Journal: Life Science Alliance

Article Title: Congress of multiple dimers is needed for cross-phosphorylation of IRE1α and its RNase activity

doi: 10.26508/lsa.202302562

Figure Lengend Snippet: (A) Cells reconstituted with FL-, dim-, dim-DDD- and dim-KB-IRE1 were induced with different concentrations of doxycycline to induce comparable low, medium, and high expression levels, as indicated. IRE1 mutant proteins were revealed by immunoblot with anti-IRE1 antibodies. (B) Cells expressing dim-DDD- and dim-KB-IRE1 at low or high levels were treated with AP20187 (green) and/or 1NM-PP1 (blue) as indicated. Immunoblot analysis with anti-IRE1 antibodies shows that none of the treatments alter the expression levels of either of the two IRE1 mutants. (C) Similar to , HeLa cell lines expressing KB- and KB-DDD-IRE1α mutants under the TetON-inducible promoter were treated with different concentrations of doxycycline to adjust the expression level of the transgenes to comparable levels between each other (low and medium levels in this case). Aliquots from the cell lysates were resolved electrophoretically and the blots decorated with anti-IRE1α. (D) Microscopy images of cells expressing GFP-tagged KB- and KB-DDD-IRE1 at the medium expression level (see (C)). The boxed area in each image is shown magnified on the right, to facilitate the comparison. KB-DDD-IRE1 forms distinct foci upon treatment with Tm and 1NM-PP1, whereas KB-IRE1 does not. All images have been acquired with the same magnification; scale bar (valid for all panels) = 10 μm.

Article Snippet: For allosteric activation of kinase bypass IRE1 mutants (KB-, KB-DDD- and dim-KB-IRE1), 1NM-PP1 (MedChemExpress) was used at the concentration of 7 μM.

Techniques: Expressing, Mutagenesis, Western Blot, Microscopy, Comparison

Journal: Life Science Alliance

Article Title: Congress of multiple dimers is needed for cross-phosphorylation of IRE1α and its RNase activity

doi: 10.26508/lsa.202302562

Figure Lengend Snippet: (A) Cells expressing endogenous IRE1 were treated with dimerizing drug AP20187, nucleotide analog 1NM-PP1, and/or Tm, as indicated. As shown by the XBP1 splicing assay, in none of the conditions AP20187 and/or 1NM-PP1 affected endonuclease activity of wt IRE1. (B) HeLa cells reconstituted with full-length (FL) or dimerizable (dim) IRE1 were treated with doxycycline to achieve low or high expression levels of the respective protein. Cells were then treated with Tm (red) or AP20187 (green). Neither of the treatments alter the expression levels of IRE1, as shown by immunoblot with anti-IRE1 antibodies. A similar result for two other IRE1 mutants is shown in . (C) Cells expressing dim-IRE1 at high or medium levels were treated with increasing concentrations of AP20187 as indicated. As shown by the XBP1 splicing assay, a concentration of 5 nM is already sufficient to activate endonuclease activity if dim-IRE1 is expressed at high levels. On the contrary, if dim-IRE1 is present in lesser amounts (medium expression) AP20187 is unable to trigger XBP1 splicing even at 320 nM concentration. Key for activation is therefore the concentration of IRE1 dimers. In the rest of the study, unless differently stated, we have used AP20187 at a concentration of 10 nM.

Article Snippet: For allosteric activation of kinase bypass IRE1 mutants (KB-, KB-DDD- and dim-KB-IRE1), 1NM-PP1 (MedChemExpress) was used at the concentration of 7 μM.

Techniques: Expressing, Splicing Assay, Activity Assay, Western Blot, Concentration Assay, Activation Assay

Journal: Life Science Alliance

Article Title: Congress of multiple dimers is needed for cross-phosphorylation of IRE1α and its RNase activity

doi: 10.26508/lsa.202302562

Figure Lengend Snippet: (A) Endonuclease activity of dimerizable IRE1α mutants at different expression levels (low, medium, high) after treatment with or without dimerizing drug (AP20187, green) and with or without 1NM-PP1 (blue). (B) Cells expressing FL-, dim-KB-, and dim-DDD-IRE1, at low or high levels as indicated, were treated with Tm (red) or AP20187 (green). The phosphorylation state of IRE1 mutants was assessed by Phos-tag gels and immunoblot with anti-IRE1 antibodies. dim-DDD (because of the substitutions of the three serines) and dim-KB (because of the inability to bind ATP and, hence, phosphotransfer) cannot be phosphorylated even if highly expressed. (C) Endonuclease activity of nucleotide binding requiring (KB-)IRE1α mutants, expressed at low and medium levels, after treatment with Tm (red) and/or NM-PP1 (blue), as indicated. KB-DDD-IRE1α was used to evaluate the possible role of phosphorylation in stabilization of the nucleotide-bound state. XBP1 splicing by both KB mutants depends on nucleotide activation (compare, for instance, lanes 1–2 with 3–4), intracellular concentration (compare lanes 3 with 7 or 4 with 8), and phosphorylation (compare lanes 3 with 11 or 4 with 12). (D) Confocal images of cells expressing GFP-tagged dim-DDD-IRE1. Even at high expression levels the mutant does not form foci upon treatment with AP20187. Scale bar = 10 μm.

