Journal: The Journal of Cell Biology

Article Title: Mechanical stress impairs pheromone signaling via Pkc1-mediated regulation of the MAPK scaffold Ste5

doi: 10.1083/jcb.201808161

Figure Lengend Snippet: Pkc1 protects cells from lysis upon mechanostress during pheromone exposure through inhibition of the mating pathway. (A) Haploid mating-type a WT cells were exposed to α-factor for 100 min, followed by 15-min pretreatment with DMSO or 7.5 µM of the Pkc1 inhibitor cercosporamide (cerc.). Then, mechanostress or no stress was applied for 30 min. Lysis of shmooing cells was visualized by phase-contrast microscopy and staining with Trypan blue dye. (B) Quantification of cell lysis in shmooing WT cells, treated as in A; >150 cells in at least three independent experiments were quantified for each condition and shown as percentage of lysed cells. Error bars indicate SEM, and significance was determined by t test (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). (C) Quantification of cell lysis in shmooing WT cells harboring or not (control) a plasmid expressing dominant-negative Pkc1 (Pkc1 K853R ) from the inducible GAL1 promoter. Cells growing in log phase in 2% raffinose were induced with 2% galactose for 2 h followed by exposure to α-factor for 100 min. Mechanostress was applied and cell lysis analyzed as described in B. (D) WT cells expressing the Fus3 SKAR and Hta2-CFP reporters were treated with pheromone and 7.5 µM cercosporamide (cerc.) as in A. Nuclear accumulation of the Fus3 SKAR reporting Fus3 activity after 30 min of mechanostress was monitored microscopically and quantified in ≥150 cells in three independent experiments. Error bars indicate SEM, and significance was determined by a t test (*, P ≤ 0.05). Representative images before and after mechanostress are shown below. (E) WT cells expressing the Fus3 SKARS and Hta2-CFP reporters were treated with pheromone and subsequently with 7.5 µM cercosporamide (cerc.) as described in A, and Fus3 activity was quantified in lysing and protected cells. Cells failing to relocalize the reporter to the nucleus under mechanostress were scored as high Fus3 activity; ≥150 cells were analyzed for each condition in three independent experiments and are shown as percentage of high or low Fus3 activity. Error bars indicate SEM. Note that cells unable to down-regulate Fus3 activity are prone to cell lysis. (F) WT cells expressing the chemically inhibitable Fus3-as allele from the endogenous locus were treated with α-factor for 100 min followed by pretreatment with 7.5 µM cercosporamide (cerc.) and 5 µM NaPP1. Then mechanostress was applied. Lysis of ≥150 pheromone-responsive cells was scored for each condition in three independent experiments and is shown as percentage of lysed cells. Error bars indicate SEM, and significance was determined by a t test (***, P ≤ 0.001). (G) Quantification of cell lysis upon mechanostress in WT or mpk1Δ cells treated as in B. At least 150 cells in at least three independent experiments were analyzed for each condition and are shown as percentage of lysed cells. Error bars indicate SEM, and significance was verified by a t test (**, P ≤ 0.01; ***, P ≤ 0.001). Genetic background and growth conditions in this experiment are the same as in B, and thus, the data for the WT conditions were combined. (H) Cells expressing Ste5-tV under its endogenous promotor in WT or mpk1Δ cells were treated as in A, and Ste5-tV was visualized microscopically in microfluidic chips during 30 min of mechanostress. Loss of Ste5-tV at shmoo tips was quantified in ≥150 cells for each condition in at least three independent experiments, and shown as percentage of cells with dispersed Ste5 localization. Error bars indicate SEM and significance was validated by a t test (**, P ≤ 0.01; ***, P ≤ 0.001). (I) Pkc1-dependent signaling protects mating cells against mechanostress. Pkc1 triggers loss of Ste5 from shmoo tips by an Mpk1-independent mechanism, leading to reduced Fus3 activity.

Article Snippet: The in vitro kinase assays for NMR analysis were performed with 150 µM Ste5 149–238 (in 250 µl) with either assay buffer (3 mM MgCl 2 and 1 mM ATP), 5 µl of PKCα (P61-10G; SignalChem), and 0.5-0.8× of Lipid Activator (L51-39, SignalChem) or 250 µl yeast extract complemented with 5 mM ATP and phosphatase inhibitors.

