pmk1Δ and sty1-T97A mutants display spindle checkpoint activation defects, while pek1DD and wis1DD mutants are defective in checkpoint silencing.

(A) Schematic of core modules of three Schizosaccharomyces pombe mitogen-activated protein kinase (MAPK) signaling pathways. Each cascade consists of three core kinases (MAP kinase kinase kinase (MAPKKK), MAP kinase kinase (MAPKK), and MAPK). CIP, the cell integrity pathway; SAP, the stress-activated pathway; PSP, pheromone signaling pathway. These pathways can be disrupted by pmk1Δ, sty1Δ or ATP analogue-sensitive mutation sty1-T97A or spk1Δ, and constitutively activated by mutations in MAPKKs Pek1 (pek1DD, pek1-S234D;T238D) or Wis1 (wis1DD, wis1-S469D;T473D) respectively.

(B) Serial dilution assay on TBZ sensitivity of all MAPK deletion mutants and pek1DD- or wis1DD-overexpressing mutants. mad2Δ is a positive control. Note Padh11 is a stronger version of Padh21 promoter.

(C) Schematic depiction of the experiment design for time-course analyses on SAC or APC/C activation. nda3-KM311 cells carrying Cdc13-GFP were grown, synchronized with HU and treated at 18 °C for 6 hours to activate SAC, and finally shifted back to the permissive temperature 30 °C. Samples were collected at 10 min intervals and subjected to microscopy analyses. Example pictures of cells with Cdc13-GFP signals enriched or disappeared at spindle pole bodies (SPBs) are shown. Scale bar, 5 μm.

(D) Time-course analyses of SAC activation and inactivation in nda3-KM311 cdc13-GFP strains with indicated genotypes. sty1-T97A was inactivated by 5μM 3-BrB-PP1.

Upon spindle checkpoint activation, Slp1Cdc20 levels are reduced in pek1DD and the MCC-APC/C association is enhanced in wis1DD cells compared to wild-type cells.

(A) Co-immunoprecipitation analysis on MCC-APC/C association. Cells with indicated genotypes were grown at 30 °C to mid-log phase and arrested at 18 °C for 6 hours. Lid1-TAP was immunoprecipitated and associated Mad2, Mad3 and Slp1Cdc20 were detected by immunoblotting. The amount of co-immunoprecipitated Mad2, Mad3 and Slp1Cdc20 was quantified by being normalized to those of total immunoprecipitated Lid1 in each sample, with the relative ratio between Mad2-GFP plus Mad3-GFP or Slp1Cdc20 and Lid1-TAP in wild-type sample set as 1.0. Blots are representative of three independent experiments. p values were calculated against wild-type cells. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001; n.s., not significant.

(B, C) Immunoblot analysis of Slp1Cdc20 abundance in nda3-KM311 cells treated at 18 °C for 6 hr. Slp1Cdc20 levels were quantified with the relative ratio between Slp1Cdc20 and Cdc2 in wild-type strain set as 1.0. Phosphorylated Pmk1 (Pmk1-P) or phosphorylated Sty1 (Sty1-P) in (A) were detected using anti-phospho p42/44 and anti-phospho p38 antibodies and represents activated CIP or SAP signaling, respectively. sty1-T97A was inactivated by 5μM 3-BrB-PP1. Blots shown are the representative of three independent experiments. p values were calculated against wild-type cells. ***, p<0.001; n.s., not significant.

(D) RT-qPCR analysis of mRNA levels of slp1+. Cells with indicated genotypes were grown and treated as in (A-C) before RNA extraction. The relative fold-change (slp1+/act1+) in mRNA expression was calculated with that in wild-type cells being normalized to 1.0. Note mRNA level of slp1+ in pek1DD mutant is not decreased. Error bars indicate mean ±standard deviation of three independent experiments. Two-tailed unpaired t-test was used to derive p values. n.s, not significant.

(E) Schematic summary of the negative effect of activated CIP and SAP signaling on APC/C activation based on primary phenotype characterization of pmk1Δ, sty1-T97A, pek1DDand wis1DD mutants.

Pmk1 directly binds Slp1Cdc20 to attenuate its levels upon spindle checkpoint activation.

(A) In vitro binding assay using bacterially expressed MBP-Slp1Cdc20 and yeast lysates prepared from nda3-KM311 pmk1-HA-6His cells arrested at 18 °C for 6 hr. Note that weak band detected in MBP sample was due to unspecific background binding to amylose beads. Coomassie blue staining shows inputs for MBP and MBP-Slp1Cdc20.

