Abstract
Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause the recessive genetic disease cystic fibrosis, where the chloride transport across the apical membrane of epithelial cells mediated by the CFTR protein is impaired. CFTR protein trafficking to the plasma membrane (PM) is the result of a complex interplay between the secretory and membrane recycling pathways that control the number of channels present at the membrane. In addition, the ion transport activity of CFTR at the PM is modulated through post-translational protein modifications. Previously we described that spleen tyrosine kinase (SYK) phosphorylates a specific tyrosine residue in the nucleotide-binding domain 1 domain and this modification can regulate the PM abundance of CFTR. Here we identified the underlying biochemical mechanism using peptide pull-down assays followed by mass spectrometry. We identified in bronchial epithelial cells that the adaptor protein SHC1 recognizes tyrosine-phosphorylated CFTR through its phosphotyrosine-binding domain and that the formation of a complex between SHC1 and CFTR is induced at the PM in the presence of activated SYK. The depletion of endogenous SHC1 expression was sufficient to promote an increase in CFTR at the PM of these cells. The results identify a SYK/SHC1 pathway that regulates the PM levels of CFTR channels, contributing to a better understanding of how CFTR-mediated chloride secretion is regulated.
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References
Bobadilla JL, Macek M, Fine JP, Farrell PM (2002) Cystic fibrosis: a worldwide analysis of CFTR mutations—correlation with incidence data and application to screening. Hum Mutat 19:575–606. https://doi.org/10.1002/humu.10041
Rowe SM, Miller S, Sorscher EJ (2005) Cystic fibrosis. N Engl J Med 352:1992–2001. https://doi.org/10.1056/NEJMra043184
Saint-Criq V, Gray MA (2017) Role of CFTR in epithelial physiology. Cell Mol Life Sci 74:93–115. https://doi.org/10.1007/s00018-016-2391-y
Donaldson SH, Boucher RC (2003) Update on pathogenesis of cystic fibrosis lung disease. Curr Opin Pulm Med 9:486–491
Ehre C, Ridley C, Thornton DJ (2014) Cystic fibrosis: an inherited disease affecting mucin-producing organs. Int J Biochem Cell Biol 52:136–145. https://doi.org/10.1016/j.biocel.2014.03.011
Riordan JR (2008) CFTR Function and Prospects for Therapy. Annu Rev Biochem 77:701–726. https://doi.org/10.1146/annurev.biochem.75.103004.142532
Liu F, Zhang Z, Csanády L et al (2017) Molecular structure of the human CFTR ion channel. Cell 169:85–95.e8. https://doi.org/10.1016/j.cell.2017.02.024
Amaral MD, Farinha CM (2013) Rescuing mutant CFTR: a multi-task approach to a better outcome in treating cystic fibrosis. Curr Pharm Des 19:3497–3508. https://doi.org/10.2174/13816128113199990318
Farinha CM, Matos P, Amaral MD (2013) Control of cystic fibrosis transmembrane conductance regulator membrane trafficking: not just from the endoplasmic reticulum to the golgi. FEBS J 280:4396–4406. https://doi.org/10.1111/febs.12392
Bell SC, De Boeck K, Amaral MD (2015) New pharmacological approaches for cystic fibrosis: promises, progress, pitfalls. Pharmacol Ther 145:19–34. https://doi.org/10.1016/j.pharmthera.2014.06.005
Clancy JP, Rowe SM, Accurso FJ et al (2012) Results of a phase IIa study of VX-809, an investigational CFTR corrector compound, in subjects with cystic fibrosis homozygous for the F508del-CFTR mutation. Thorax 67:12–18. https://doi.org/10.1136/thoraxjnl-2011-200393
Sala MA, Jain M (2018) Tezacaftor for the treatment of cystic fibrosis. Expert Rev Respir Med 12:725–732. https://doi.org/10.1080/17476348.2018.1507741
Zhang W, Zhang X, Zhang YH et al (2016) Lumacaftor/ivacaftor combination for cystic fibrosis patients homozygous for Phe508del-CFTR. Drugs Today 52:229–237. https://doi.org/10.1358/dot.2016.52.4.2467205