Techniques: Lysis, Inhibition, Microscopy, Staining, Plasmid Preparation, Expressing, Dominant Negative Mutation, Activity Assay

Journal: The Journal of Cell Biology

Article Title: Mechanical stress impairs pheromone signaling via Pkc1-mediated regulation of the MAPK scaffold Ste5

doi: 10.1083/jcb.201808161

Figure Lengend Snippet: Pkc1-dependent phosphorylation of Ste5-Ser185 inhibits signaling and prevents lysis during mechanostress. (A and B) Expression of the FIG1-qV reporter was measured by FACS in ste5Δ cells expressing GFP-tagged WT Ste5 (diamond), Ste5 S185A (square), or Ste5 S185D (triangle) and plotted in arbitrary units at the times after addition of α-factor. The cells were transformed with a plasmid expressing either WT (Pkc1, A) or dominant-active (Pkc1 R398A , B) Pkc1 from the inducible GAL1-promoter for 2 h. Expression of Pkc1 R398A inhibits pheromone signaling in a Ste5 Ser185 -dependent manner. (C) Cell lysis upon mechanostress in cells expressing WT Ste5 or Ste5 S185A mutant was analyze in cells treated as in ; ≥150 shmooing cells from three independent experiments were analyzed for each condition and are shown as percentage of lysed cells. The error bars indicate SEM, and significance was validated with a t test (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). (D) Dispersal of either tV-tagged WT Ste5 or Ste5 S185A from shmoo tips upon mechanostress in cells treated as in . Loss of Ste5-tV at shmoo tips was quantified in ≥150 cells from three independent experiments for each condition and is shown as percentage of cells with dispersed Ste5 localization. The error bars indicate SEM, and significance was validated with a t test (**, P ≤ 0.01; ***, P ≤ 0.001).

Article Snippet: The in vitro kinase assays for NMR analysis were performed with 150 µM Ste5 149–238 (in 250 µl) with either assay buffer (3 mM MgCl 2 and 1 mM ATP), 5 µl of PKCα (P61-10G; SignalChem), and 0.5-0.8× of Lipid Activator (L51-39, SignalChem) or 250 µl yeast extract complemented with 5 mM ATP and phosphatase inhibitors.

Techniques: Lysis, Expressing, Transformation Assay, Plasmid Preparation, Mutagenesis

Journal: The Journal of Cell Biology

Article Title: Mechanical stress impairs pheromone signaling via Pkc1-mediated regulation of the MAPK scaffold Ste5

doi: 10.1083/jcb.201808161

Figure Lengend Snippet: Serine 185 within the RING-H2 domain of Ste5 is phosphorylated by Pkc1. (A) Schematic of Ste5 functional domains. MAPK, MAPK docking site; VWA, von Willebrand factor type A domain. Phosphorylated sites identified by MS analysis (localization probability >0.75 according to MaxQuant) in cells exposed to α-factor after phosphopeptide enrichment on a TiO 2 column are indicated by tick marks. The RING domain sequence is aligned with those of different yeast Ste5 proteins and the RING domain of S. cerevisiae Far1. Ser 185 of S. cerevisiae is indicated in bold red and conserved in other yeast species. An analogous serine residue (Ser 210 ) located within the Far1 RING domain (bold red) is also phosphorylated, together with two lower confidence sites (red; Ser 208 and Ser 211 ). (B) Recombinant 6His-tagged Ste5- and Far1-RING-H2 fragments were incubated for 30 min at 30°C with GST-Pkc1 purified from yeast extracts containing γ 32 P-ATP with (+) or without (−) 30 µM cercosporamide (cerc.). Phosphorylated proteins were visualized by autoradiography. An aliquot of purified Ste5- and Far1-RING-H2 fragments were separated by SDS-PAGE and stained with Coomassie blue. The bar points to the 35-kD size marker. (C–F) In vitro kinase assays using either WT or the nonphosphorylatable S185A mutant RING-H2 domain with amino acids 149–238 of Ste5 (Ste5 149−238 ) as a substrate and incubated as indicated with yeast extracts in the absence (DMSO) or presence of cercosporamide were analyzed by 600 MHz 1 H- 15 N correlation NMR spectra (SOFAST HMQC; ). The resonance position of the phosphorylated Ser185 amide group is magnified in C and highlighted in the dashed circle in D–F. The spectra in C are an overlay of the Ste5-RING-H2 fragment incubated with (black) or without (red) Pkc1. (G) M-track protein–protein proximity assays using extracts of cells expressing HKMT-myc–tagged Ste5 (bait) and protA-H3–tagged Pkc1 (prey). Cells were treated with α-factor for the times indicated (hours). Proximity signals were detected by Western blotting using an antibody against triple-methylated lysine of histone H3 (α-me). Loading was controlled via a hemagglutinin epitope embedded in the protA-H3 tag (α-HA). The asterisk marks an unspecific band.

