Hcm1 integrates signals from Cdk1 and calcineurin to control cell proliferation

Abstract

Cyclin-dependent kinase (Cdk1) orchestrates progression through the cell cycle by coordinating the activities of cell-cycle regulators. Although phosphatases that oppose Cdk1 are likely to be necessary to establish dynamic phosphorylation, specific phosphatases that target most Cdk1 substrates have not been identified. In budding yeast, the transcription factor Hcm1 activates expression of genes that regulate chromosome segregation and is critical for maintaining genome stability. Previously we found that Hcm1 activity and degradation are stimulated by Cdk1 phosphorylation of distinct clusters of sites. Here we show that, upon exposure to environmental stress, the phosphatase calcineurin inhibits Hcm1 by specifically removing activating phosphorylations and that this regulation is important for cells to delay proliferation when they encounter stress. Our work identifies a mechanism by which proliferative signals from Cdk1 are removed in response to stress and suggests that Hcm1 functions as a rheostat that integrates stimulatory and inhibitory signals to control cell proliferation.

FIGURE 1:

Hcm1 phosphorylation affects cellular fitness. (A) Positions of Cdk1 phosphosites that activate (green) and destabilize Hcm1 (red). Mutations that block degradation lead to an increase in protein level (15A, 3N, 3N/16E); phosphomimetic mutations (16E, 3N/16E) lead to constitutive activity (CA). (B) Strains expressing PGK1-URA3 (blue lines) and PGK1-GFP-URA3 (red lines) were cocultured and the percentage of each strain in the population determined at the indicated time points. Average values ±1 SD of five to eight replicates.

FIGURE 2:

Hcm1 is dephosphorylated by calcineurin in vivo. (A) Diagram of Hcm1 mutants assayed in B–D, along with the number of Cdk1 (S/T-P) sites retained in each protein. (B) Yeast two-hybrid assay showing interaction between Hcm1 and Cna1. Deletion of the docking site (hcm1ΔPSIEIQ) disrupts the interaction. (C) Western blots comparing separation of Hcm1 proteins that contain different numbers of Cdk1 sites on a Phos-tag gel (top) and standard SDS–PAGE (bottom). (D) Phos-tag Western blot of strains expressing Hcm1-15A (15A, no Cdk1 sites), wild-type Hcm1 (WT), Hcm1ΔPSIEIQ (ΔP), or wild-type Hcm1 in a cnb1Δ strain assayed after the addition of CaCl₂ for 10 min and compared with untreated control cells. (E) Hcm1 phosphorylation assayed as in D, except that cells were treated with FK506 or ET buffer for 1 h and then CaCl₂ was added for 15 min.

FIGURE 3:

Calcineurin removes activating phosphorylations from Hcm1. (A) Strains expressing indicated Hcm1 proteins were treated with CaCl₂ for 10 min and phosphorylation assayed by Phos-tag Western blot. Note that the top band in the 12C sample is also present in the 15A sample, suggesting that it is the result of Cdk1-independent phosphorylation. (B) Cdk1-phosphorylated Hcm1 proteins were incubated with or without CN for 45 min. Average percentage phosphorylation remaining is shown ± 1 SD (n = 3). Hcm1ΔPSIEIQ is significantly different from wild-type Hcm1 (*p < 0.05, Student's t test). Also see Supplemental Figure S1B. (C) Cycloheximide-chase assays comparing the stability of Hcm1 proteins with or without CaCl₂ treatment. (D) Average data from five experiments were used to calculate half-life values (indicated in parentheses) of each protein with or without CaCl₂.

FIGURE 4:

Calcineurin inactivates Hcm1. (A) Changes in expression of predicted Hcm1 target genes (Pramila et al., 2006) in response to CaCl₂ treatment with or without the CN inhibitor FK506. Data are from Yoshimoto et al., (2002). Cluster 1 includes 49 of 91 genes that are down-regulated (less than [−0.5 log 2]-fold change) at the 30-min time point. Cluster 2 includes the remaining 42 genes. (B) Average expression of genes in each cluster over time with or without FK506. For cluster 1, average values with or without FK506 are significantly different at each time point (p < 0.0001, paired t test); for cluster 2, average values with or without FK506 are not significantly different at any time point. (C) Reverse transcription quantitative PCR of Hcm1 target genes 30 min after the addition of CaCl₂. Fold change values were calculated by comparing expression after 30 min to expression in the same strain before treatment. Average log 2–fold change values ±1 SD (n = 6). Down-regulation was significantly impaired in each mutant strain compared with wild type. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05.

FIGURE 5:

Hcm1 inactivation limits proliferation in response to environmental stress. (A) Indicated strains were cocultured and the percentage of each population at each time point quantified by flow cytometry. Average values ± 1 SD (n = 13). (B) Strains expressing the indicated Hcm1 proteins were treated with Congo red (cell wall stress) for 10 min or LiCl for 5 min and phosphorylation assayed by Phos-tag Western blot. (C) Fivefold dilutions of the indicated strains were spotted onto synthetic complete medium (SC) with or without Congo red. (D) Fivefold dilutions of the indicated strains were spotted onto rich medium (YPD) with or without LiCl.