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

Abstract

The cyclin-dependent kinases (CDKs) are the major cell-cycle regulators that phosphorylate hundreds of substrates, controlling the onset of S phase and M phase [1-3]. However, the patterns of substrate phosphorylation increase are not uniform, as different substrates become phosphorylated at different times as cells proceed through the cell cycle [4, 5]. In fission yeast, the correct ordering of CDK substrate phosphorylation can be established by the activity of a single mitotic cyclin-CDK complex [6, 7]. Here, we investigate the substrate-docking region, the hydrophobic patch, on the fission yeast mitotic cyclin Cdc13 as a potential mechanism to correctly order CDK substrate phosphorylation. We show that the hydrophobic patch targets Cdc13 to the yeast centrosome equivalent, the spindle pole body (SPB), and disruption of this motif prevents both centrosomal localization of Cdc13 and the onset of mitosis but does not prevent S phase. CDK phosphorylation in mitosis is compromised for approximately half of all mitotic CDK substrates, with substrates affected generally being those that require the highest levels of CDK activity to become phosphorylated and those that are located at the SPB. Our experiments suggest that the hydrophobic patch of mitotic cyclins contributes to CDK substrate selection by directing the localization of Cdc13-CDK to centrosomes and that this localization of CDK contributes to the CDK substrate phosphorylation necessary to ensure proper entry into mitosis. Finally, we show that mutation of the hydrophobic patch prevents cyclin B1 localization to centrosomes in human cells, suggesting that this mechanism of cyclin-CDK spatial regulation may be conserved across eukaryotes.

Keywords: CDK; SPB; centrosome; cyclin; localization; mitosis.

Graphical abstract

Figure 1

The Cdc13 Hydrophobic Patch Is Not Necessary for S Phase (A) Promoter systems used in (B)–(G) to repress endogenous cdc13⁺ through the thiamine-repressible nmt41 promoter. An extra copy of cdc13 is inserted into the leu1 (exogenous) locus, maintaining all endogenous UTR regions. (B) Cells lacking the G1/S cyclin genes cig1⁺ and cig2⁺, with cdc13^HPM/WT in the leu1 locus, were arrested in G1 through nitrogen starvation. Thiamine was added to cultures 1 h before release. Cells were then released into S phase at 32°C through refeeding minimal media containing nitrogen and examined for DNA content (see STAR Methods). S.O., shut off. (C) Upper: experimental schematic for (D)–(G). Cells are blocked in G2 initially using 1 μM of the ATP analog 1-NmPP1. Thiamine is added to repress endogenous cdc13 1 h before release (see STAR Methods). Cells are then washed of 1-NmPP1 to release into mitosis before addition of a range of 1-NmPP1 concentrations 14 min after the washout to study S phase by DNA content analysis. Lower: western blots show complete degradation of endogenous Cdc13. Cdc13 levels (upper) and total protein amounts (Ponceau-S, lower) before the addition of 1-NmPP1, 20 min after mitosis and 120 min after mitosis are shown from left to right, respectively. Western blots are of cells released into 3 μM 1-NmPP1. Empty corresponds to cells lacking any exogenous cdc13 construct in the leu1 locus. All Cdc13 and Ponceau-S panels are from the same exposure of a single membrane. (D and E) G1 cells after S phase release as a percentage of the G1 cell population at 54 min after mitosis (see STAR Methods). Before 54 min, cells are present as binucleate septated cells and not uninucleate G1 cells. (D) Cdc13^WT and (E) Cdc13^HPM are shown. Curves are a sigmoid fit through the data. (F and G) Comparison of (F) the population-level G1/S transition rate, measured as the exponent of the sigmoid fit curves in (D) and (E), and (G) the time at which 50% of cells have executed S phase as measured by G1₅₀ values extracted from the sigmoid fit curves in (D) and (E). See also Figure S1 and Table S2.

Figure 2

Cdc13^HPM Cannot Localize to the SPB in Interphase (A) Top: serial dilution assays of cells with thiamine-repressible endogenous Cdc13 and either Cdc13^WT or Cdc13^HPM in the presence (endogenous Cdc13 OFF) or absence (endogenous Cdc13 ON) of thiamine. Bottom: calcofluor staining of cells expressing Cdc13^HPM and repressible endogenous Cdc13 in the presence (right) or absence (left) of thiamine is shown. Scale bars, 10 μm. (B) Representative maximum projection images of different cells of increasing size from an asynchronous population expressing exogenous Cdc13^WT/HPM-sfGFP. Endogenous Cdc13 is still expressed but is not fused to a fluorophore. Sid4-mRFP marks the SPB. Arrows show first appearance of a Cdc13-sfGFP SPB focus. The same pixel range has been applied to all images from the same channel. Scale bar, 2 μm. (C) Uninucleate cells as in (B) were analyzed for Cdc13 signal at single (interphase) or separated (mitotic) SPBs. The mean and SD of 3 replicates are shown. Uninucleate population n > 200 cells per replicate; total n = 824 total for Cdc13^WT and 954 for Cdc13^HPM. (D) Cell lengths of the entire uninucleate population and of cells with a Cdc13-sfGFP SPB focus from one replicate of (C). Error bars represent median, with whiskers delimiting the 25^th and 75^th percentiles. n = 212 total for Cdc13^WT and 214 for Cdc13^HPM. (E and F) Cell length compared to presence of Plo1-mCherry and (E) Cdc13^WT-sfGFP or (F) Cdc13^HPM-sfGFP foci at the SPB. Endogenous Cdc13 is still expressed. Data are pooled from 3 replicates; mean cell length per cohort is plotted against % of cells within that cohort with Plo1-mCherry or Cdc13-sfGFP foci at the SPB. n ≥ 89 cells per cohort. Total n = 899 cells for Cdc13^WT and 903 cells for Cdc13^HPM. (G) Cell length compared to Cdc13^HPM-sfGFP SPB foci in strains that accelerate the accumulation of Polo kinase at the SPB and in a wild-type background. Data are pooled from 3 replicates and sorted into 1-μm bins. Data are given as percentage of cells in a given bin with a Cdc13^HPM-sfGFP focus. n > 100 cells per replicate per strain. Total n = 310 cells for cut12.s11, 350 cells for cut12.T75DT78D, 244 cells for plo1.S402E, and 332 cells for wild-type (WT) cells. (H) Percentage of cells with Cdc13-sfGFP foci when released into mitosis in the presence of the temperature-sensitive plo1-24c allele. cdc2^asplo1-24c cells carrying either Cdc13^WT-sfGFP or Cdc13^HPM-sfGFP were arrested in G2 by the addition of 1 μM 1-NmPP1 for 3.5 h. Cells labeled 36°C were shifted to the plo1-24c restrictive temperature 90 min before washing out 1-NmPP1 to release into mitosis. Cells were imaged 6 min after release from the 1-NmPP1 block. The mean and SD of 3 replicates are shown. n > 75 cells per replicate per strain with a minimum of 225 cells analyzed in total. See also Figure S2 and Table S2.