Matos A, Matos P (2018) Combination therapy in Phe508del CFTR: how many will be enough? J Lung Health Dis 2:9–16
Amaral MD (2015) Novel personalized therapies for cystic fibrosis: treating the basic defect in all patients. J Intern Med 277:155–166. https://doi.org/10.1111/joim.12314
Farinha CM, Matos P (2016) Repairing the basic defect in cystic fibrosis—one approach is not enough. FEBS J 283:246–264. https://doi.org/10.1111/febs.13531
Guggino WB, Stanton BA (2006) New insights into cystic fibrosis: molecular switches that regulate CFTR. Nat Rev Mol Cell Biol 7:426–436. https://doi.org/10.1038/nrm1949
Moniz S, Sousa M, Moraes BJ et al (2013) HGF stimulation of Rac1 signaling enhances pharmacological correction of the most prevalent cystic fibrosis mutant F508del-CFTR. ACS Chem Biol 8:432–442. https://doi.org/10.1021/cb300484r
Farinha CM, Swiatecka-Urban A, Brautigan DL, Jordan P (2016) Regulatory crosstalk by protein kinases on CFTR trafficking and activity. Front Chem 4:1. https://doi.org/10.3389/fchem.2016.00001
Farinha CM, Canato S (2017) From the endoplasmic reticulum to the plasma membrane: mechanisms of CFTR folding and trafficking. Cell Mol Life Sci 74:39–55. https://doi.org/10.1007/s00018-016-2387-7
Farinha CM, Matos P (2018) Rab GTPases regulate the trafficking of channels and transporters—a focus on cystic fibrosis. Small GTPases 9:136–144. https://doi.org/10.1080/21541248.2017.1317700
Billet A, Jia Y, Jensen TJ et al (2016) Potential sites of CFTR activation by tyrosine kinases. Channels 10:247–251. https://doi.org/10.1080/19336950.2015.1126010
Billet A, Jia Y, Jensen T et al (2015) Regulation of the cystic fibrosis transmembrane conductance regulator anion channel by tyrosine phosphorylation. FASEB J 29:3945–3953. https://doi.org/10.1096/fj.15-273151
Mendes AI, Matos P, Moniz S et al (2011) Antagonistic regulation of cystic fibrosis transmembrane conductance regulator cell surface expression by protein kinases WNK4 and spleen tyrosine kinase. Mol Cell Biol 31:4076–4086. https://doi.org/10.1128/MCB.05152-11
Mócsai A, Ruland J, Tybulewicz VLJ (2010) The SYK tyrosine kinase: a crucial player in diverse biological functions. Nat Rev Immunol 10:387–402. https://doi.org/10.1038/nri2765
Ulanova M, Puttagunta L, Marcet-Palacios M et al (2005) Syk tyrosine kinase participates in beta1-integrin signaling and inflammatory responses in airway epithelial cells. Am J Physiol Lung Cell Mol Physiol 288:L497–507. https://doi.org/10.1152/ajplung.00246.2004
Wang X, Lau C, Wiehler S et al (2006) Syk is downstream of intercellular adhesion molecule-1 and mediates human rhinovirus activation of p38 MAPK in airway epithelial cells. J Immunol 177:6859–6870. https://doi.org/10.4049/jimmunol.177.10.6859
Woodside DG, Obergfell A, Leng L et al (2001) Activation of Syk protein tyrosine kinase through interaction with integrin β cytoplasmic domains. Curr Biol 11:1799–1804. https://doi.org/10.1016/S0960-9822(01)00565-6
Illek B, Maurisse R, Wahler L et al (2008) Cl transport in complemented CF bronchial epithelial cells correlates with CFTR mRNA expression levels. Cell Physiol Biochem 22:57–68. https://doi.org/10.1159/000149783
Botelho HM, Uliyakina I, Awatade NT et al (2015) Protein traffic disorders: an effective high-throughput fluorescence microscopy pipeline for drug discovery. Sci Rep 5:9038. https://doi.org/10.1038/srep09038
Galietta LJV, Haggie PM, Verkman AS (2001) Green fluorescent protein-based halide indicators with improved chloride and iodide affinities. FEBS Lett 499:220–224. https://doi.org/10.1016/S0014-5793(01)02561-3
Matos AM, Pinto FR, Barros P et al (2019) Inhibition of calpain 1 restores plasma membrane stability to pharmacologically rescued Phe508del-CFTR variant. J Biol Chem. https://doi.org/10.1074/jbc.RA119.008738
Loureiro CA, Matos AM, Dias-Alves  et al (2015) A molecular switch in the scaffold NHERF1 enables misfolded CFTR to evade the peripheral quality control checkpoint. Sci Signal 8:ra48. https://doi.org/10.1126/scisignal.aaa1580
