Requirement of PP2A-B56Par1 for the Stabilization of the CDK Inhibitor Rum1 and Activation of APC/CSte9 during Pre-Start G1 in S. pombe

Abstract

Exit from the cell cycle during the establishment of quiescence and upon cell differentiation requires the sustained inactivation of CDK complexes. Fission yeast cells deprived of nitrogen halt cell cycle progression in pre-Start G1, before becoming quiescent or undergoing sexual differentiation. The CDK inhibitor Rum1 and the APC/C activator Ste9 are fundamental for this arrest, but both are down-regulated by CDK complexes. Here, we show that PP2A-B56^Par1 is instrumental for Rum1 stabilization and Ste9 activation. In the absence of PP2A-B56^Par1, cells fail to accumulate Rum1, and this results in persistent CDK activity, Ste9 inactivation, retention of the mitotic cyclin Cdc13, and impaired withdrawal from the cell cycle during nitrogen starvation. Importantly, mutation of a putative B56 interacting motif in Rum1 recapitulates these defects. These results underscore the relevance of CDK-counteracting phosphatases in cell differentiation, establishment of the quiescent state, and escape from it in cancer cells.

Keywords: Biochemistry; Biological Sciences; Cell Biology.

Graphical abstract

Figure 1

PP2A-B56^Par1 Activity Is Required for an Adequate Mating and Cell-Cycle Arrest in G1 upon Nitrogen Depletion (A) Homothallic WT, pab1Δ, par1Δ, clp1Δ, and dis2Δ cells were incubated at 25°C in the absence of nitrogen, and their mating ability was determined at 0, 8, 24, and 48 h. Mean values of five biological replicates ± SD are shown. Statistical significance of the difference between strains was assessed with a t test assuming two-tailed distribution and unequal variance. ∗∗p < 0.01, and ∗∗∗p < 0.001. (B) Homothallic WT and par1Δ cells were maintained at 25°C in the absence of nitrogen. Cells were fixed at indicated time points, and pictures were taken after staining the cells with DAPI. Differential interference contrast images were overlaid to determine the cell outline. Arrowheads indicate zygotes and tetrads. (C) Homothallic WT and par1Δ cells were incubated at 25°C in the absence of nitrogen, and samples were collected at the indicated time points. Phosphorylation at Ser546 of the AKT homolog Gad8 was used as a readout of TORC2 activity. Phosphorylation of Psk1 (S6 kinase in S. pombe) and Rps6 (ribosomal protein S6) were used as a readout of the activity of TORC1, and total Cdc2 (PSTAIR) served as loading control. (D) Flow cytometric analysis of the DNA content of isolated nuclei from heterothallic WT and par1Δ cells collected at the indicated time points during a time course in the absence of nitrogen. See also Figure S1.

Figure 2

Depletion of Par1 Activity Prevents the Complete Degradation of Cdc13 (A) Homothallic WT and par1Δ cells were incubated at 25°C in the absence of nitrogen, and samples were collected at the indicated time points. Protein levels of Cdc13, Cig2, and phosphorylation of Cdc2 on Tyr15 were followed over the time course by western blot. Total Cdc2 (PSTAIR) served as loading control. (B) Control cells containing the auxin-inducible degron background (Padh15-skp1-At-Tir1-2NLS Padh15-sk1-Os-Tir1) and nmt41-3PK-miniAID-par1 par2Δ cells in the same genetic background (referred to as AID-par1 par2Δ) were treated or not (as indicated in the figure) with thiamine (0.5 μM) and 1-Naphthaleneacetic acid potassium salt (NAA; 1 mM) 2 h before being incubated in the absence of nitrogen. Samples were collected at the indicated time points. Protein levels of Cdc13 and Cig2, and phosphorylation of Cdc2 on Tyr15, were determined by western blot. B56^Par1 depletion was assessed by western blot against its N-terminal 3PK tag. Total Cdc2 (PSTAIR) was used as loading control. (C) Flow cytometric analysis of the DNA content of control cells containing the auxin-inducible degron background (Padh15-skp1-At-Tir1-2NLS Padh15-sk1-Os-Tir1) and nmt41-3PK-miniAID-par1 par2Δ treated or not with thiamine (0.5 μM) and NAA (1 mM) and collected at the indicated time points during a time course in the absence of nitrogen. At least 7,000 gated single nuclei are represented at each time point. (D) Heterothallic h-WT, par1Δ, cdc2-3w, and cdc2-3w par1Δ cells were incubated at 30°C in EMM (control) or in EMM-N for 4 h. Fixed cells were measured after staining them with Calcofluor. (E) Heterothallic h-WT, par1Δ, cdc2-3w, and cdc2-3w par1Δ cells were incubated at 30°C in the absence of nitrogen, and samples were collected at the indicated time points. Protein levels of Cdc13 were assessed by western blot. Total Cdc2 (PSTAIR) was used as loading control. (F) cdc10-V50 and cdc10-V50 par1Δ cells were incubated at 32°C for the indicated time. Control cells were incubated at 25°C. Western blots show Cdc13 and Cig2 levels, phosphorylation of Cdc2, and total Cdc2 (PSTAIR) as loading control. (G) Flow cytometric analysis of the DNA content of isolated nuclei from cdc10-V50 and cdc10-V50 par1Δ cells collected at the indicated time points during a time course at 32°C. See also Figure S2.

