The Hydrophobic Patch Directs Cyclin B to Centrosomes to Promote Global CDK Phosphorylation at Mitosis

Basu, Souradeep; Roberts, Emma L.; Jones, Andrew W.; Swaffer, Matthew P.; Snijders, Ambrosius P.; Nurse, Paul

doi:10.1016/j.cub.2019.12.053

Cited by 21 publications

(39 citation statements)

References 46 publications

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“…Regulation of microtubule dynamics and spindle morphogenesis by cyclin B-CDK may be conserved throughout eukaryotes (Basu et al, 2020; Verde et al, 1992) – our results extend this conservation to the deeply diverged plant kingdom. Overall, we observe strong conservation between Chlamydomonas and yeast and animals of the roles of cyclin B/CDK and the APC with respect to their inter-regulation and their overall effects on cell cycle biology including spindle morphogenesis.…”

Section: Discussionsupporting

confidence: 67%

“…Localization of CYCB1 to the region of the spindle poles 1-2 min before spindle formation is consistent with a direct regulation of the microtubule-organizing activity of spindle poles. In animal cells and in yeast, cyclin B localizes to centrosomes via the cyclin B ‘hydrophobic patch’ docking motif (Basu et al 2020 and references therein); and Chlamydomonas CYCB1 retains all key residues making up the hydrophobic patch. In contrast to Chlamydomonas , though, this localization may occur long before actual spindle formation.…”

Section: Discussionmentioning

confidence: 99%

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Control of division and microtubule dynamics in Chlamydomonas by cyclin B/CDKB1 and the anaphase-promoting complex

Cross

Pecani

Lieberman

et al. 2021

Preprint

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In yeast and animals, cyclin B binds and activates the cyclin-dependent kinase ('CDK') CDK1 to drive entry into mitosis. We show that CYCB1, the sole cyclin B in Chlamydomonas, activates the plant-specific CDKB1 rather than the CDK1 ortholog CDKA1. Time-lapse microscopy shows that CYCB1 is synthesized before each division in the multiple fission cycle, then is rapidly degraded 3-5 minutes before division occurs. CYCB1 degradation is dependent on the anaphase-promoting complex (APC). Like CYCB1, CDKB1 is not synthesized until late G1; however, CDKB1 is not degraded with each division within the multiple fission cycle. The microtubule plus-end-binding protein EB1 labeled with mNeonGreen (EB1-NG) allowed detection of mitotic events in live cells. The earliest detectable step in mitosis, splitting of polar EB1-NG signal into two foci, likely associated with future spindle poles, was dependent on CYCB1. CYCB1-GFP localized close to these foci immediately before spindle formation. Spindle breakdown, cleavage furrow formation and accumulation of EB1 in the furrow were dependent on the APC. In interphase, rapidly growing microtubules are marked by 'comets' of EB1; comets are absent in the absence of APC function. Thus CYCB1/CDKB1 and the APC mitosis modulate microtubule dynamics while regulating mitotic progression.

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Section: Discussionsupporting

confidence: 67%

Section: Discussionmentioning

confidence: 99%

Control of division and microtubule dynamics in Chlamydomonas by cyclin B/CDKB1 and the anaphase-promoting complex

Cross

Pecani

Lieberman

et al. 2021

Preprint

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“…Where the sfGFP labelled C-CDK was used, the sfGFP was present internally within the Cdc13 component 48,49 . Cut3-mCherry was generated by C-terminal tagging 50 and Cut3-GFP was developed previously 29 .…”

Section: S Pombe Genetics and Cell Culturementioning

confidence: 99%

CDK control pathways integrate cell size and ploidy information to control cell division