Article Snippet: For allosteric activation of kinase bypass IRE1 mutants (KB-, KB-DDD- and dim-KB-IRE1), 1NM-PP1 (MedChemExpress) was used at the concentration of 7 μM.

Techniques: Activity Assay, Expressing, Western Blot, Binding Assay, Activation Assay, Concentration Assay, Mutagenesis

Journal: bioRxiv

Article Title: Negative regulation of APC/C activation by MAPK-mediated attenuation of Cdc20 Slp1 under stress

doi: 10.1101/2024.04.02.587770

Figure Lengend Snippet: ( 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 ( pek1 DD , pek1-S234D;T238D ) or Wis1 ( wis1 DD , wis1-S469D;T473D ) respectively. ( B) Serial dilution assay on TBZ sensitivity of all MAPK deletion mutants and pek1 DD - or wis1 DD -overexpressing mutants. mad2 Δ is a positive control. Note P adh11 is a stronger version of P adh21 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.

Article Snippet: For cdc2-asM17 strains, 1 μM 1-NM-PP1 (Toronto Research Chemicals; A603003) was added into one of the cultures to inactivate Cdc2 (Cdk1) 20 min before being collected.

Techniques: Mutagenesis, Serial Dilution Assay, Positive Control, Activation Assay, Microscopy

Journal: bioRxiv

Article Title: Negative regulation of APC/C activation by MAPK-mediated attenuation of Cdc20 Slp1 under stress

doi: 10.1101/2024.04.02.587770

Figure Lengend Snippet: ( 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 Slp1 Cdc20 were detected by immunoblotting. The amount of co-immunoprecipitated Mad2, Mad3 and Slp1 Cdc20 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 Slp1 Cdc20 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 Slp1 Cdc20 abundance in nda3-KM311 cells treated at 18 °C for 6 hr. Slp1 Cdc20 levels were quantified with the relative ratio between Slp1 Cdc20 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 pek1 DD 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 , pek1 DD and wis1 DD mutants.

Article Snippet: For cdc2-asM17 strains, 1 μM 1-NM-PP1 (Toronto Research Chemicals; A603003) was added into one of the cultures to inactivate Cdc2 (Cdk1) 20 min before being collected.

Techniques: Immunoprecipitation, Western Blot, Quantitative RT-PCR, RNA Extraction, Expressing, Mutagenesis, Standard Deviation, Two Tailed Test, Activation Assay

Journal: bioRxiv

Article Title: Negative regulation of APC/C activation by MAPK-mediated attenuation of Cdc20 Slp1 under stress

doi: 10.1101/2024.04.02.587770

Figure Lengend Snippet: (A) Non-radioactive in vitro phosphorylation assays with bacterially expressed recombinant GST-Slp1 Cdc20 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-Pek1 DD does not affect Slp1 Cdc20 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 Slp1 Cdc20 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 Slp1 Cdc20 fragment (456-488aa), GST-Pmk1 and GST-Pek1 DD . The reactions were blotted with pT480 antibodies. Asterisks indicate bands corresponding to unspecific or likely degraded proteins. (D) Immunoblot detection of Slp1 Cdc20 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 pek1 DD cells relative to that in wild type cells. (E) In vitro phosphorylation assays with bacterially expressed recombinant MBP-fusion of Slp1 Cdc20 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 Slp1 Cdc20 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 Slp1 Cdc20 abundance in nda3-KM311 cells treated at 18 °C for 6 hr. Slp1 Cdc20 levels were quantified as in and . 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 . (I) Schematic summarizing the negative effect of Slp1 Cdc20 phosphorylation by Pmk1 on its abundance and APC/C activation.

Article Snippet: For cdc2-asM17 strains, 1 μM 1-NM-PP1 (Toronto Research Chemicals; A603003) was added into one of the cultures to inactivate Cdc2 (Cdk1) 20 min before being collected.

Techniques: In Vitro, Recombinant, Purification, Positive Control, Mass Spectrometry, In Vivo, Western Blot, Immunoprecipitation, Activation Assay