Figure 3

Rescue of the Defect in the G1 Arrest, Mating Efficiency, and Degradation of Cdc13 of a par1Δ Mutant by Deletion of the G1/S Cyclins (A) Homothallic WT, par1Δ, cig1Δ cig2Δ, and cig1Δ cig2Δ par1Δ cells were incubated at 25°C in the absence of nitrogen; cells were collected at the indicated time points, and after processing their DNA content was measured by flow cytometry. (B) Efficiency of mating of the homothallic strains WT, par1Δ, cig1Δ cig2Δ, and cig1Δ cig2Δ par1Δ after 0, 8, 24, and 48 h upon nitrogen depletion. Mean values of three biological repeats ± SD are shown. Statistical significance of the difference between strains was assessed with a t test assuming two-tailed distribution and unequal variance. ∗p < 0.05, ∗∗∗p < 0.001. (C) Homothallic WT, par1Δ, cig1Δ cig2Δ, and cig1Δ cig2Δ par1Δ cells were incubated at 25°C in the absence of nitrogen, and samples were collected at the indicated time points. Protein levels of Cdc13 and Cig2 and phosphorylation of Cdc2 on Tyr15 were assessed by western blot. Total Cdc2 (PSTAIR) served as loading control. (D) Homothallic WT, par1Δ, cig1Δ cig2Δ, and cig1Δ cig2Δ par1Δ cells were maintained in the absence of nitrogen for 8 h. Pictures of fixed cells were taken after staining them with DAPI. Differential interference contrast images were overlaid to determine the cell outline. Arrows indicate zygotes. See also Figure S3.

Figure 4

Deletion of par1 Causes a Defect in Ste9 Dephosphorylation and Rum1 Stabilization (A) Homothallic HA-ste9, HA-ste9 par1Δ, HA-ste9 cig1Δ cig2Δ, and HA-ste9 cig1Δ cig2Δ par1Δ cells were incubated at 25°C in the absence of nitrogen, and samples were collected at the indicated time points. Protein levels of Cdc13 were followed over the time course by western blot. Ste9 was detected by western blot against its N-terminal HA-tag. β-Actin served as loading control. HA, hemagglutinin. (B) Homothallic WT, par1Δ, cig1Δ cig2Δ, and cig1Δ cig2Δ par1Δ cells were incubated at 25°C in the absence of nitrogen, and samples were collected at the indicated time points. Protein levels of Rum1 were assessed by western blot. β-Actin was used as loading control. (C) WT, par1Δ, wee1-50, and wee1-50 par1Δ cells were grown in liquid rich medium (yeast extract with supplements (YES)) medium at 30°C and then spotted onto YES plates at serial 10-fold dilutions (from left to right). Plates were incubated at 25°C, 32°C, 34°C, and 36°C for 3 days before pictures were taken. (D) wee1-50 cells and wee1-50 par1Δ cells were incubated at 36°C, and samples were collected at the indicated time points. Control cells (0 time point) were incubated at 25°C. Western blots show protein levels of Cdc13, Rum1 together with β-actin and Cdc2 (PSTAIR) as loading controls. See also Figure S4.

Figure 5

The Defect in the Degradation of Cdc13 in a par1Δ Mutant Is Due to Inability of PP2A to Interact with B56-Specific Substrates (A) WT and par1Δ cells, and WT cells transformed with either pREP1-GFP-B56-SLiM-AA (mock-inhibitor) or pREP1-GFP-B56-SLiM (Par1-inhibitor), were incubated in EMM in the absence of thiamine for 40 h at 25°C. Subsequently, cells were shifted to fresh EMM (control point) and to EMM-N for 6 h. Cells were fixed and stained with DAPI and Calcofluor before obtaining images. (B) Overexpressed GFP-B56-SLiM (Par1-inhibitor) and GFP-B56-SLiM-AA (mock-inhibitor) were purified by means of their N-terminal GFP-tag using a GFP trap from cells containing a TAP-tagged allele of par1. A representative experiment shows the co-purifying and the unspecifically pulled down TAP-Par1 detected through western blot against the CBP epitope in the TAP-tag. Western blot against GFP served as control of the GFP pull down. (C) WT and par1Δ cells, and WT cells transformed with either pREP1-GFP-B56-SLiM-AA (mock-inhibitor) or pREP1-GFP-B56-SLiM (Par1-inhibitor) were incubated in EMM in the absence of thiamine for 40 h at 25°C. Cells were shifted to EMM-N, and samples were collected at the indicated time points. Western blots show protein levels of Cdc13 and Cig2, phosphorylation of Cdc2 on Tyr15, and total Cdc2 (PSTAIR) as loading control. See also Figure S5.