Patterson

Basu

Rees

et al. 2021

eLife

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Maintenance of cell size homeostasis is a property that is conserved throughout eukaryotes. Cell size homeostasis is brought about by the co-ordination of cell division with cell growth, and requires restriction of smaller cells from undergoing mitosis and cell division, whilst allowing larger cells to do so. Cyclin-CDK is the fundamental driver of mitosis and therefore ultimately ensures size homeostasis. Here we dissect determinants of CDK activity in vivo to investigate how cell size information is processed by the cell cycle network in fission yeast. We develop a high-throughput single-cell assay system of CDK activity in vivo and show that inhibitory tyrosine phosphorylation of CDK encodes cell size information, with the phosphatase PP2A aiding to set a size threshold for division. CDK inhibitory phosphorylation works synergistically with PP2A to prevent mitosis in smaller cells. Finally, we find that diploid cells of equivalent size to haploid cells exhibit lower CDK activity in response to equal cyclin-CDK enzyme concentrations, suggesting that CDK activity is reduced by increased DNA levels. Therefore, scaling of cyclin-CDK levels with cell size, CDK inhibitory phosphorylation, PP2A, and DNA-dependent inhibition of CDK activity, all inform the cell cycle network of cell size, thus contributing to cell-size homeostasis.

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“…Multiple members of the CMGC kinase family possess well-documented Pro-directed substrate specificity. Although CDK complexes such as Cyclin B-CDK1 phosphorylate a plethora of crucial M-phase substrates, containing minimal [S/T]P and full consensus [S/T]PXX[L/R] motifs [ 94–97 ], it is now clear that at least one CDK enzyme complex can also phosphorylate non-[S/T]P consensus motifs [ 98 ], operating through a combined ‘multisite’ code [ 99 ], differential substrate co-localisation and ordered phosphorylation that is dependent on substrate affinity [ 100 , 101 ]. Similar findings have also been made with the abscission-controlling PKCε-Aurora B complex, whose substrate specificity can switch at different phases of the cell cycle [ 102 ].…”

Section: Discussionmentioning

confidence: 99%

Use of the Polo-like kinase 4 (PLK4) inhibitor centrinone to investigate intracellular signalling networks using SILAC-based phosphoproteomics

Byrne

Clarke

Brownridge

et al. 2020

Biochemical Journal

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Polo-like kinase 4 (PLK4) is the master regulator of centriole duplication in metazoan organisms. Catalytic activity and protein turnover of PLK4 are tightly coupled in human cells, since changes in PLK4 concentration and catalysis have profound effects on centriole duplication and supernumerary centrosomes, which are associated with aneuploidy and cancer. Recently, PLK4 has been targeted with a variety of small molecule kinase inhibitors exemplified by centrinone, which rapidly induces inhibitory effects on PLK4 and leads to on-target centrosome depletion. Despite this, relatively few PLK4 substrates have been identified unequivocally in human cells, and PLK4 signalling outside centriolar networks remains poorly characterised. We report an unbiased mass spectrometry (MS)-based quantitative analysis of cellular protein phosphorylation in stable PLK4-expressing U2OS human cells exposed to centrinone. PLK4 phosphorylation was itself sensitive to brief exposure to the compound, resulting in PLK4 stabilisation. Analysing asynchronous cell populations, we report hundreds of centrinone-regulated cellular phosphoproteins, including centrosomal and cell cycle proteins and a variety of likely ‘non-canonical’ substrates. Surprisingly, sequence interrogation of ∼300 significantly down-regulated phosphoproteins reveals an extensive network of centrinone-sensitive [Ser/Thr]Pro phosphorylation sequence motifs, which based on our analysis might be either direct or indirect targets of PLK4. In addition, we confirm that NMYC and PTPN12 are PLK4 substrates, both in vitro and in human cells. Our findings suggest that PLK4 catalytic output directly controls the phosphorylation of a diverse set of cellular proteins, including Pro-directed targets that are likely to be important in PLK4-mediated cell signalling.

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The Hydrophobic Patch Directs Cyclin B to Centrosomes to Promote Global CDK Phosphorylation at Mitosis

Abstract: Highlights d The hydrophobic patch of human and yeast cyclin B directs it to the centrosome d Loss of the yeast cyclin B hydrophobic patch allows S phase but prevents mitosis d Compartmentalized mitotic CDK phosphorylation relies on the hydrophobic patch

Cited by 21 publications

References 46 publications

Control of division and microtubule dynamics in Chlamydomonas by cyclin B/CDKB1 and the anaphase-promoting complex

Control of division and microtubule dynamics in Chlamydomonas by cyclin B/CDKB1 and the anaphase-promoting complex

CDK control pathways integrate cell size and ploidy information to control cell division

Use of the Polo-like kinase 4 (PLK4) inhibitor centrinone to investigate intracellular signalling networks using SILAC-based phosphoproteomics

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