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. 2015;14(19):3138-45.
doi: 10.1080/15384101.2015.1078036.

Phosphorylation and dephosphorylation regulate APC/C(Cdh1) substrate degradation

Kobi J Simpson-Lavy  1 , Drora Zenvirth  1 , Michael Brandeis  1
Affiliations

Phosphorylation and dephosphorylation regulate APC/C(Cdh1) substrate degradation

Kobi J Simpson-Lavy et al. Cell Cycle. 2015.

Abstract

The Anaphase Promoting Complex/Cyclosome (APC/C) ubiquitin ligase activated by its G1 specific adaptor protein Cdh1 is a major regulator of the cell cycle. The APC/C(Cdh1) mediates degradation of dozens of proteins, however, the kinetics and requirements for their degradation are largely unknown. We demonstrate that overexpression of the constitutive active CDH1(m11) mutant that is not inhibited by phosphorylation results in mitotic exit in the absence of the FEAR and MEN pathways, and DNA re-replication in the absence of Cdc7 activity. This mode of mitotic exit also reveals additional requirements for APC/C(Cdh1) substrate degradation, which for some substrates such as Pds1 or Clb5 is dephosphorylation, but for others such as Cdc5 is phosphorylation.

Keywords: APC/C, Cdc5, Cdc14, Cdh1, Clb5, Dbf4, DNA replication, exit from mitosis, Pds1, substrate phosphorylation, yeast.

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Figures

Figure 1.
Figure 1.
CDH1m11 expression enables mitotic exit with bypass of the MEN and FEAR pathways. (A) Cells were arrested with nocodazole for 3 hours and CDH1m11 expression induced by addition of 4% galactose. Cells were stained with calcofluor white to visualize cell walls and septa. (B) Diploid cells were arrested with nocodazole and galactose was added to induce CDH1m11 expression. Samples were taken at indicated times and processed for immunoblotting. (C) Cells were arrested with nocodazole at 37°C as indicated and galactose was added to induce CDH1m11 expression. (D) Cells were arrested with nocodazole for 2.5 hours and CDH1m11 expression induced by addition of 4% galactose. Cells were stained with calcofluor white to visualize cell walls and septa. In all strains, rebudding occurred. (E) Cells expressing Cdc20 under the control of the MET25 promoter were grown overnight in SC-Ura-Met 2% raffinose media. 80mg/liter methionine was added to repress Cdc20 expression. After 5 hours, cells were transferred to glass bottom Petri dishes and immobilized using Concavalin A. Galactose was added to induce CDH1m11 expression. Images of Myo1-GFP were taken at indicated times. Although rebudding occurs, Myo1-GFP remains at the bud neck.
Figure 2.
Figure 2.
Expression of CDH1m11 results in mitotic exit without Cdc14 release or cytokinesis. (A) prMET25:CDC20 prGAL:CDH1m11 cells bearing plasmids for RFP-Pus1 (nuclear marker) and Myo1-GFP (contractile ring) were arrested for 5 hours with methionine in raffinose containing medium and galactose was added to induce CDH1m11 expression. The images are stills from Movie S1. (B) Cdc14-GFP cells were arrested with nocodazole for 3 hours, washed and released into drug-free medium with galactose. The images are stills from Movie S2. (C) Cdc14-GFP prGAL:CDH1m11 cells were arrested with nocodazole for 3 hours. Galactose was added to induce CDH1m11 expression. The images are stills from Movie S3.
Figure 3.
Figure 3.
Expression of CDH1m11 overrides the requirement for Cdc7 for DNA replication. (A) Cells were arrested with nocodazole for 2.5 hours and then BrdU was added for an additional hour. Galactose was added to induce CDH1m11 expression. Samples were taken for processing at the indicated times. One sample was left for the duration of the experiment without galactose to determine the incorporation of BrdU into non-replicating cells (X120). (B) Cells were arrested with nocodazole for 2.5 hours and galactose was added to induce CDH1m11 expression. Samples were taken at indicated times and processed for immunoblotting. One sample was left without galactose treatment for the duration of the experiment as a control against prolonged exposure to nocodazole (X60). (C) Cells were arrested with nocodazole for 2.5 hours and then BrdU was added for an additional hour, during which the temperature was raised slowly to 37°C. Galactose was added to induce CDH1m11 expression. Samples were taken for processing at indicated times. One sample was left for the duration of the experiment without galactose to determine the incorporation of BrdU into non-replicating cells (X120). Another sample was grown at 37°C for the duration of the experiment without nocodazole to test the efficacy of the cdc7-1 mutation.
Figure 4.
Figure 4.
Kinetics of APC/C substrate degradation. (A) Cells were arrested with nocodazole for 3 hours and galactose was added to induce CDH1m11 expression. Samples were taken at indicated times and processed for immunoblotting. One sample was left without galactose treatment for the duration of the experiment as a control against prolonged exposure to nocodazole (X60). (B) Cells were arrested with nocodazole for 3 hours and galactose added to induce CDH1m11 expression. Samples were taken at indicated times and processed for immunoblotting.
Figure 5.
Figure 5.
Degradation of Clb5 requires its dephosphorylation. (A) Cells were arrested in metaphase with nocodazole for 2.5h, and GST6H-Clb5 expressed from a GAL promoter for an additional hour. Cells were lysed and GST6H-Clb5 pulled down using glutathione beads. Half of the extract was treated with λ-phosphatase for one hour. (B) cdc14-3 and cdc15-2 cells expressing Clb5-6HA were arrested in telophase by elevation of the temperature to 37°C for 3 hours. (C) cdc14-3 cells expressing either wild-type Clb5-6HA or Clb5T60A-6HA were either arrested at metaphase with nocodazole for 3 hours at 23°C, or in telophase by elevation of the temperature to 37°C for 3 hours.
Figure 6.
Figure 6.
Phosphorylation of Cdc5 is required for its degradation. (A) Cells expressing Cdc5 (and mutants) from a CEN plasmid were arrested with nocodazole for 3 hours and galactose was added to induce CDH1m11 expression. Samples were taken at indicated times and processed for immunoblotting. One sample was left without galactose treatment for the duration of the experiment as a control against prolonged exposure to nocodazole (X60). Another sample was treated with rapamycin (and was not treated with nocodazole) for the duration of the experiment to provide a control for G1 arrest. Both Cdc5T29A and Cdc5K26A are stable in rapamycin. (B) Cells were arrested with nocodazole for 2.5h and released into rapamycin containing media with 1M sorbitol, with and without staurosporine (40 μg/ml) for 1 hour. (C) Cells containing a plasmid with prGAL:GST6H-Cdc5 were arrested in rapamycin for 2.5 hours, and pulsed with galactose to express GST-Cdc5 or 1 additional hour during which the temperature was slowly raised to 37°C. The media was switched to media containing rapamycin (1μg/ml), glucose (4%) and cycloheximide (200 μg/ml) to cease GST-Cdc5 production. During the chase, samples were withdrawn at the indicated time points.
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