Carsetti L, Laurenti L, Gobessi S et al (2009) Phosphorylation of the activation loop tyrosines is required for sustained Syk signaling and growth factor-independent B-cell proliferation. Cell Signal 21:1187–1194. https://doi.org/10.1016/j.cellsig.2009.03.007
Schulze WX, Mann M (2004) A novel proteomic screen for peptide–protein interactions. J Biol Chem 279:10756–10764. https://doi.org/10.1074/jbc.M309909200
Rozakis-Adcock M, Fernley R, Wade J et al (1993) The SH2 and SH3 domains of mammalian Grb2 couple the EGF receptor to the Ras activator mSos1. Nature 363:83–85. https://doi.org/10.1038/363083a0
Yaffe MB (2002) Phosphotyrosine-binding domains in signal transduction. Nat Rev Mol Cell Biol 3:177–186. https://doi.org/10.1038/nrm759
Rozakis-Adcock M, McGlade J, Mbamalu G et al (1992) Association of the Shc and Grb2/Sem5 SH2-containing proteins is implicated in activation of the Ras pathway by tyrosine kinases. Nature 360:689–692. https://doi.org/10.1038/360689a0
Schlessinger J, Lemmon MA (2003) SH2 and PTB domains in tyrosine kinase signaling. Sci STKE 2003:RE12. https://doi.org/10.1126/stke.2003.191.re12
Jabril-Cuenod B, Zhang C, Scharenberg AM et al (1996) Syk-dependent phosphorylation of Shc: a potential link between FcϵRI and the Ras/mitogen-activated protein kinase signaling pathway through SOS and Grb2. J Biol Chem 271:16268–16272. https://doi.org/10.1074/jbc.271.27.16268
van der Geer P, Wiley S, Gish GD et al (1996) Identification of residues that control specific binding of the Shc phosphotyrosine-binding domain to phosphotyrosine sites. Proc Natl Acad Sci USA 93:963–968. https://doi.org/10.1073/pnas.93.3.963
Sakaguchi K, Okabayashi Y, Kido Y et al (1998) Shc phosphotyrosine-binding domain dominantly interacts with epidermal growth factor receptors and mediates Ras activation in intact cells. Mol Endocrinol 12:536–543. https://doi.org/10.1210/mend.12.4.0094
Vanderlaan RD, Hardy WR, Kabir MG et al (2011) The ShcA phosphotyrosine docking protein uses distinct mechanisms to regulate myocyte and global heart function. Circ Res 108:184–193. https://doi.org/10.1161/CIRCRESAHA.110.233924
Denning GM, Anderson MP, Amara JF et al (1992) Processing of mutant cystic fibrosis transmembrane conductance regulator is temperature-sensitive. Nature 358:761–764. https://doi.org/10.1038/358761a0
Kunzelmann K, Mall M (2002) Electrolyte transport in the mammalian colon: mechanisms and implications for disease. Physiol Rev 82:245–289. https://doi.org/10.1152/physrev.00026.2001
Wang X, Venable J, LaPointe P et al (2006) Hsp90 cochaperone Aha1 downregulation rescues misfolding of CFTR in cystic fibrosis. Cell 127:803–815. https://doi.org/10.1016/j.cell.2006.09.043
Pankow S, Bamberger C, Calzolari D et al (2015) ∆F508 CFTR interactome remodelling promotes rescue of cystic fibrosis. Nature 528:510–516. https://doi.org/10.1038/nature15729
Luz S, Kongsuphol P, Mendes AI et al (2011) Contribution of casein kinase 2 and spleen tyrosine kinase to CFTR trafficking and protein kinase A-induced activity. Mol Cell Biol 31:4392–4404. https://doi.org/10.1128/MCB.05517-11
Pelicci G, Lanfrancone L, Grignani F et al (1992) A novel transforming protein (SHC) with an SH2 domain is implicated in mitogenic signal transduction. Cell 70:93–104. https://doi.org/10.1016/0092-8674(92)90536-L
Bonfini L, Migliaccio E, Pelicci G et al (1996) Not all Shc’s roads lead to Ras. Trends Biochem Sci 21:257–261. https://doi.org/10.1016/S0968-0004(96)10033-5
Migliaccio E, Mele S, Salcini AE et al (1997) Opposite effects of the p52shc/p46shc and p66shc splicing isoforms on the EGF receptor-MAP kinase-fos signalling pathway. EMBO J 16:706–716. https://doi.org/10.1093/emboj/16.4.706
Migliaccio E, Giorgio M, Mele S et al (1999) The p66shc adaptor protein controls oxidative stress response and life span in mammals. Nature 402:309–313. https://doi.org/10.1038/46311