Figure 6

PP2A-B56 ^Par1 Interacts with the CDK Inhibitor Rum1 (A) Amino acid sequence of Rum1. The putative SLiMs for PP2A-B56 are in blue and the sites phosphorylated by CDK are in red. (B) TAP-Par1 was purified using the tandem affinity purification method. 100 ng of Ste9 homogeneous recombinant protein was added to the purified Par1, and after washing and eluting CBP-Par1 with TEV protease (which cleaves the TEV site between the protein A and CBP moieties of the TAP-tag), the bound (elution) and unbound (supernatants) Ste9 was detected by western blot. Untagged WT extract was used as a negative control. Note that, as Ste9 and Par1 have very similar molecular masses, and the protein A present in the TAP-tag is also bound by the rabbit IgGs, signal corresponding to the uncleaved TAP-Par1 appears in the Ste9 blot (marked with an asterisk). In the lower panel, signal coming from the previous incubation with the Ste9 antibody remains in the CBP western blot. (C) TAP-Par1 and TAP-Par1F314Q were purified using the tandem affinity purification method. 50 ng of Rum1 homogeneous recombinant protein was added to the purified Par1 and TAP-Par1F314Q; after washing and eluting CBP-Par1 with TEV protease, the unbound (supernatants) and the interacting and unspecifically pulled down (elution) Rum1 was detected by western blot. Untagged WT extract was used as a negative control. The amount of pulled down CBP-Par1 and CBP-Par1F314Q were detected by western blot against the CBP-tag. See also Figure S6.

Figure 7

Mutation of a Putative SLiM Motif of PP2A-B56 in Rum1 Phenocopies the Deletion of par1 (A) Homothallic WT, par1Δ, and rum1 BM1AA cells were incubated at 25°C in the absence of nitrogen, and their mating efficiency was determined after 0, 8, 24, and 48 h. Mean values of three biological repeats ± SD is shown. Statistical significance of the difference between strains was assessed with a t test assuming two-tailed distribution and unequal variance. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. (B) Flow cytometric analysis of the DNA content of WT and rum1 BM1AA cells collected every 30 min during a time course of 6 h in the absence of nitrogen. (C) WT and rum1 BM1AA cells were incubated at 25°C in the absence of nitrogen, and samples were collected at the indicated time points. Western blots show protein levels of Cdc13 and Rum1. α-Tubulin was used as loading control. (D) HA-ste9 and HA-ste9 rum1 BM1AA cells were maintained at 25°C without nitrogen, and samples were collected at the indicated time points. Ste9 was detected by western blot against its N-terminal HA-tag, and β-actin served as loading control. HA, hemagglutinin. (E) HA-Ste9, HA-Ste9 par1Δ, HA-Ste9 nmt81::rum1 T58A T62A, and HA-Ste9 nmt81::rum1 T58A T62A par1Δ cells were incubated for 24 h in the absence of thiamine for the expression of Rum1 T58A T62A. Cells were washed with four volumes of EMM-N before resuspending them in EMM-N, and samples were collected at the indicated time points. Protein levels of Cdc13 and Cig2, and phosphorylation of Cdc2 Tyr15 were determined by western blot. Total Cdc2 (PSTAIR) was used as loading control. See also Figure S7.

Figure 8

Model (A) (Left) During the nitrogen starvation response rum1 mRNA expression is induced and Rum1 protein is produced. CDK complexes phosphorylate and target newly synthesized Rum1 for degradation, but this phosphorylation is opposed by PP2A-B56^Par1, which leads to the stabilization of Rum1. As Rum1 accumulates, this eventually results in the sustained inactivation of CDK complexes. (Right) Owing to the presence of double-negative feedback loops between CDK complexes and their inhibitors, inactivation of CDK complexes leads to the further stockpiling of Rum1 and the termination of the inhibition of Ste9. (B) (Left) In the absence of PP2A-B56 activity, Rum1 phosphorylation mediated by CDK complexes cannot be counteracted and Rum1 does not accumulate in the cell. (Right) In consequence CDK complexes are not inhibited and can repress APC/C-Ste9 activity, resulting in impaired degradation of Cdc13.