Journal: Biomolecules

Article Title: YES1 Kinase Mediates the Membrane Removal of Rescued F508del-CFTR in Airway Cells by Promoting MAPK Pathway Activation via SHC1

doi: 10.3390/biom13060949

Figure Lengend Snippet: Interaction with YES1 decreases the PM abundance and function of rF508del-CFTR in airway cells. ( A ) Intact CFBE cells expressing extracellularly Flag-tagged mCherry-wt-CFTR (Wt) or mCherry-F508del-CFTR (rΔF, after rescue to the PM by 48 h treatment with 5 µM VX-661), were labelled with either an anti-Flag antibody or a non-specific IgG prior to non-denaturing lysis, and antibody-bound CFTR complexes were precipitated using protein G-coupled magnetic beads. Input protein levels were adjusted so that equivalent amounts of wt- and rF508del-CFTR were precipitated. Both input lysates and co-precipitates were analyzed using WB to assess the levels of CFTR and YES1. Tubulin (known to not interact with CFTR at the PM ) was used as an additional co-precipitation control. ( B ) Efficiency of siRNA-mediated depletion in CFBE cells expressing mCherry-F508del-CFTR, rescued as in ( A ), transfected with either a mock siRNA (siCtrl) or a commercial triple oligonucleotide mix against YES1 (siYES1). Representative WBs of CFTR, YES1, and tubulin (used as loading control) are shown (left panels), as well as the quantifications as means ± SEM from four independent experiments (right panel). ( C ) Immunofluorescence images of CFBE cells expressing Flag-tagged mCherry-F508del-CFTR rescued to the PM by 48 h treatment with 5 µM VX-661. The cells were transfected as in ( B ) and treated for 3 h with either vehicle (DMSO) or 10 µM of YES1 inhibitor SU6656. rF508del-CFTR at the surface of intact cells was immunolabelled on ice using an anti-Flag antibody followed by an Alexa 488-conjugated secondary antibody. Cells were then fixed, and the nuclei were stained with DAPI. Confocal images of the cells’ surface, showing surface CFTR staining in green (left panels) and a merged overlay image of surface (green) and total CFTR signals (mCherry, red) along with the stained nuclei (DAPI, blue) are shown in the right panels. White scale bars represent 10 µm. ( D ) Representative traces of ion transport activity measured through iodide-induced HS-YFP sensor fluorescence decay of untagged F508del-CFTR CFBE cells treated with 5 μM VX-661 for 48 h. The cells were transfected and treated as in ( C ) and co-treated with or without 25 μM of inh172 15 min prior to stimulation for 30 min in PBS with Fsk (5 µM) and Gen (10 µM), in the presence or absence of inh172. This was followed by continuous fluorescence recording and the addition of I - (represented by the black arrow, final concentration 100 mM). ( E ) Quantification of HS-YFP fluorescence quenching rates (QR) of at least five independent assays for each condition, calculated by fitting the iodide assay results to exponential decay curves. The means ± SEM are shown. ns—not significant, ** p < 0.01, and *** p < 0.001 between conditions indicated by the horizontal lines.

Article Snippet: An siRNA oligonucleotide against luciferase (siCtrl, 5′-CGUACGCGGAAUACUUCGA) from Eurofins Genomics was used as a mock control, and the siRNAs used to deplete YES1 (sc-29860), YAP1 (sc-38637) or SHC1 (sc-29480) were commercial mixes (each composed of three different oligonucleotides) from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Expressing, Lysis, Magnetic Beads, Control, Transfection, Immunofluorescence, Staining, Activity Assay, Fluorescence, Concentration Assay

Journal: Biomolecules

Article Title: YES1 Kinase Mediates the Membrane Removal of Rescued F508del-CFTR in Airway Cells by Promoting MAPK Pathway Activation via SHC1

doi: 10.3390/biom13060949

Figure Lengend Snippet: YES1 inhibition increases rF508del-CFTR retention at the PM upon thermal destabilization. ( A ) Diagram depicting the parallel cell surface protein biotinylation assays used to assess rF508del-CFTR thermal stability and internalization. Replicate dishes of CFBE cells expressing F508del-CFTR were incubated for 48 h at 30 °C in the presence of 5 µM of VX-661. One of the replicates was directly placed on ice, then the surface proteins were labeled with sulfo-NHS-SS-biotin, lysed, and the surface-labeled proteins were captured using streptavidin beads. These precipitates represented the input amount of CFTR at the PM without any thermal destabilization (DMSO 30 °C). A second set of replicates were moved to 37 °C in the absence (DMSO) or presence of YES1 inhibitors (10 µM SU6656 or 1 µM P505-15). Three hours later, surface proteins were labelled and isolated, as described above. This second set revealed the amount of CFTR remaining at the PM after thermal destabilization (TS) and inhibitor treatment. A third set of replicates was first labeled with biotin, then placed at 37 °C for 3 h in the presence or absence of YES inhibitors. These samples were then returned to ice, and the labeled CFTR remaining at the cell surface was stripped from biotin with glutathione prior to lysis and isolation of the labeled proteins that entered the cells. This third set represents the amount of CFTR internalized from the surface upon TS and inhibitor treatment. ( B ) Analysis of input lysates and biotin-labelled fractions obtained as described in ( A ). WBs representative of five independent assays, probed with antibodies against the indicated proteins, are shown. Glucose transporter 1 (Glut-1) and tubulin were used as controls for the equivalence and purity of the biotinylated fractions, respectively. ( C ) Quantification of CFTR abundance in the biotinylated fraction (mean ± SEM) in ( B ) after normalization to Glut-1 levels and to the respective controls. *** p < 0.001 relative to DMSO (30 °C), ## p < 0.01 and ### p < 0.001, both relative to DMSO in the internalized set.