Luzi L, Confalonieri S, Di Fiore PP, Pelicci PG (2000) Evolution of Shc functions from nematode to human. Curr Opin Genet Dev 10:668–674. https://doi.org/10.1016/S0959-437X(00)00146-5
Uhlik MT, Temple B, Bencharit S et al (2005) Structural and evolutionary division of phosphotyrosine binding (PTB) domains. J Mol Biol 345:1–20. https://doi.org/10.1016/j.jmb.2004.10.038
Shoelson SE (1997) SH2 and PTB domain interactions in tyrosine kinase signal transduction. Curr Opin Chem Biol 1:227–234. https://doi.org/10.1016/S1367-5931(97)80014-2
Wagner MJ, Stacey MM, Liu BA, Pawson T (2013) Molecular mechanisms of SH2- and PTB-domain-containing proteins in receptor tyrosine kinase signaling. Cold Spring Harb Perspect Biol 5:a008987. https://doi.org/10.1101/cshperspect.a008987
Pawson T, Gish GD, Nash P (2001) SH2 domains, interaction modules and cellular wiring. Trends Cell Biol 11:504–511. https://doi.org/10.1016/S0962-8924(01)02154-7
Batzer AG, Rotin D, Ureña JM et al (1994) Hierarchy of binding sites for Grb2 and Shc on the epidermal growth factor receptor. Mol Cell Biol 14:5192–5201. https://doi.org/10.1128/mcb.14.8.5192
Mandiyan V, O’Brien R, Zhou M et al (1996) Thermodynamic studies of SHC phosphotyrosine interaction domain recognition of the NPXpY motif. J Biol Chem 271:4770–4775. https://doi.org/10.1074/jbc.271.9.4770
Zhou M-M, Ravichandran KS, Olejniczak ET et al (1995) Structure and ligand recognition of the phosphotyrosine binding domain of Shc. Nature 378:584–592. https://doi.org/10.1038/378584a0
Farooq A, Zhou M-M (2004) PTB or not to be: promiscuous, tolerant and Bizarro domains come of age. IUBMB Life 56:547–557. https://doi.org/10.1080/15216540400013895
Farooq A, Zeng L, Yan KS et al (2003) Coupling of folding and binding in the PTB domain of the signaling protein Shc. Structure 11:905–913. https://doi.org/10.1016/S0969-2126(03)00134-5
Amaral MD (2005) Processing of CFTR: traversing the cellular maze—how much CFTR needs to go through to avoid cystic fibrosis? Pediatr Pulmonol 39:479–491. https://doi.org/10.1002/ppul.20168
Hutt DM, Loguercio S, Campos AR, Balch WE (2018) A proteomic variant approach (ProVarA) for personalized medicine of inherited and somatic disease. J Mol Biol 430:2951–2973. https://doi.org/10.1016/j.jmb.2018.06.017
Plasschaert LW, Žilionis R, Choo-Wing R et al (2018) A single-cell atlas of the airway epithelium reveals the CFTR-rich pulmonary ionocyte. Nature 560:377–381. https://doi.org/10.1038/s41586-018-0394-6
Liu D, Mamorska-Dyga A (2017) Syk inhibitors in clinical development for hematological malignancies. J Hematol Oncol 10:145. https://doi.org/10.1186/s13045-017-0512-1
Masuda ES, Schmitz J (2008) Syk inhibitors as treatment for allergic rhinitis. Pulm Pharmacol Ther 21:461–467. https://doi.org/10.1016/j.pupt.2007.06.002
Belcher CN, Vij N (2010) Protein processing and inflammatory signaling in cystic fibrosis: challenges and therapeutic strategies. Curr Mol Med 10:82–94
Acknowledgements
This work was supported by Fundação para a Ciência e Tecnologia (FCT), Portugal, through Grants PTDC/BIA-CEL/28408/2017 to PJ and UID/MULTI/04046/2019 to the research unit BioISI, and fellowship SFRH/BD/52488/2014 from the BioSYS Ph.D. programme PD65-2012 to CAL and SFRH/BPD/94322/2013 to PB. The authors acknowledge the following colleagues for providing reagents used in this study: A. M. Matos, Lisbon; J. P. Clancy, University of Alabama, USA; Dimitar G. Efremov, ICGEB, Rome; Enrica Migliaccio, Campos IFOM-IEO, Milan, Italy.
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Loureiro, C.A., Pinto, F.R., Barros, P. et al. A SYK/SHC1 pathway regulates the amount of CFTR in the plasma membrane. Cell. Mol. Life Sci. 77, 4997–5015 (2020). https://doi.org/10.1007/s00018-020-03448-4
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DOI: https://doi.org/10.1007/s00018-020-03448-4