YES1 Kinase Mediates the Membrane Removal of Rescued F508del-CFTR in Airway Cells by Promoting MAPK Pathway Activation via SHC1
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Culture, Treatment and Transfection
2.2. Protein Thermal Destabilization Assay (Thermal Shift Assay—TS)
2.3. Immunoblotting and Immunofluorescence
2.4. Biotinylation of Cell Surface Proteins and Internalization Protocol
- (1)
- Cell surface protein analysis: The cells were lysed in a pull-down (PD) buffer (50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 10% (v/v) glycerol, 1% (v/v) Nonidet P-40, 0.1% (v/v) SDS, protease inhibitor cocktail (Sigma-Aldrich, Saint Louis, MO, USA), phosphatase inhibitor cocktail C (Santa Cruz Biotechnology, Dallas, TX, USA)). The cell lysates were cleared at 10,000× g for 5 min at 4 °C, and an aliquot was removed while the remaining supernatant was added to streptavidin-agarose beads (Sigma-Aldrich, Saint Louis, MO, USA) that were previously blocked for 1 h with 2% (w/v) milk in PD-buffer to represent the lysate input. The lysate and beads were rotated for 1 h at 4 °C, centrifuged for 1 min at 3000× g, and washed five times with an excess of wash buffer (100 mM Tris-HCl (pH 7.5), 300 mM NaCl, 1% (v/v) Triton X-100). The captured proteins were eluted in 2× Laemmli buffer with 100 mM of DTT and analyzed using immunoblotting, as described above.
- (2)
- Internalization studies: Biotin-labeled cells were incubated for 3 h at 37 °C with warm media containing the indicated treatments. Next, the cells were replaced on ice, rinsed with ice-cold PBS-CM, and left for 5 min to block endocytosis. After two ice-cold PBS-CM washes, the cells were incubated for 30 min (2 × 15 min) with ice-cold stripping buffer (60 mM glutathione, 90 mM NaCl, 0.9 mM CaCl2, 0.5 mM MgCl2, 90 mM NaOH, 10% (v/v) FBS). The cells were then washed three times with ice-cold PBS-CM and processed as in step 1.
2.5. Co-Immunoprecipitation of Membrane CFTR-Associated Complexes
2.6. Co-Immunoprecipitation of Membrane Coaxed rF508del-CFTR
2.7. CFTR Functional Assay by Halide-Sensitive YFP (HS-YFP)
2.8. Statistical Analysis
3. Results
3.1. Interaction with YES1 Decreases rF508del-CFTR Functional Permanence at the PM
3.2. Inhibition of YES1 Impairs Thermal Destabilization and Internalization of PM-Bound rF508del-CFTR
3.3. The Interaction between YES1 and rF508del-CFTR Is Mediated by YAP1
3.4. YES1 Participates in the Removal of rF508del-CFTR from the PM via the MEK/ERK1/2 MAPK Pathway
3.5. Phosphorylation of SHC1 by YES1 Links rF508del-CFTR Internalization to MAPK Pathway Activation
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Barros, P.; Matos, A.M.; Matos, P.; Jordan, P. YES1 Kinase Mediates the Membrane Removal of Rescued F508del-CFTR in Airway Cells by Promoting MAPK Pathway Activation via SHC1. Biomolecules 2023, 13, 949. https://doi.org/10.3390/biom13060949
Barros P, Matos AM, Matos P, Jordan P. YES1 Kinase Mediates the Membrane Removal of Rescued F508del-CFTR in Airway Cells by Promoting MAPK Pathway Activation via SHC1. Biomolecules. 2023; 13(6):949. https://doi.org/10.3390/biom13060949
Chicago/Turabian StyleBarros, Patrícia, Ana M. Matos, Paulo Matos, and Peter Jordan. 2023. "YES1 Kinase Mediates the Membrane Removal of Rescued F508del-CFTR in Airway Cells by Promoting MAPK Pathway Activation via SHC1" Biomolecules 13, no. 6: 949. https://doi.org/10.3390/biom13060949