Figure 3

Loss of Centrosomal Cyclin-CDK Results in Impaired Global Mitotic Phosphorylation (A) Cell length measurements of either wild-type (left, +G1/S cyclins) or Δcig1 Δcig2 Δpuc1 (right, ΔG1/S cyclins) cells expressing either exogenous Cdc13^WT/HPM or with no additional Cdc13 construct. p < 0.0001 for all comparisons using unpaired Student’s t test with Welch’s correction. Box is delimited by 25^th and 75^th percentiles and shows the mean. Whiskers delimit the 10th to 90th percentiles. n > 50 cells per condition. (B) Heatmap of mitotic CDK phosphosites after mitotic release. Each row represents a single CDK phosphosite. All measurements are normalized to phosphorylation intensity of the phosphosite at 12 min after release in the Cdc13^WT condition, and therefore, all Cdc13^WT measurements are 0 at this time point (this time point represents maximum mitotic phosphorylation). n = 105 phosphosites. Only sites that were found in all time points across both conditions are shown. Sites shown are hierarchically clustered (see STAR Methods). Right-hand bar represents sites that encompasses HP-dependent (red) and HP-independent (blue) categories. (C) Gene enrichment analysis of mitotic CDK phosphosites, showing p values obtained from Gene Ontology cellular compartment enrichment analysis (see STAR Methods). Analysis was conducted using Fisher’s exact test, using false discovery rate correction. (D) Phosphosite phosphorylation profiles for substrates considered to be either localized to the nucleus, SPB, nuclear envelope, or cytoplasm (see STAR Methods). Median phosphorylation values are plotted as a percentage of phosphorylation at t = 12 for Cdc13^WT phosphosites. Error bars represent the interquartile range; n numbers represent number of phosphosites detected at a given location. (E) Cumulative frequency curves of HP-dependent and HP-independent phosphosites against their average IC₅₀ to 1-NmPP1 inhibition of CDK [5]. 0–400 nM 1-NmPP1 is shown on x axis; inset shows entire range of data (0–3,000 nM 1-NmPP1). One data point is beyond axis limits but included in statistical analysis. Mann-Whitney rank comparison p < 0.0001. (F) Cumulative frequency of differentially localized phosphosites against their average IC₅₀ to 1-NmPP1 inhibition of CDK [5]. 0–1,500 nM 1-NmPP1 is shown on x axis; inset shows entire range of data (0–5,000 nM 1-NmPP1). 3 data points are beyond axis limits but included in statistical analysis. Difference between nucleus/NE versus cytoplasm: p < 0.01. Difference between nucleus/NE versus SPB: p < 0.001. Both are calculated by Mann-Whitney rank comparison. For determination of substrate location, see STAR Methods. See also Figures S3 and S4 and Table S2. See Table S1 for phosphoproteomic data.

Figure 4

The Hydrophobic Patch Directs Human Cyclin B1 to Centrosomes (A) Alignment of major B-type cyclin sequences from evolutionarily distant eukaryotes. Boxed residues correspond to those mutated in this study to construct Cdc13^HPM and human cyclin B1^HPM. (B) U2OS cells stably expressing γ-tubulin-EGFP (to mark the centrosome) were transfected with cyclin B1^WT/HPM-mCherry. Cells from an asynchronous population were analyzed for centrosomal cyclin B1-mCherry signal. Mean and SD of 3 replicates are shown. n > 70 cells per replicate per condition. Total n = 294 cells for cyclin B1^WT-mCherry and 287 cells for cyclin B1^HPM-mCherry. (C) Representative cells as in (B) followed through division with time-lapse microscopy. Only the in-focus slice (judged by γ-tubulin-EGFP signal) is shown. All frames are the first time point with nuclear cyclin B1-mCherry indicating mitotic entry. Arrows show position of prominent γ-tubulin-EGFP foci, co-localization with cyclin B1^WT, and lack of co-localization with cyclin B1^HPM. Graphs show average pixel intensity of a line 3 pixels in height drawn through the centrosome. Scale bars, 20 μm. See also Videos S1 and S2.