Article Snippet: The in vitro kinase assays for NMR analysis were performed with 150 µM Ste5 149–238 (in 250 µl) with either assay buffer (3 mM MgCl 2 and 1 mM ATP), 5 µl of PKCα (P61-10G; SignalChem), and 0.5-0.8× of Lipid Activator (L51-39, SignalChem) or 250 µl yeast extract complemented with 5 mM ATP and phosphatase inhibitors.

Techniques: Functional Assay, Sequencing, Recombinant, Incubation, Purification, Autoradiography, SDS Page, Staining, Marker, In Vitro, Mutagenesis, Expressing, Western Blot, Methylation

Journal: The Journal of Cell Biology

Article Title: Mechanical stress impairs pheromone signaling via Pkc1-mediated regulation of the MAPK scaffold Ste5

doi: 10.1083/jcb.201808161

Figure Lengend Snippet: Phosphorylation of the RING-H2 domains of Ste5 and Far1 inhibits signaling and oriented cell polarity. (A) Extracts prepared from ste5Δ cells expressing 3xGFP-tagged WT Ste5, nonphosphorylatable Ste5 S185A , or phospho-mimicking Ste5 S185D or harboring an empty control plasmid were analyzed by immunoblotting using anti-GFP antibodies. Pgk1 controls equal loading. (B) Halo assays were used to assess cell cycle arrest in response to α-factor for ste5Δ cells expressing Ste5 and control constructs as in A. (C) Pheromone-dependent gene expression of ste5Δ cells harboring a FIG1-qV reporter and the indicated WT Ste5 (diamond), Ste5 S185A (square), or Ste5 S185D (triangle) was determined by FACS analysis and plotted in arbitrary units at the times indicated (minutes) after α-factor addition. (D) Microfluidic chambers generated a 0–80-nM α-factor gradient. The angle of the polarity site with respect to the α-factor gradient (schema) was measured for far1Δ strains expressing WT Far1, nonphosphorylatable Far1 3A , or phospho-mimicking Far1 3E . Results are expressed as percentages and binned into 45° incremental distances .

Article Snippet: The in vitro kinase assays for NMR analysis were performed with 150 µM Ste5 149–238 (in 250 µl) with either assay buffer (3 mM MgCl 2 and 1 mM ATP), 5 µl of PKCα (P61-10G; SignalChem), and 0.5-0.8× of Lipid Activator (L51-39, SignalChem) or 250 µl yeast extract complemented with 5 mM ATP and phosphatase inhibitors.

Techniques: Expressing, Plasmid Preparation, Western Blot, Construct, Generated

Journal: The Journal of Cell Biology

Article Title: Mechanical stress impairs pheromone signaling via Pkc1-mediated regulation of the MAPK scaffold Ste5

