Sequential counteracting kinases restrict an asymmetric gene expression program to early G1

Abstract

Gene expression is restricted to specific times in cell division and differentiation through close control of both activation and inactivation of transcription. In budding yeast, strict spatiotemporal regulation of the transcription factor Ace2 ensures that it acts only once in a cell's lifetime: at the M-to-G1 transition in newborn daughter cells. The Ndr/LATS family kinase Cbk1, functioning in a system similar to metazoan hippo signaling pathways, activates Ace2 and drives its accumulation in daughter cell nuclei, but the mechanism of this transcription factor's inactivation is unknown. We found that Ace2's nuclear localization is maintained by continuous Cbk1 activity and that inhibition of the kinase leads to immediate loss of phosphorylation and export to the cytoplasm. Once exported, Ace2 cannot re-enter nuclei for the remainder of the cell cycle. Two separate mechanisms enforce Ace2's cytoplasmic sequestration: 1) phosphorylation of CDK consensus sites in Ace2 by the G1 CDKs Pho85 and Cdc28/CDK1 and 2) an unknown mechanism mediated by Pho85 that is independent of its kinase activity. Direct phosphorylation of CDK consensus sites is not necessary for Ace2's cytoplasmic retention, indicating that these mechanisms function redundantly. Overall, these findings show how sequential opposing kinases limit a daughter cell specific transcriptional program to a brief period during the cell cycle and suggest that CDKs may function as cytoplasmic sequestration factors.

Figure 1.

Ace2 is exported from nuclei in early G1, concurrent with loss of Cbk1 phosphorylation and inactivation of transcriptional activity. (A) Diagram of GAL-CDC20 metaphase arrest and release. Illustrations summarize Ace2-GFP localization throughout the course of the release. (B) GAL-CDC20 Ace2-GFP Myo1-CHERRY cells were arrested in M phase by glucose incubation for 2 h and then released into galactose medium. Images were collected every 3 min. At 30 min after release, Ace2-GFP accumulated in daughter nuclei, and actomyosin ring contraction occurred at 36 min (signifying completion of cytokinesis). Loss of nuclear fluorescence was evident around 60 min. Ace2-GFP nuclear fluorescence was completely lost by 84 min, whereas cytoplasmic fluorescence of Ace2-GFP increased. (C) Quantification of 10 GAL-CDC20 Ace2-GFP Myo1-CHERRY time courses. The ratio of the average nuclear-to-cytoplasmic fluorescence is displayed for each time point. Time points of one are highlighted in black. Gray box represents distribution of timing of actomyosin ring contraction for all cells quantified. (D) Quantitative RT-PCR of Ace2 transcriptional target CTS1 after GAL-CDC20 arrest and release. Ace2 transcriptional activity peaked at 50 min after release and fell at 60–70 min. Averages of two independent trials are shown. Error bars, SEM. (E) GAL-CDC20 Ace2-HA cells were arrested and released as in A, and Ace2-HA was immunoprecipitated every 10 min after release. Blots were probed for phosphorylated S122 with a phospho-specific antibody (α-pS122) and α-HA. S122 phosphorylation was evident at 30 min and disappeared by 50 min.

Figure 2.

Ace2 requires continuous Cbk1 activity to remain in the nucleus. (A) GAL-CDC20 Ace2-GFP cbk1-as2 cells were arrested in M phase and released into galactose medium. 25 μM 1NA-PP1 was added 35 min after release, and images were collected every minute after inhibition. Nontreated control cells are shown for comparison (above). Nuclear Ace2-GFP fluorescence decreased 2 min after Cbk1 inhibition and was absent after 5 min. (B) Quantification of time courses performed in A. For each time point, the ratio of nuclear-to-cytoplasmic fluorescence is shown. Top graph shows quantification of untreated control cells, bottom graph shows 1NA-PP1–treated cells. (C) GAL-CDC20 Ace2-HA cbk1-as2 cells arrested and released as in A. 1NA-PP1 was added after sample collection at 30 min after release. Ace2-HA immunoprecipitates probed with α-p122 and α-HA show that S122 phosphorylation was undetectable after addition of 1NA-PP1.

Figure 3.

Ace2 nuclear import is blocked during most of the cell cycle. (A) Asynchronous crm1-T539C Ace2-GFP cells were pulse-labeled with Rhodamine-ConA for 5 min and grown in fresh media without ConA for approximately one doubling. Unlabeled G1 daughter cells were scored for Ace2 nuclear fluorescence with or without 30 min LMB treatment. The increase in Ace2 nuclear localization upon LMB addition is not statistically significant (p = 0.3, Student's two-tailed t test). Graph shows percentage of daughter cells with Ace2 nuclear localization. Each bar denotes the average of four independent trials; error bars, SEM. (B) Small and large-budded crm1-T539C Ace2-GFP cells from an asynchronous culture were scored for the presence or absence of Ace2 in nuclei with and without LMB treatment.

