Cdk1 regulates centrosome separation by restraining proteolysis of microtubule-associated proteins

Abstract

In yeast, separation of duplicated spindle pole bodies (SPBs) (centrosomes in higher eukaryotes) is an indispensable step in the assembly of mitotic spindle and is triggered by severing of the bridge that connects the sister SPBs. This process requires Cdk1 (Cdc28) activation by Tyrosine 19 dephosphorylation. We show that cells that fail to activate Cdk1 are devoid of spindles due to persistently active APCCdh1, which targets microtubule-associated proteins Cin8, Kip1 and Ase1 for degradation. Tyrosine 19 dephosphorylation of Cdk1 is necessary to specifically prevent proteolysis of these proteins. Interestingly, SPB separation is dependent on the microtubule-bundling activity of Cin8 but not on its motor function. Since ectopic expression of proteolysis-resistant Cin8, Kip1 or Ase1 is sufficient for SPB separation even in the absence of Cdc28-Clb activity, we suggest that stabilization of these mechanical force-generating proteins is the predominant role of Cdc28-Clb in centrosome separation.

Figure 1

Low endogenous levels of microtubule-associated proteins in cdc28–as1 and cdc28Y19E cells. (A) cdc28Y19E (top panel) and cdc28-as1 (middle panel) cells were arrested with α factor and released at 37°C and 24°C in presence of 1NM-PP1, respectively. Electron micrographs show state of SPBs at 240 min. Plots show spindle and budding index. (Lower panel) Western blots show histone H1 kinase activity and tyrosine phosphorylation status. (B) Wild-type (WT), cdc28-as1, cdc28-Y19E and cdc28-1N cells carrying endogenously tagged Cin8-HA₃, Kip1-cmyc₃ or Ase1-cmyc₃ were synchronized in G1 with α factor treatment and then released into at 24°C in medium containing 1NM-PP1 (for cdc28-as1) or 37°C (for cdc28Y19E and cdc28-1N). Samples for Western blotting were taken at 15-min intervals for WT and 30-min intervals for cdc28 mutants for comparison (doubling time in cdc28 mutants is approximately twice that of WT). (C) G1-synchronised cdc28-as1 cells were released in the presence of 1NM-PP1. Samples were collected at 30-min intervals for RNA isolation and subsequent Northern blotting.

Figure 2

Proteasomal degradation of microtubule-associated proteins in cdc28 mutants (A) WT, cdc28Y19E, cdc28-as1 and cdc28-1N carrying either GAL-CIN8-cmyc_3, GAL-KIP1-cmyc₃ or GAL-ASE1-cmyc₃ on a CEN plasmid were arrested in YEP+Raff containing α factor and then released into YEP+Raff+Gal at respective arrest conditions (as in Figure 1) for 90 min to induce protein expression. Cells were then transferred to YEP+Glu containing 1 mg/ml cycloheximide for shut-off of protein synthesis and the fate of the protein pulse was monitored by Western blotting. Numbers below Western blots indicate Protein/Cdc28 ratio measured by densitometry. (B) G1-synchronized cdc28-as1 cells carrying endogenous Cin8-HA₃ and SPC42-GFP integrated at TRP1 locus were released at 24°C into medium containing 1NM-PP1. Proteasome inhibitor MG132 was added 100 min after release from α factor and samples were collected for Western blot and microscopic analysis. (C) (Top left panel) Scheme illustrating principle of BRET between Cin8-Rluc and GFP-ubiquitin. (Left graph) Plot for BRET measured in cdc28-as1 cells in 1NM-PP1-expressing Cin8-Rluc alone, Cin8-Rluc and GFP-monoUbi (from GAL1 promoter) or Cin8-Rluc and GFP-Ubi_AA (from GAL1 promoter). Sample at 150 min after the release from alpha-factor is shown. (Right graph) Plot of BRET ratios of cells co-expressing Cin8-Rluc and GFPmonoUbi (from GAL1 promoter) or Cin8-Rluc and GFP-Ubi_AA (from GAL1 promoter). BRET ratios were calculated as defined in Materials and methods. Data represent the mean of three independent experiments. Samples were also analyzed by Western blotting for Cin8 levels in cdc28-as1 cells expressing Cin8-Rluc and cdc28-as1 cells co-expressing Cin8-Rluc and GFP-UbiAA (from GAL1 promoter) (Top left panel).

Figure 3

Cin8-bundling activity, not its motor activity or phosphorylation, is required for SPB separation. (A) G1-synchronized cdc28-as1 expressing nondegradable versions of Cin8, Kip1 and Ase1, from their respective native promoter-driven constructs on a CEN vector, were released in the presence of 1NM-PP1. Cells at 240 min after release are shown. The Western blot (rightmost panel) shows stability of the nondegradable versions in G1. (B) clb3Δ clb4Δ clb5Δ GAL-CLB5 cells carrying MET3-CIN8-HA₃ were synchronized in G1 in Raff+Gal+Methionine medium and released into methionine-deficient glucose medium. (C) G1-synchronised cdc28Y19E carrying SPC42-GFP at the TRP1 locus and expressing from CEN vector GAL-CIN8-cmyc₃, GAL-CIN8 (R196K)-cmyc₃ (affecting motor activity), GAL-CIN8 (F467A)-cmyc₃ (affecting microtubule binding) or GAL-CIN8 (R394A, H396A, E871A)-cmyc₃ (affecting bundling) were released into YEP+Raff+Gal at 37°C. Numbers indicate percentage of cells with two SPC42-GFP dots at 240 min. Western blots show the extent of Cin8 expression. (D) (Top panel) Kinetics of spindle formation in cdc28Y19E expressing GAL-CIN8-cmyc₃, GAL-KIP1-cmyc₃ or GAL-ASE1-cmyc₃ on a CEN plasmid or versions carrying mutations at Cdc28 consensus sites mimicking phosphorylated (E) or unphosphorylated forms (A). (Lower panel) Spindle kinetics in cin8-3 kip1Δ and cin8Δ ase1Δ cells expressing phospho-deficient versions at the Cdc28 consensus sites (A).