doi: 10.1083/jcb.201808161

Figure Lengend Snippet: Phosphorylation of serine 185 interferes with membrane recruitment of Ste5 and prevents Gβγ binding. (A) The localization of 3xGFP-tagged WT Ste5 (upper panel), Ste5 S185A (middle panel), or Ste5 S185D (lower panel) expressed in WT cells exposed to α-factor was analyzed by light microscopy. The percentage of cells with accumulated GFP-signal at shmoo tips was quantified by counting ≥100 cells for each strain. (B) Recruitment of 3xGFP-tagged WT Ste5, Ste5 S185A or Ste5 S185D was quantified in single cells at the indicated times upon addition of α-factor (t = 0). The membrane-to-cytoplasmic ratio was calculated using YeastQuant using TMD-mCherry to segment the plasma membrane. The solid line represents the median of the single-cell traces and the shaded area the 25th–75th percentiles. (C) FIG1-qV reporter expression was measured by FACS in ste5Δ cells expressing from the estradiol-inducible GAL promoter WT Ste5 (diamond), nonphosphorylatable Ste5 S185A (square), or phospho-mimicking Ste5 S185D (triangle) fused to a TMD and plotted in arbitrary units for different estradiol concentrations. For control, WT Ste5 (x) and Ste5 S185D (*) were also expressed without TMD fusion. (D) GST-Ste4–Ste18 purified from yeast was immobilized on glutathione-Sepharose beads and incubated in vitro with either 6His-tagged Ste5 WT (Ste5 149–238 ) or S185D (Ste5 149–238 S185D ) RING domains expressed in E. coli . Bound and flow-through fractions were collected and analyzed by Western blotting with the indicated antibodies. +, protein added; −, protein omitted. An aliquot of the input fraction controls for the presence of the specified proteins. GST-Ste4–Ste18 preferentially binds to the nonphosphorylated Ste5-RING domain. (E) Overlay of [ 1 H, 15 N] correlation spectra of WT (left panel), S185A (middle panel), and S185D (right panel) Ste5 RING-H2 domain in free form (black contours) and in complex with Gβγ subunits (red). Ste5 adopts a better-defined fold upon binding Gβγ binding with larger peak dispersion. In contrast to WT and the S185A mutant, the S185D RING-H2 domain fails to interact with Gβγ heterodimers.

Article Snippet: The in vitro kinase assays for NMR analysis were performed with 150 µM Ste5 149–238 (in 250 µl) with either assay buffer (3 mM MgCl 2 and 1 mM ATP), 5 µl of PKCα (P61-10G; SignalChem), and 0.5-0.8× of Lipid Activator (L51-39, SignalChem) or 250 µl yeast extract complemented with 5 mM ATP and phosphatase inhibitors.

Techniques: Binding Assay, Light Microscopy, Expressing, Purification, Incubation, In Vitro, Western Blot, Mutagenesis

Journal: The Journal of Cell Biology

Article Title: Mechanical stress impairs pheromone signaling via Pkc1-mediated regulation of the MAPK scaffold Ste5

doi: 10.1083/jcb.201808161

Figure Lengend Snippet: Pkc1 prevents cell lysis by promoting membrane dissociation of Ste5 and Far1 upon cell–cell fusion. (A) The relative mating efficiency of ste5Δ cells expressing either WT Ste5 or Ste5 S185A was measured in media where only diploid cells can grow. The SD was determined from at least three experiments. (B) Schematic of the microfluidic device used to observe the mating process. Cells were captured in pockets (magnified inset) and imaged by live-cell microscopy. The pillar array (1,008 traps/array) allows one to trap cells and visualize mating. (C and D) Zygotes resulting from the indicated crosses were followed after fusion and scored for cell lysis by microscopy. (C) The percentage of viable diploids is plotted as a function of time after cell–cell fusion; ≥100 zygotes from three different experiments were analyzed for each cross. The error bars indicate SD of three independent experiments, and significance was determined for the 150-min time point by a t test (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). (D) Zygotes were assessed for emergence of their first bud after fusion event. Box and whisker plots show median and first and third quartiles, with the outlier 5th and 95th percentiles indicated as symbols (filled circles). Significance was determined by ANOVA and a t test (*, P ≤ 0.05; **, P ≤ 0.01). (E) 3xGFP-tagged Ste5 expressed in MAT a cells was localized by fluorescence microscopy during cell–cell fusion with unlabeled MATα-partners. Images were taken at the indicated times with t = 0 defined when cell–cell contact is detected. Successful cell–cell fusion was monitored by the appearance of GFP-tagged proteins in the unlabeled mating partner. (F and G) The levels of 3xGFP-tagged WT Ste5 and Ste5 S185A at the site of cell–cell fusion were quantified, and relative intensity with SD compared with the cytoplasmic signal as a function of time was plotted (F). t = 0 was defined as cell–cell fusion, monitored by the appearance of GFP-tagged proteins in the unlabeled mating partner. Zygotes were assessed for the time of localized GFP residence. The error bars indicate SD of three independent experiments, and significance was determined by a t test at the indicated time points (*, P ≤ 0.05). (G) Box and whisker plots show median and first and third quartiles, with the outlier 5th and 95th percentiles indicated as symbols (filled circles); ≥70 cells were analyzed, and a t test was used to determine significance (*, P ≤ 0.05).