Article Snippet: An siRNA oligonucleotide against luciferase (siCtrl, 5′-CGUACGCGGAAUACUUCGA) from Eurofins Genomics was used as a mock control, and the siRNAs used to deplete YES1 (sc-29860), YAP1 (sc-38637) or SHC1 (sc-29480) were commercial mixes (each composed of three different oligonucleotides) from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Inhibition, Expressing, Incubation, Labeling, Isolation, Lysis

Journal: Biomolecules

Article Title: YES1 Kinase Mediates the Membrane Removal of Rescued F508del-CFTR in Airway Cells by Promoting MAPK Pathway Activation via SHC1

doi: 10.3390/biom13060949

Figure Lengend Snippet: YAP1 is required for the binding of YES1 to rF508del-CFTR complexes at the PM. ( A ) STRING-based analysis ( https://string-db.org/ , accessed on 19 October 2021) of the strength of annotated evidence on the interaction between YES1 and the CFTR/NHERF1 (SLC9A3R1)/Ezrin (EZR) membrane anchoring complex (the thickness of the grey lines is proportional to the degree of confidence for the interaction between the two proteins they connect, extrapolated from text mining, experimental, and database-collected evidence). ( B ) A STRING-generated expanded interaction network, extended (green nodes) around the core complex in ( A ) (red nodes). ( C ) CFTR-containing complexes were immunoprecipitated from the PM of intact CFBE cells expressing extracellularly Flag-tagged mCherry-wt-CFTR (Wt) or mCherry-F508del-CFTR, as described for A. Both input lysates and co-precipitates were analyzed using WB to assess the levels of CFTR and YAP1. Tubulin was used as an additional co-precipitation control. ( D ) CFBE cells expressing untagged F508del-CFTR transfected with either mock siRNA (siCtrl) or a commercial siRNA mix targeting YAP1 (siYAP1), were incubated with 5 µM of VX-661 for 48 h at 30 °C to coax most of the mutant channel to the PM. The cells were then lysed in non-denaturing conditions and YES1 immunoprecipitated with a specific antibody (a non-specific IgG was used as a control) from whole cell lysates (WCL). Both input lysates and co-precipitates were analyzed using WB to assess the levels of precipitated YES1 and co-precipitated rF508del-CFTR. ( E ) Thermal shift (TS) assay, as described in , to assess the thermal stability of untagged rF508del-CFTR in CFBE cells transfected with mock siRNA (siCtrl) or one of two commercial siRNA mixes targeting either YAP1 (siYAP1) or YES1 (siYES1). WBs representative of at least four independent assays, probed with antibodies against the indicated proteins, are shown. Glucose transporter 1 (Glut-1) and tubulin were used as controls for the equivalence and purity of the biotinylated fractions, respectively. ( F ) Quantification of CFTR abundance in the biotinylated fraction (mean ± SEM) in ( E ) after normalization to Glut-1 levels and to siCtrl (30 °C). *** p < 0.001 relative to siCtrl (30 °C), ## p < 0.01 and ### p < 0.001, both relative to siCtrl (TS).

Article Snippet: An siRNA oligonucleotide against luciferase (siCtrl, 5′-CGUACGCGGAAUACUUCGA) from Eurofins Genomics was used as a mock control, and the siRNAs used to deplete YES1 (sc-29860), YAP1 (sc-38637) or SHC1 (sc-29480) were commercial mixes (each composed of three different oligonucleotides) from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Binding Assay, Membrane, Generated, Immunoprecipitation, Expressing, Control, Transfection, Incubation, Mutagenesis

Journal: Biomolecules

Article Title: YES1 Kinase Mediates the Membrane Removal of Rescued F508del-CFTR in Airway Cells by Promoting MAPK Pathway Activation via SHC1

doi: 10.3390/biom13060949

Figure Lengend Snippet: The MAPK pathway participates in YES1-mediated internalization of rF508del-CFTR at the PM. The thermal shift (TS) assay, as described in , was used to assess the thermal stability of untagged rF508del-CFTR in CFBE cells. ( A ) The cells were treated for 3 h with either vehicle (DMSO) or 10 µM of selumetinib, or ( C ) transfected with empty vector or Myc-H-RAS-V12 (HRAS) and then treated for 3 h with vehicle (DMSO) or 10 µM of SU6656, as indicated. WBs representative of input and cell surface fractions from four independent assays, probed with antibodies against the indicated proteins, are shown. Glucose transporter 1 (Glut-1) and tubulin were used as controls for the equivalence and purity of the biotinylated fractions, respectively. H-RAS V12 was detected using an anti-Myc antibody, and an anti-phosphorylated ERK1/2 antibody was used to monitor MAPK pathway activity, which was quantified and shown below the respective blot lanes. ( B , D ) Corresponding quantification of CFTR abundance in the biotinylated fractions (mean ± SEM) from four independent assays after normalization to Glut-1 levels and DMSO (30 °C). * p < 0.05, ** p < 0.01, and *** p < 0.001, relative to DMSO (30 °C) in ( B ), and as indicated by the horizontal lines in (C); ## p < 0.01, relative to DMSO (TS) in ( B ).

