the regulatory complexes assemble and disassemble is a crucial barrier to our understanding of how the cell cycle is controlled that until now has been difficult to address. This considerable gap in our understanding is due to the difficulty of reconciling biochemical assays with single cell-based techniques, but recent advances in microscopy and gene editing techniques now enable the measurement of the kinetics of protein–protein interaction in living cells. Here, we apply fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy to study the dynamics of the cell cycle machinery, beginning with Cyclin B1 and its binding to its partner kinase Cdk1 that together form the major mitotic kinase. Although Cyclin B1 and Cdk1 are known to bind with high affinity, our results reveal that in living cells there is a pool of Cyclin B1 that is not bound to Cdk1. Furthermore, we provide evidence that the affinity of Cyclin B1 for Cdk1 increases during the cell cycle, indicating that the assembly of the complex is a regulated step. Our work lays the groundwork for studying the kinetics of protein complex assembly and disassembly during the cell cycle in living cells.

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“…Co-expression of CDK1, Cyclin-B, and CKS1 in insect cells was used in a very recent study that revealed how recombinant CCC complexes form a stoichiometric complex with Separase (Yu et al, 2021). In that study, the CCC assembly complexed to Separase had been obtained by insect cell co-expression, but had not been co-expressed with CAK or treated with CAK activity during purification.…”

Section: Discussionmentioning

confidence: 99%

Reconstitution and use of highly active human CDK1:Cyclin-B:CKS1 complexes

Veld

Wohlgemuth

Koerner

et al. 2021

Preprint

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As dividing cells transition into mitosis, hundreds of proteins are phosphorylated by a complex of cyclin-dependent kinase 1 (CDK1) and Cyclin-B, often at multiple sites. CDK1:Cyclin-B phosphorylation patterns alter conformations, interaction partners, and enzymatic activities and need to be recapitulated in vitro for the structural and functional characterization of the mitotic protein machinery. This requires a pure and active recombinant kinase complex. The kinase activity of CDK1 critically depends on the phosphorylation of a Threonine residue in its activation loop by a CDK1 activating kinase (CAK). We developed protocols to activate CDK1:Cyclin-B either in vitro with purified CDK1 activating kinases (CAK) or in insect cells through CDK-CAK co-expression. To boost kinase processivity, we reconstituted a tripartite complex consisting of CDK1, Cyclin-B, and CKS1. In this work, we provide and compare detailed protocols to obtain and use highly active CDK1:Cyclin-B (CC) and CDK1:Cyclin-B:CKS1 (CCC).

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Structural basis of human separase regulation by securin and CDK1–cyclin B1

Cited by 65 publications

References 55 publications

Multisite phosphorylation by Cdk1 initiates delayed negative feedback to control mitotic transcription

Multisite phosphorylation by Cdk1 initiates delayed negative feedback to control mitotic transcription

Cell cycle-dependent binding between Cyclin B1 and Cdk1 revealed by time-resolved fluorescence correlation spectroscopy

Reconstitution and use of highly active human CDK1:Cyclin-B:CKS1 complexes

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