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CDK activity at the centrosome regulates the cell cycle

ABSTRACT: Cyclin-dependent kinases (CDKs) complexed with cyclins drive progression through the eukaryotic cell cycle. From yeast to human cells, cyclin-CDK localises to the centrosome, but the importance of this localisation is unclear. The conserved ‘hydrophobic patch’ substrate docking site on human Cyclin B1 and on the equivalent fission yeast Cdc13 mediates their localisation to the centrosome and the spindle-pole body (SPB, yeast centrosome equivalent). A hydrophobic patch mutant (HPM) of Cdc13 cannot enter mitosis, but whether this mitotic defect is due to defective SPB localisation or defective cyclin-substrate docking is unknown. Here we show that artificially restoring Cdc13HPM SPB localisation in fission yeast partially rescues both mitosis and defective CDK substrate phosphorylation at both the SPB and within the cytoplasm. In addition, we found that an HPM of the S-phase cyclin Cig2 has defective SPB localisation but is still able to perform bulk DNA synthesis. Our results demonstrate that the hydrophobic patch mediates the SPB localisation of both S- and M-phase cyclins, and that Cdc13 SPB localisation is essential for mitotic entry and for full phosphorylation of CDK substrates, supporting the view that the centrosome plays a role as a signalling hub regulating CDK cell cycle control.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Schizosaccharomyces Pombe 927

TISSUE(S): Permanent Cell Line Cell

SUBMITTER: Tania Auchynnikava

LAB HEAD: Paul Nurse

PROVIDER: PXD044732 | Pride | 2024-04-04

REPOSITORIES: Pride

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Project description:CP190 is a Drosophila protein of unknown function that exhibits cell cycle-specific localisation to the centrosome during mitosis and the nucleus during the interphase. CP190 associates with multiple sites on polytene chromosomes and its predicted amino acid sequence identifies four C2h2 zinc finger motifs along with an N-terminal BTB/POZ domain (similar to GAGA factor). Experimental studies have identified a 128 amino acid domain that direct centrosome association and a bipartite NLS. Interest in the function of CP190 was initially driven by an interest in centrosome function. Is is also fascinating to know why a protein that forms a core component of the centrosome also shares the attributes and distribution expected of a regulator of gene expression. CP190 mutants (EMS generated) were recently isolated whose recessive lethal phenotype indicates that CP190 is essential. The two best-characterised isolates, CP190[1] and CP190[2], are either nulls or severe hypomorphs (CP190 protein is undetectable on Western blots of total protein from homozygous mutatant third instar larvae). Both mutants can be rescued as homozygotes by expression of wild type CP190 from a transgenic second chromosome carrying a CP190 cDNA under the control of the polyubiquitin promoter. Stocks homozygous or hemizygous for either of these mutant alleles die as pharate adults that show no obvious externally visible defects. Mutant larvae show a slight developmental delay compared to their heterozygous siblings, but their brains and discs are of normal size and brain squash preparation show no obvious mitotic phenotype. Since CP190 is a chromatin-associated protein and has motifs that are typical of proteins involved in regulating gene expression, it seems likely that the gene expression profile of CP190 mutants will be aberrant. This experiment hopes to identify genes that exhibit aberrant expression in the mutant compared to the wild type. Target RNA were isolated from: homozygous CP190[1] 0 hour white prepupae (non tubby, red malpighian tubules) homozygous CP190[2] 0 hour white prepupae (non tubby, red malpighian tubules) homozygous red[1] e[1] 0 h white prepupae (source from which the mutated chromosome was obtained) For each CP190 mutant, total RNA preparations were made from three separate groups of 100 prepupae selected from independent cultures, similarly, six independent RNA samples were prepared from red[1] e[1] stock as 'wild type' controls, giving a total of 12 target RNA samples. Prepupae were collected from bottles every 30 min and frozen directly in liquid N2. Frozen samples were pooled and ground to a powder in liquid N2 before resuspension in TRIZOL and shipment to Flychip facility at the University of Cambridge, UK.

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CDK activity at the centrosome regulates the cell cycle

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