Article Snippet: The in vitro kinase assays for NMR analysis were performed with 150 µM Ste5 149–238 (in 250 µl) with either assay buffer (3 mM MgCl 2 and 1 mM ATP), 5 µl of PKCα (P61-10G; SignalChem), and 0.5-0.8× of Lipid Activator (L51-39, SignalChem) or 250 µl yeast extract complemented with 5 mM ATP and phosphatase inhibitors.

Techniques: Lysis, Expressing, Microscopy, Whisker Assay, Fluorescence

Journal: The Journal of Cell Biology

Article Title: Mechanical stress impairs pheromone signaling via Pkc1-mediated regulation of the MAPK scaffold Ste5

doi: 10.1083/jcb.201808161

Figure Lengend Snippet: Yeast strains

Article Snippet: The in vitro kinase assays for NMR analysis were performed with 150 µM Ste5 149–238 (in 250 µl) with either assay buffer (3 mM MgCl 2 and 1 mM ATP), 5 µl of PKCα (P61-10G; SignalChem), and 0.5-0.8× of Lipid Activator (L51-39, SignalChem) or 250 µl yeast extract complemented with 5 mM ATP and phosphatase inhibitors.

Techniques:

Journal: The Journal of Cell Biology

Article Title: Mechanical stress impairs pheromone signaling via Pkc1-mediated regulation of the MAPK scaffold Ste5

doi: 10.1083/jcb.201808161

Figure Lengend Snippet: Plasmids

Article Snippet: The in vitro kinase assays for NMR analysis were performed with 150 µM Ste5 149–238 (in 250 µl) with either assay buffer (3 mM MgCl 2 and 1 mM ATP), 5 µl of PKCα (P61-10G; SignalChem), and 0.5-0.8× of Lipid Activator (L51-39, SignalChem) or 250 µl yeast extract complemented with 5 mM ATP and phosphatase inhibitors.

Techniques:

Journal: PLoS ONE

Article Title: Nucleolin Regulates Phosphorylation and Nuclear Export of Fibroblast Growth Factor 1 (FGF1)

doi: 10.1371/journal.pone.0090687

Figure Lengend Snippet: A) U2OSR1 cells were transfected with siRNA as indicated, serum starved for 24 h and labelled by [ 33 P]phosphate, and thereafter stimulated with 100 ng/ml unlabelled, recombinant FGF1 in the presence of 10 U/ml heparin, and 10 nM BafA1 where indicated, for 6 h. FGF1 was extracted from total cell lysates by adsorption to Heparin-Sepharose and analyzed by SDS PAGE and fluorography to detect in vivo phosphorylation, and immunoblotting (IB) to detect FGF1. The lysates were also analyzed for nucleolin and HSP90 by IB. B) U2OSR1 cells transfected with siRNA as indicated were serum starved for 24 h, stimulated with FGF1 and heparin for the indicated time and then lysed and fractionated into a cytoplasmic (CP) and a nuclear (N) fraction before analysis by SDS-PAGE and IB for phospho-PKCδ and total PKCδ. The fractions were also analyzed for nucleolin, lamin A, and ERK1/2 by IB. C) Recombinant FGF1 or FGF1 mutants as indicated, were incubated with recombinant, active PKCδ and [γ- 33 P]ATP and thereafter analyzed for in vitro phosphorylation by fluorography, and IB to show loading. D) U2OSR1 cells were serum starved and labelled with [ 33 P]phosphate and stimulated with FGF1 or FGF1 mutants as indicated for 6 h. The cells were fractionated into cytoplasmic and nuclear fractions. FGFs were extracted from the fractions by binding to Heparin-Sepharose and analyzed by fluorography to detect in vivo phosphorylation and immunoblotting (IB) to detect FGFs. Fractions were also analyzed for marker proteins by IB as indicated. E) U2OSR1 cells were transfected with siRNA as indicated, serum starved for 24 h and labelled with [ 33 P]phosphate and stimulated with FGF1 in the absence or presence of 1 µg/ml thapsigargin. The cells were fractionated and analyzed as described in (D).