Figure 4

APC^Cdh1-mediated degradation of microtubule-associated proteins prevents spindle assembly. (A) (Left panel) cdc28Y19E cdh1Δ cells or cdc28Y19E cdc20-1 were arrested with α-factor and released at 37°C. (Right panel) cdc28-as1 cells with similar mutations were released in the presence of 1NM-PP1. Cells are shown 240 min after release. (B) cdc28-as1 and cdc28-as1 cdh1Δ cells carrying endogenously tagged Cin8-HA₃, Kip1-HA₃ or Ase1-HA₃ were arrested in telophase at 37°C by a cdc15-2 mutation, and released in the presence of 1NM-PP1 at 24°C. (C) G1-synchronised WT cells tagged endogenously with Cin8-HA₃ with or without GAL-Cdh1-cmyc₃ on a CEN plasmid were released from YEP+Raff into YEP+Raff+Gal medium. (D) cdc15-2 and cdc15-2 cdh1Δ cells expressing Spc29-CFP were synchronized in telophase at 37°C, released into fresh medium at 24°C and were monitored for kinetics of spindle assembly. ‘^*'Indicate the time at which a significant difference in the percentage of cells-with-spindles was observed between the two strains.

Figure 5

Phosphorylation status of Cdh1 determines its subcellular localization and Cin8 ubiquitylation. (A) Extracts were prepared from cmyc₉-Cdh1 carrying WT cells grown in α factor (α) and nocodazole (Noc), and from clb3Δ clb4Δ clb5Δ GAL-CLB5, cdc28-as1, cdc28Y19E, cdc28-1N and cdc28Y19F arrested at their terminal points. Cdh1 was immunoprecipitated using anti-myc agarose beads, treated with and without alkaline phosphatase (CIP) and separated on a 10% SDS gel. Extracts from untagged WT strains (WT) as well as from nocodazole-arrested WT strain carrying cmyc₉-Cdh1 immunoprecipitated with IgG beads (IgG) were used as negative controls. (B) cim5-1 cdc23-1 (lanes 2 and 7), cim5-1 cdc28Y19E (lanes 4 and 9), cim5-1 cdc28-1N (lanes 5 and 10) cells were synchronized in G1 with α factor and then released into fresh YPD medium at 37°C to reach their respective terminal phenotype. Since cim5-1 cdc28Y19E and cim5-1 cdc28-1N are synthetic lethal, they were kept alive by GAL-CDC28. Cycling (lanes 1 and 6) or α factor arrested (lanes 3 and 8) cim5-1 cells were used as controls. All strain carried endogenously tagged Cin8-HA₃ or Clb2-HA₃. Extracts were prepared, Clb2 and Cin8 were immunoprecipitated with anti-HA agarose beads, and ubiquitin conjagates were detected with anti-ubiquitin antibodies. Total amounts of Clb2-HA₃ or Cin8-HA₃ were detected using anti-HA antibodies. (C) WT, cdc28-as1, cdc28Y19E, cdc28-1N and WT overexpressing Cdh1-m11_A (under the weak GALL promoter), all carrying Cdh1-GFP, were released from G1 arrest at their respective nonpermissive growth condition.

Figure 6

Phosphorylation sites in Cdh1 essential for spindle formation. Cdc28 phosphorylation sites in Cdh1 are schematically shown. ‘P' denotes residues whose phosphorylation is essential for spindle formation. WT and WT expressing Cdh1-m11_A, Cdh1-m7_A, Cdh1-m7_E under the weak GALL promoter (all carrying endogenously tagged Cin8-HA₃) were synchronized in G1 in YEP+Raff and released into YEP+Raff+Gal. Cin8 and Clb2 levels were determined by Western blotting. Graph shows proportion of cells with short spindles.

Figure 7

Tyrosine dephosphorylation correlates with the timing of SPB separation during normal cell cycle. (A) G1-synchronized WT cells carrying SPC42-GFP were released into fresh medium at 20°C. Samples were withdrawn at 10 min interval and analyzed for tyrosine dephosphorylation of Cdc28, Cdh1 phosphorylation and Cin8, Clb2 levels. ‘^*'Denotes timing of Tyr19-Cdc28 dephosphorylation. The photomicrograph below shows cells at intermediate time points and the graph represents extent of tyrosine 19 phosphorylation. (B) A scheme illustrating the role of activated Cdc28 in SPB separation (see Discussion).