PP1 promotes cyclin B destruction and the metaphase-anaphase transition by dephosphorylating CDC20

Bancroft, James; Holder, James; Geraghty, Zoë; Alfonso-Pérez, Tatiana; Murphy, Daniel; Barr, Francis A.; Grüneberg, Ulrike

doi:10.1101/2020.06.22.164251

Cited by 7 publications

(15 citation statements)

References 92 publications

(142 reference statements)

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“…Based on our results, we favour that this is PP2A-B55. A recent study also implicated PP1 in Cdc20 T70 dephosphorylation in human cells in line with work from C. elegans showing PP1 as a Cdc20 phosphatase (Bancroft et al, 2020;Kim et al, 2017). The results from human somatic cells could be an indirect effect of PP1 inhibition leading to delayed PP2A-B55 activation.…”

Section: Cdk1 Phosphorylation Of Cdc20 Is Required For a Prolonged MIsupporting

confidence: 62%

Coupling of Cdc20 inhibition and activation by BubR1

Hein

Garvanska

Nasa

et al. 2020

Preprint

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Tight regulation of the APC/C-Cdc20 ubiquitin ligase that targets Cyclin B1 for degradation is important for mitotic fidelity. The spindle assembly checkpoint (SAC) inhibits Cdc20 through the mitotic checkpoint complex (MCC). In addition, phosphorylation of Cdc20 by Cyclin B1-Cdk1 independently inhibits APC/C-Cdc20 activation. This creates a conundrum for how Cdc20 gets activated prior to Cyclin B1 degradation. Here we show that the MCC component BubR1 harbours both Cdc20 inhibition and activation activities, allowing for cross-talk between the two Cdc20 inhibition pathways. Specifically BubR1 acts as a substrate specifier for PP2A-B56 to enable efficient Cdc20 dephosphorylation in the MCC. A mutant Cdc20 mimicking the dephosphorylated state escapes a mitotic checkpoint arrest arguing that restricting Cdc20 dephosphorylation to the MCC is important. Collectively our work reveals how Cdc20 can be dephosphorylated in the presence of Cyclin B1-Cdk1 activity without causing premature anaphase onset.

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Section: Cdk1 Phosphorylation Of Cdc20 Is Required For a Prolonged MIsupporting

confidence: 62%

Coupling of Cdc20 inhibition and activation by BubR1

Hein

Garvanska

Nasa

et al. 2020

Preprint

View full text Add to dashboard Cite

show abstract

“…In the global analysis presented here the most basic phospho-sites were dephosphorylated the most rapidly, suggesting that a preference for basicity is an inherent characteristic of PPPs and a general principle of phosphatase-mediated anaphase regulation. To test whether PP1 shares this same preference, samples from a high-resolution analysis of mitotic exit under PP1 depleted conditions ( Bancroft et al, 2020 ) were subjected to mass spectrometry analysis. To identify PP1-dependent clusters, the average phospho-site half-life was calculated for both control and PP1 depletion within each cluster.…”

Section: Resultsmentioning

confidence: 99%

“… HeLa cell samples depleted of either control or PPP1CA/C were processed for mass spectrometry and analysed as described previously ( Bancroft et al, 2020 ). ( A ) Clustering heat-map comparing the trends in phosphorylation over time in control and PPP1CA/C depleted cells following CDK1 inhibition ( Figure 7—source data 1 ).…”

Section: Resultsmentioning

confidence: 99%

“…Typically, 6 µg was loaded on each western blot but when necessary increased to 10 µg. Western blot data from time course samples depleted of PP1 subunits were published previously ( Bancroft et al, 2020 ), here the remaining lysate was subjected to trypsin digestion for subsequent proteomic analysis. Low-resolution time courses for western blot were set up as above, however cells were lysed directly in 3x sample buffer.…”

Section: Methodsmentioning

confidence: 99%

“…Clusters where phospho-site half-life in the PP1 depleted condition was >50% longer than that of control were taken forward for further analysis. Depletion of PPP1CB had little effect on bulk dephosphorylation HeLa cell samples depleted of either control or PPP1CA/C were processed for mass spectrometry and analysed as described previously (Bancroft et al, 2020). Figure 7A).…”

Section: Pp1 and Pp2a-b55 Share A Preference For Basic Phospho-threonmentioning

confidence: 99%

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Ordered dephosphorylation initiated by the selective proteolysis of cyclin B drives mitotic exit

Holder

Mohammed

Barr

2020

eLife

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APC/C-mediated proteolysis of cyclin B and securin promotes anaphase entry, inactivating CDK1 and permitting chromosome segregation, respectively. Reduction of CDK1 activity relieves inhibition of the CDK1-counteracting phosphatases PP1 and PP2A-B55, allowing wide-spread dephosphorylation of substrates. Meanwhile, continued APC/C activity promotes proteolysis of other mitotic regulators. Together, these activities orchestrate a complex series of events during mitotic exit. However, the relative importance of regulated proteolysis and dephosphorylation in dictating the order and timing of these events remains unclear. Using high temporal-resolution proteomics, we compare the relative extent of proteolysis and protein dephosphorylation. This reveals highly-selective rapid proteolysis of cyclin B, securin and geminin at the metaphase-anaphase transition, followed by slow proteolysis of other substrates. Dephosphorylation requires APC/C-dependent destruction of cyclin B and was resolved into PP1-dependent categories with unique sequence motifs. We conclude that dephosphorylation initiated by selective proteolysis of cyclin B drives the bulk of changes observed during mitotic exit.

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Ordered dephosphorylation initiated by the selective proteolysis of cyclin B drives mitotic exit

Holder

Mohammed

Barr

2020

Preprint

Self Cite

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APC/C-mediated proteolysis of cyclin B and securin promotes entry into anaphase, inactivating CDK1 and permitting chromosome segregation, respectively. Reduction of CDK1 activity relieves inhibition of the CDK1-opposing phosphatases PP1 and PP2A-B55 leading to dephosphorylation of substrates crucial for mitotic exit.Meanwhile, continued APC/C activity is required to target various proteins, including Aurora and Polo kinases, for degradation. Together, these activities orchestrate a complex series of events during mitotic exit. However, the relative importance of regulated proteolysis and dephosphorylation in dictating the order and timing of these events remains unclear. Using high temporal-resolution mass spectrometry, we compare the relative extent of proteolysis and protein dephosphorylation. This reveals highly-selective rapid (~5min half-life) proteolysis of cyclin B, securin and geminin at the metaphase to anaphase transition, followed by slow proteolysis (>60 min half-life) of other mitotic regulators. Protein dephosphorylation requires APC/Cdependent destruction of cyclin B and was resolved into PP1-dependent fast, intermediate and slow categories with unique sequence motifs. We conclude that dephosphorylation initiated by the selective proteolysis of cyclin B drives the bulk of changes observed during mitotic exit.

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PP1 promotes cyclin B destruction and the metaphase-anaphase transition by dephosphorylating CDC20

Cited by 7 publications

References 92 publications

Coupling of Cdc20 inhibition and activation by BubR1

Coupling of Cdc20 inhibition and activation by BubR1

Ordered dephosphorylation initiated by the selective proteolysis of cyclin B drives mitotic exit

Ordered dephosphorylation initiated by the selective proteolysis of cyclin B drives mitotic exit

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