Article Snippet: An siRNA oligonucleotide against luciferase (siCtrl, 5′-CGUACGCGGAAUACUUCGA) from Eurofins Genomics was used as a mock control, and the siRNAs used to deplete YES1 (sc-29860), YAP1 (sc-38637) or SHC1 (sc-29480) were commercial mixes (each composed of three different oligonucleotides) from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Transfection, Plasmid Preparation, Activity Assay

Journal: Biomolecules

Article Title: YES1 Kinase Mediates the Membrane Removal of Rescued F508del-CFTR in Airway Cells by Promoting MAPK Pathway Activation via SHC1

doi: 10.3390/biom13060949

Figure Lengend Snippet: SHC1 phosphorylation by YES1 mediates rF508del-CFTR internalization via MAPK pathway signaling. ( A ) Effects of YES1 inhibitors. Lysates from F508del-CFTR expressing CFBE cells were incubated with 5 µM of VX-661 for 48 h at 30 °C, treated with either vehicle (DMSO), SU6656 (10 µM), or P505-15 (1 µM) for 3 h at 37 °C, then were analyzed using WB. Immunoblots representative of three independent experiments, probed with antibodies against the indicated proteins, are shown. p-SHC1 indicates the level of SHC1 phosphorylation at Tyr239/240 in the different conditions, and an anti-phosphorylated ERK1/2 antibody (p-ERK1/2) was used to monitor MAPK pathway activity (both show quantified band intensities below their respective blots). ( B ) Thermal shift (TS) assay described in to assess how much rF508del-CFTR remained at the PM after thermal destabilization in CFBE cells transfected either with a mock siRNA (siCtrl) or a siRNA against SHC1 (siSHC1). WBs representative of input and cell surface fractions, probed with antibodies against the indicated proteins, are shown. Glucose transporter 1 (Glut-1) and tubulin were used as controls for the equivalence and purity of the biotinylated fractions, respectively. Quantifications of SCH1 depletion efficiency and ERK1/2 phosphorylation levels are shown below their respective blots. ( C ) Corresponding quantification of CFTR abundance in the biotinylated fractions (mean ± SEM) from three independent assays after normalization to Glut-1 levels and to siCtrl (30 °C). *** p < 0.001 relative to siCtrl (30 °C), ## p < 0.01 relative to siCtrl (TS).

Article Snippet: An siRNA oligonucleotide against luciferase (siCtrl, 5′-CGUACGCGGAAUACUUCGA) from Eurofins Genomics was used as a mock control, and the siRNAs used to deplete YES1 (sc-29860), YAP1 (sc-38637) or SHC1 (sc-29480) were commercial mixes (each composed of three different oligonucleotides) from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Phospho-proteomics, Expressing, Incubation, Western Blot, Activity Assay, Transfection

Journal: Biomolecules

Article Title: YES1 Kinase Mediates the Membrane Removal of Rescued F508del-CFTR in Airway Cells by Promoting MAPK Pathway Activation via SHC1

doi: 10.3390/biom13060949

Figure Lengend Snippet: Proposed model for SHC1-mediated removal of CFTR from the PM through activation of the MAPK pathway. ( A ) Phosphorylation of PM-anchored wt-CFTR at Tyr512 by SYK kinase leads to its recognition and binding by the adaptor protein SHC1. This links CFTR internalization to the activation of the MAPK pathway downstream of receptor tyrosine kinases. ( B ) F508del-CFTR pharmacologically rescued to the PM is not phosphorylated by SYK, but its deficient anchoring to the actin cytoskeleton allows its interaction with the YES1 kinase via the adaptor protein YAP1 and the scaffold protein NHERF1. SHC1 is a substrate for YES1 at the PM, and its phosphorylation by YES1 increases its affinity to membrane receptors in the vicinity, enhancing their activation of the MAPK pathway. This could contribute to the much faster internalization rate of rF508del-CFTR compared to the wild-type protein. RTK—receptor tyrosine kinases; EB—Ezrin binding domain; 1-2—NHERF1’s PDZ1 and PDZ2.

Article Snippet: An siRNA oligonucleotide against luciferase (siCtrl, 5′-CGUACGCGGAAUACUUCGA) from Eurofins Genomics was used as a mock control, and the siRNAs used to deplete YES1 (sc-29860), YAP1 (sc-38637) or SHC1 (sc-29480) were commercial mixes (each composed of three different oligonucleotides) from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Activation Assay, Phospho-proteomics, Binding Assay, Membrane