(B) Schematic depiction of the S. pombe Slp1Cdc20 protein structure with the positions of two confirmed basic-residue patches mediating Slp1Cdc20-Pmk1 association indicated by pink bars. Alignment highlights the conservation of basic-residue patches within 4 Schizosaccharomyces species. The deduced Pmk1-docking motifs and different versions of motif mutations are shown. MIM, Mad2-interaction motif; IR, isoleucine-arginine tail.

(C) In vitro GST pull-down assays with bacterially expressed recombinant GST-Pmk1 and MBP-fusions of wild-type Slp1Cdc20 or Slp1Cdc20 mutants harboring Pmk1-docking motif mutations. An aliquot of the same amount of MBP-Slp1Cdc20 as that added in each GST pull-down reactions was immobilized by amylose resin as the input control. Asterisks indicate unspecific or degraded protein bands.

(D) Immunoblot analysis of Slp1Cdc20 abundance in nda3-KM311 cells with indicated genotypes treated at 18 °C for 6 hr. Slp1Cdc20 levels were quantified as in Figure 2B and 2C. The experiment was repeated 3 times and the mean value for each sample was calculated. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001; n.s., not significant.

(E) Time-course analyses of SAC activation and inactivation in nda3-KM311 cdc13-GFP strains with indicated genotypes. The experiment was repeated 3 times and the mean value for each sample was calculated as in Figure 1D.

(F) Schematic summarizing the negative effect of Pmk1-Slp1Cdc20 association on Slp1Cdc20 abundance and APC/C activation.

Pmk1 synergizes with Cdk1 to phosphorylate and reduce Slp1Cdc20 abundance.

(A) Non-radioactive in vitro phosphorylation assays with bacterially expressed recombinant GST-Slp1Cdc20 and Pmk1-HA-His purified from yeast cells. The incorporation of the thiophosphate group was determined using anti-thiophosphate ester antibodies (anti-thioP) as indicative of phosphorylation. Note that the presence or absence of GST-Pek1DD does not affect Slp1Cdc20 phosphorylation efficiency. A known Pmk1 substrate Atf1 was used as a positive control (Atf1-P). Asterisks indicate bands corresponding to unspecific or likely degraded proteins.

(B) Summary of mass spectrometry data on Slp1Cdc20 phosphorylation and ubiquitylation in vivo in nda3-KM311-arrested cells. Red arrows and filled blue circles denote all detected phosphorylated or ubiquitylated sites respectively, while gray arrows and unfilled circles indicate the absence of phosphorylation or ubiquitylation in some of these sites respectively. Alignment highlights the conservation of most of the detected phosphorylation and ubiquitylation sites within 4 Schizosaccharomyces species.

(C) In vitro phosphorylation assays with bacterially expressed recombinant GST-fusion of Slp1Cdc20 fragment (456-488aa), GST-Pmk1 and GST-Pek1DD. The reactions were blotted with pT480 antibodies. Asterisks indicate bands corresponding to unspecific or likely degraded proteins.

(D) Immunoblot detection of Slp1Cdc20 phosphorylation at T480 in vivo. GST-slp1(456-488aa) was purified from mts3-1 cells with indicated genotypes arrested at 36℃ for 3.5 hours, and detected with anti-GST and anti-pThr480 antibodies. Note that pThr480 is absent in pmk1Δ cells, and enhanced in pek1DDcells relative to that in wild type cells.

(E) In vitro phosphorylation assays with bacterially expressed recombinant MBP-fusion of Slp1Cdc20 fragment (1-190aa) and Cdc13 (cyclin B)-containing Cdk1 complexes purified from metaphase-arrested nda3-KM311 yeast cells. 1-NM-PP1 was added as inhibitor for analogue-sensitive Cdc2-as. The reactions were blotted with pS28/pT31 antibodies. Asterisks indicate bands corresponding to unspecific or likely degraded proteins.

(F) Immunoblot detection of Slp1Cdc20 phosphorylation at S28/T31 in vivo. Apc15-13myc was immunoprecipitated from nda3-KM311 cells treated at 18 °C for 6 hr and the samples were blotted with pS28/pT31 antibodies. One IP sample from wild type background was treated with λ-phosphatase. For cdc2-asM17 cells, 1-NM-PP1 was added to inactivate Cdc2 during culturing.

(G) Immunoblot analysis of Slp1Cdc20 abundance in nda3-KM311 cells treated at 18 °C for 6 hr. Slp1Cdc20 levels were quantified as in Figure 2B and 2C. The experiment was repeated 3 times and the mean value for each sample was calculated. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001; n.s., not significant.