Article Snippet: Recombinant active PKCδ was from SignalChem and full length FGF1 was from Abcam.

Techniques: Transfection, Recombinant, Adsorption, SDS Page, In Vivo, Phospho-proteomics, Western Blot, Incubation, In Vitro, Binding Assay, Marker

Journal: FEBS Open Bio

Article Title: Protein kinase C regulates AMPA receptor auxiliary protein Shisa9/ CKAMP 44 through interactions with neuronal scaffold PICK 1

doi: 10.1002/2211-5463.12261

Figure Lengend Snippet: Shisa9/ CKAMP 44 interacts with PICK 1 and is phosphorylated by PKC α. (A) Schematic overview of Shisa9/ CKAMP 44 constructs used in this study (including the location of the respective tag): in CKAMP 44‐ FL (full length), the tag is C‐terminal to the signal peptide in the extracellular domain ( TM , yellow). Constructs with only the cytoplasmic tail of Shisa9/ CKAMP 44 are N‐terminally tagged (with either EGFP or GST ). PDZ ligand sequences are shown in red and are deleted in constructs labelled with ΔC ( PDZ ‐binding‐deficient variant). A conserved region of Shisa9/ CKAMP 44 (blue; N‐terminal region of the cytoplasmic tail) is deleted in the construct CKAMP 44‐ΔNΔC. Constructs are drawn to scale. (B) Comparative coimmunoprecipitation experiments of PICK 1 ( FLAG ‐ PICK 1) with Shisa9/ CKAMP 44 C‐terminal variants EGFP ‐ CKAMP 44, −ΔC, or −ΔNΔC). Proteins were precipitated using GFP antibody or mIgG (negative control) and detected using the antibodies indicated. Inputs are shown on the left. (C) Full‐length Shisa9/ CKAMP 44 ( FLAG ‐ CKAMP 44‐ FL ), expressed in COS ‐7 cells, is phosphorylated following induction by PMA (1 μ m , 30 min). Phosphorylated proteins (indicated) are separated from nonphosphorylated proteins (indicated) via Phos‐tag‐ SDS / PAGE and observed via WB; phosphatase treatment with alkaline phosphatase is used as a negative control, thereby highlighting the phosphorylation dependence of the mobility shift. (D) Phosphorylation of Shisa9/ CKAMP 44 ( FLAG ‐ CKAMP 44‐ FL ) is induced with PMA and inhibited by application of the PKC inhibitor GF 109203X in COS ‐7 cells. Again phosphorylated proteins are observable via Phos‐tag‐ SDS / PAGE . PKC inhibitor concentrations are indicated above. (E) An in vitro PKC α kinase assay followed by Phos‐tag SDS / PAGE analysis highlights a PKC α‐dependent mobility shift of the phosphorylated substrate GST ‐ CKAMP 44 (top panel, Phos‐tag SDS / PAGE ). Presence of PKC α and kinase substrate present in the same samples is shown below (lower panel, standard Laemmli SDS / PAGE and WB; antibodies indicated).

Article Snippet: Purified and desalted GST‐CKAMP44 protein was used for an in vitro PKCα kinase assay (SignalChem, Richmond, Canada; #P61–18G) according to the manufacturer's instructions.

Techniques: Construct, Binding Assay, Variant Assay, Negative Control, SDS Page, Phospho-proteomics, Mobility Shift, In Vitro, Kinase Assay