Figure 4.

Ace2's nuclear import is unrestricted when PHO85 is deleted and Ace2's CDK sites are eliminated. (A) G1 crm1-T539C daughter cells were scored for nuclear localization of Ace2-GFP with or without addition of LMB. Cdc28-as1 cells were treated for 2 h with 5 μM 1NM-PP1 to inhibit kinase activity. Note that inhibition of cdc28-as1 results in cell cycle arrest at late G1. Mother and daughter cells were distinguished as in Figure 3A. Pho85-as1 cells were treated for 2 h with 10 μM 1NA-PP1, and importantly, this does not cause cell cycle arrest. Dotted line denotes percentage of wild-type G1 cells with Ace2 localized upon LMB addition as shown in Figure 3A. Error bars, SEM. (B) G1 daughter cells, differentiated from mothers as in A, were scored for Ace2 nuclear localization with and without simultaneous inhibition of cdc28-as1 and pho85-as1 with 5 μM 1NM-PP1 and 10 μM 1NA-PP1. Note that uninhibited cells are asynchronous, whereas doubly inhibited cells are all arrested in late G1. Dotted line denotes percentage of wild-type G1 cells with Ace2 localized upon LMB addition. Each bar denotes average of two independent trials; error bars, SEM. Significant increase in the number of cells with nuclear Ace2-GFP was evident upon inhibition of both kinases. (C) Cdc28 and Pho85 kinase activity is required for Ace2 cytoplasmic retention. (D) G1 crm1-T539C daughter cells of indicated genotypes from asynchronous cultures were scored for ace2-AP-GFP nuclear fluorescence with and without 30-min LMB treatment. Mother and daughter cells were distinguished, and cdc28-as1 and pho85-as1 cells were inhibited as in A. Graph shows percentage of daughter cells with ace2-AP-GFP nuclear localization for each strain. Each bar denotes the average of 3–5 independent trials; error bars, SEM. Dotted line denotes percentage of wild-type G1 cells with Ace2 localized upon LMB addition. (E) CDK phosphorylation on Ace2 does not fully block nuclear import, suggesting an additional mechanism independent of direct phosphorylation. (F) G1 crm1-T539C daughter cells were scored for nuclear Ace2 localization with and without LMB treatment as in A. Graph shows percentage of daughter cells with Ace2 localized to nuclei. Each bar denotes the average of 3–4 independent trials; error bars, SEM. Black dotted line indicates percentage of wild-type G1 cells with Ace2 localized after LMB treatment. Red dotted lines denotes percentage of G1 cells with ace2-AP localized to nuclei upon LMB addition as in D. (G) Ace2 cytoplasmic sequestration requires both direct phosphorylation at CDK sites (red lines) and an unknown phosphorylation-independent mechanism mediated by Pho85 (blue line), which is activated by Cdc28 or Pho85 kinase activity.

Figure 5.

Pho85 does not phosphorylate non-consensus motifs within Ace2. (A) Diagram of the ace2-AP allele, denoting positions of all 21 putative CDK consensus motifs ([S/T]-P), in which serines and threonines were changed to alanine. Sites with evidence for in vivo phosphorylation by mass spectrometry are noted, with an asterisk (*), indicating sites specifically identified, and dagger (†), indicating ambiguity of phosphorylation among sites within an examined peptide. (B) Ace2-HA and ace2-AP-HA substrates were immunoprecipitated from yeast cells and in vitro phosphorylated by bacterially expressed His-Pho85as/Pcl1 complex and γ-³²P-ATP for 1 h. Protein levels are shown. (C) Quantification of assays shown in B. Phosphorylation was normalized to total protein, and background levels were subtracted. Graph shows average of three independent trials; error bars, SEM. Ace2-HA is efficiently phosphorylated, whereas ace2-AP-HA cannot be statistically significantly phosphorylated over background.

Figure 6.

Temporal restriction of Ace2 activity through sequential opposing regulation of its nucleo-cytoplasmic trafficking. (A) In early mitosis, Ace2 nuclear import is restricted by phosphorylation of its CDK sites. After mitotic exit, release of Cdc14 results in dephosphorylation of these sites, allowing nuclear entry in mother and daughter cells. (B) Cbk1 is activated at the M/G1 transition and phosphorylates Ace2 in the daughter cell, promoting its activation and accumulation in its nucleus. Ace2's localization requires continuous Cbk1 phosphorylation that is constitutively counteracted by an opposing phosphatase. Ace2 export occurs upon Cbk1 inactivation or phosphatase up-regulation. (C) Once exported, Ace2's nuclear import is restricted by phosphorylation of its CDK sites by Pho85 and/or Cdc28 (red lines). In addition to direct regulation by phosphorylation of Ace2 CDK sites, Pho85 likely activates a mechanism that sequesters Ace2 in the cytoplasm independent of direct phosphorylation (blue lines). This additional mechanism requires Pho85 or Cdc28 kinase activity to function (red lines).