(H) Time-course analyses of SAC activation and inactivation in nda3-KM311 cdc13-GFP strains with indicated genotypes. The experiment was repeated 3 times and the mean value for each sample was calculated as in Figure 1D.

(I) Schematic summarizing the negative effect of Slp1Cdc20 phosphorylation by Pmk1 on its abundance and APC/C activation.

Pmk1-but not Cdk1-mediated Slp1Cdc20 phosphorylation promotes its ubiquitylation.

(A) Immunoblot analysis of Slp1Cdc20 abundance in apc15+ or apc15Δ background cells with indicated genotypes after being treated at 18 °C for 6 hr. Slp1Cdc20 levels were quantified as in Figure 2B. The experiment was repeated 3 times and the mean value for each sample was calculated. ***, p<0.001.

(B) Time-course analyses of SAC activation and inactivation in nda3-KM311 cdc13-GFP strains with indicated genotypes. The experiments were performed as in Figure 1D.

(C) Immunoblot analysis of Slp1Cdc20 abundance in K472R, K479R or K472R/K479R mutants. The experiment was repeated 3 times and the mean value for each sample was calculated. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

(D) Time-course analyses of SAC activation and inactivation in nda3-KM311 cdc13-GFP strains with K472R, K479R or K472R/K479R mutations. The experiments were performed as in (B).

(E) Schematic depiction of affinity pull-down assays using TUBE (Tandem Ubiquitin Binding Entity) agarose beads to detect Slp1Cdc20 ubiquitylation.

(F) TUBE pull-down assays in mts3-1 strains carrying sfGFP-tagged wild type or mutants of Slp1Cdc20 or pmk1Δ or pek1DDmutations. The TUBE beads-bound samples were blotted with anti-GFP antibodies. Asterisk indicate unspecific bands recognized by anti-GFP antibodies.

(G) Schematic summarizing the negative effect of the coupling of Pmk1 phosphorylation and K472/K479-mediated ubiquitylation on Slp1Cdc20 abundance and APC/C activation.

Osmotic stress and cell wall damage trigger rapid Pmk1 phosphorylation-dependent Slp1Cdc20 downregulation and mitotic exit delay.

(A) Schematic depiction of the experimental design for treatment with KCl or caspofungin during nda3-mediated spindle checkpoint activation to activate MAPKs. Samples were collected at indicated time points for subsequent analyses including immunoblotting, co-IP and time-course analysis on SAC or APC/C activation.

(B) Immunoblot analysis of activation of MAPKs and Slp1Cdc20 protein levels. Samples with or without indicated treatments were blotted with anti-phospho p42/44 and anti-phospho p38 antibodies as indicative of phosphorylated Pmk1 (Pmk1-P) or phosphorylated Sty1 (Sty1-P), respectively. Slp1Cdc20 levels were detected with anti-Slp1 antibodies and anti-Cdc2 was used as loading control.

(C) Co-immunoprecipitation analysis of APC/C-MCC association upon environmental stress. Lid1-TAP was immunoprecipitated from nda3-KM311-arrested cells and associated Mad2-GFP, Mad3-GFP and Slp1Cdc20 were detected as in Figure 2A. Note that APC/C-MCC association was disrupted when 0.6 M KCl was present during cell culturing or during immunoprecipitation procedures.

(D) Time-course analyses of SAC activation and inactivation in nda3-KM311 cdc13-GFP strains with indicated genotypes after arrest at 18 °C and KCl or caspofungin treatments.

(E, F) Immunoblot analyses of Slp1Cdc20 abundance and time-course analyses of SAC activation and inactivation efficiency in phosphorylation- and ubiquitylation-deficient mutants upon SAC activation and environmental stress.

Summary of the mechanisms that how MAPKs negatively regulate APC/C activity in fission yeast.

(A) Schematic depiction of a possible dual mechanism that how activated MAPK signaling pathways are involved in delaying APC/C activation and SAC inactivation. Upon SAC and MAPK signaling activation, division of labor between the CIP and the SAP pathways enables the phosphorylation of Slp1Cdc20 and unidentified substrate(s) by Pmk1 and Sty1 respectively, which leads to lowered Slp1Cdc20 level and enhanced MCC affinity for APC/C.

(B) Summary of the alteration of Slp1Cdc20 protein levels and MCC-APC/C association strength under various conditions examined in this study. White wavy line indicates the absence of Apc15.