Inhibitory phosphorylation of the APC regulator Hct1 is controlled by the kinase Cdc28 and the phosphatase Cdc14

Jaspersen, Sue L.; Charles, Julia F.; Morgan, David

doi:10.1016/s0960-9822(99)80111-0

Cited by 396 publications

(403 citation statements)

References 43 publications

(77 reference statements)

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“…Clb2 was expressed from a 2μ P GAL1 -TAP plasmid, and Clb2-Cdk1 complexes were purified using a C-terminal TAP tag on Clb2 29. GST-Cdc14 was purified from bacteria 15 . Securin and Cdc20 were expressed in the TNT reticulocyte lysate coupled transcription/translation system (Promega) with a C-and N-terminal TEV-ZZ tag, respectively, purified rapidly on M-270 Epoxy dynabeads (Invitrogen) coupled to IgG, and eluted with TEV protease.…”

Section: Protein Purificationmentioning

confidence: 99%

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Positive feedback sharpens the anaphase switch

Holt

Krutchinsky

Morgan

2008

Nature

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174

182

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At the onset of anaphase, sister-chromatid cohesion is dissolved abruptly and irreversibly, ensuring that all chromosome pairs disjoin almost simultaneously. The regulatory mechanisms that generate this switch-like behavior are unclear. Anaphase is initiated when a ubiquitin ligase, the Anaphase-Promoting Complex (APC), triggers the destruction of securin, thereby allowing the protease, separase, to disrupt sister-chromatid cohesion 1-4. Here we demonstrate that Cdk1-dependent phosphorylation of securin near its destruction-box motif inhibits securin ubiquitination by the APC. The phosphatase Cdc14 reverses securin phosphorylation, thereby increasing the rate of securin ubiquitination. Because separase is known to activate Cdc14 5 , 6, our results support the existence of a positive feedback loop that increases the abruptness of anaphase. Consistent with this model, we show that mutations that disrupt securin phosphoregulation decrease the synchrony of chromosome segregation. Our results also suggest that coupling securin degradation with changes in Cdk1 and Cdc14 activities helps coordinate the initiation of sister-chromatid separation with changes in spindle dynamics.Securin is known to integrate multiple signaling pathways to delay anaphase in response to perturbations such as DNA damage 7,8 . We hypothesized that securin might also receive regulatory inputs that make separase activation more switch-like. To address this possibility, we used mass spectrometry to analyze the phosphorylation state of securin from mitotic budding yeast cells (Fig. 1a) 9 . We identified six phosphorylation sites, including four of the five sites corresponding to the consensus sequence of the cyclin-dependent-kinase, Cdk1 (S/ T*-P) (see also Suppl. Fig. 1). Three Cdk1 sites were found in securin's C-terminal domain, which is known to interact with separase 10 ; mutations at these sites are known to modulate securin's ability to bind separase and import it into the nucleus 11 . The fourth Cdk1 phosphopeptide, near the N terminus of securin, has not been characterized.The N-terminal Cdk1 site is near the destruction-box of securin, a motif important for recognition of substrates by the APC 2-4, 12 . To assess whether phosphorylation of this site affects securin ubiquitination by the APC, we tested the ability of purified APC to ubiquitinate purified securin before and after phosphorylation in vitro by Cdk1. We found that if securin was phosphorylated by Cdk1, the rate of ubiquitination by APC Cdc20 (the APC-activator complex that controls anaphase onset) was reduced 10-fold (Fig. 1b, c) and the rate of ubiquitination by APC Cdh1 was reduced 5-fold (Suppl. Fig. 2).This regulatory mechanism may be conserved -Drosophila and human homologues of securin have S/T-P motifs near their destruction-boxes, and both were ubiquitinated less efficiently by the APC after phosphorylation by Cdk1 (Suppl. Fig. 3 Cdc14 is a phosphatase that removes phosphates from Cdk1 substrates during anaphase and late mitosis 4,[13][14][15][16] . We found t...

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Section: Protein Purificationmentioning

confidence: 99%

“…Cdc14 is a phosphatase that removes phosphates from Cdk1 substrates during anaphase and late mitosis 4,[13][14][15][16] . We found that Cdc14 removed Cdk1-dependent phosphates from securin, as judged by the loss of a gel-mobility shift that results from phosphorylation ( Fig.…”

mentioning

confidence: 99%

Positive feedback sharpens the anaphase switch

Holt

Krutchinsky

Morgan

2008

Nature

Self Cite

174

182

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“…Studies with purified APC from budding yeast and human cells demonstrate that both Cdc20 and Cdh1/ Hct1 activate the 'in vitro' ubiquitination of cyclin B (Kramer et al, 1998;Jaspersen et al, 1999). Moreover, the APC purified from Xenopus egg extracts in which Cdc20 function is blocked presents a clearly inhibited ubiquitination activity (Lorca et al, 1998).…”

Section: Apc Activators: Cdc20 Cdh1 and Ama1mentioning

confidence: 99%

The anaphase-promoting complex: a key factor in the regulation of cell cycle

et al. 2005

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Events controlling cell division are governed by the degradation of different regulatory proteins by the ubiquitin-dependent pathway. In this pathway, the attachment of a polyubiquitin chain to a substrate by an ubiquitin-ligase targets this substrate for degradation by the 26S proteasome. Two different ubiquitin ligases play an important role in the cell cycle: the SCF (Skp1/Cullin/ F-box) and the anaphase-promoting complex (APC). In this review, we describe the present knowledge about the APC. We pay particular attention to the latest results concerning APC structure, APC regulation and substrate recognition, and we discuss the implication of these findings in the understanding the APC function.

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“…The form of eqn (2) suggests that A is a phosphatase, removing from Cdh1 the inhibitory phosphate groups placed there by CycB/Cdk. Recent experimental evidence in budding yeast (Jasperson et al, 1999;Visintin et al, 1998) identi-"es A as the product of the CDC14 gene. At the metaphasePanaphase transition, Cdc14 phosphatase is activated indirectly by Cdc20/APC, which destroys an inhibitor of Cdc14.…”

Section: Activation Of Cdh1/apc At Anaphasementioning

confidence: 99%

Regulation of the Eukaryotic Cell Cycle: Molecular Antagonism, Hysteresis, and Irreversible Transitions

Tyson

Novák

2001

Journal of Theoretical Biology

335

343

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In recent years, molecular biologists have uncovered a wealth of information about the proteins controlling cell growth and division in eukaryotes. The regulatory system is so complex that it de"es understanding by verbal arguments alone. Quantitative tools are necessary to probe reliably into the details of cell cycle control. To this end, we convert hypothetical molecular mechanisms into sets of nonlinear ordinary di!erential equations and use standard analytical and numerical methods to study their solutions. First, we present a simple model of the antagonistic interactions between cyclin-dependent kinases and the anaphase promoting complex, which shows how progress through the cell cycle can be thought of as irreversible transitions (Start and Finish) between two stable states (G1 and S}G2}M) of the regulatory system. Then we add new pieces to the &&puzzle'' until we obtain reasonable models of the control systems in yeast cells, frog eggs, and cultured mammalian cells.

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Inhibitory phosphorylation of the APC regulator Hct1 is controlled by the kinase Cdc28 and the phosphatase Cdc14

Cited by 396 publications

References 43 publications

Positive feedback sharpens the anaphase switch

Positive feedback sharpens the anaphase switch

The anaphase-promoting complex: a key factor in the regulation of cell cycle

Regulation of the Eukaryotic Cell Cycle: Molecular Antagonism, Hysteresis, and Irreversible Transitions

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