title: LMTK2-mediated phosphorylation regulates CFTR endocytosis in human airway epithelial cells.
abstract: Cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl(-)-selective ion channel expressed in fluid-transporting epithelia. Lemur tyrosine kinase 2 (LMTK2) is a transmembrane protein with serine and threonine but not tyrosine kinase activity. Previous work identified CFTR as an in vitro substrate of LMTK2, suggesting a functional link. Here we demonstrate that LMTK2 co-immunoprecipitates with CFTR and phosphorylates CFTR-Ser(737) in human airway epithelial cells. LMTK2 knockdown or expression of inactive LMTK2 kinase domain increases cell surface density of CFTR by attenuating its endocytosis in human airway epithelial cells. Moreover, LMTK2 knockdown increases Cl(-) secretion mediated by the wild-type and rescued ΔF508-CFTR. Compared with the wild-type CFTR, the phosphorylation-deficient mutant CFTR-S737A shows increased cell surface density and decreased endocytosis. These results demonstrate a novel mechanism of the phospho-dependent inhibitory effect of CFTR-Ser(737) mediated by LMTK2 via endocytosis and inhibition of the cell surface density of CFTR Cl(-) channels. These data indicate that targeting LMTK2 may increase the cell surface density of CFTR Cl(-) channels and improve stability of pharmacologically rescued ΔF508-CFTR in patients with cystic fibrosis.
authors.affiliation: From the Centre for Biodiversity, Functional and Integrative Genomics, University of Lisboa, 1749-016 Lisboa, Portugal.
authors.forename: Simão
authors.initials: S
authors.lastname: Luz
authors.affiliation: the Department of Nephrology, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania 15201.
authors.forename: Kristine M
authors.initials: KM
authors.lastname: Cihil
authors.affiliation: the Center for Cell Signaling and Department of Microbiology, Immunology, and Cancer Biology, University of Virginia School of Medicine, Charlottesville, Virginia 22908, and.
authors.forename: David L
authors.initials: DL
authors.lastname: Brautigan
authors.affiliation: From the Centre for Biodiversity, Functional and Integrative Genomics, University of Lisboa, 1749-016 Lisboa, Portugal.
authors.forename: Margarida D
authors.initials: MD
authors.lastname: Amaral
authors.affiliation: From the Centre for Biodiversity, Functional and Integrative Genomics, University of Lisboa, 1749-016 Lisboa, Portugal.
authors.forename: Carlos M
authors.initials: CM
authors.lastname: Farinha
authors.affiliation: the Department of Nephrology, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania 15201, the Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261 asurban@pitt.edu.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Swiatecka-Urban
mesh_terms: D002460:Cell Line; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D004705:Endocytosis; D004847:Epithelial Cells / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D012137:Respiratory System / Q000166:cytology; D012694:Serine / Q000235:genetics / Q000378:metabolism
chemical_list: D002712:Chlorides; D008565:Membrane Proteins; C093349:cystic fibrosis transmembrane conductance regulator delta F508; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D012694:Serine; C470798:LMTK2 protein, human; D017346:Protein Serine-Threonine Kinases
keywords: CFTR; Chloride Transport; Cystic Fibrosis; Endocytosis; Epithelial Cell; Protein Phosphorylation
references.citation: J Biol Chem. 2002 Dec 20;277(51):49605-12
references.pmid: 12393858
references.citation: J Neurosci. 2003 Jun 15;23(12):4975-83
references.pmid: 12832520
references.citation: J Biol Chem. 2007 Aug 10;282(32):23725-36
references.pmid: 17462998
references.citation: J Cell Biol. 2004 Mar 15;164(6):923-33
references.pmid: 15007060
references.citation: Nucleic Acids Res. 2003 Jan 1;31(1):383-7
references.pmid: 12520028
references.citation: Biol Reprod. 2000 Feb;62(2):412-9
references.pmid: 10642581
references.citation: Proc Natl Acad Sci U S A. 2011 Nov 15;108(46):18843-8
references.pmid: 21976485
references.citation: Science. 1989 Sep 8;245(4922):1066-73
references.pmid: 2475911
references.citation: Genome Res. 2012 Nov;22(11):2109-19
references.pmid: 22975805
references.citation: Am J Physiol Cell Physiol. 2000 Aug;279(2):C375-82
references.pmid: 10913004
references.citation: J Biol Chem. 2002 Dec 20;277(51):49952-7
references.pmid: 12376531
references.citation: Methods Mol Biol. 2011;741:471-88
references.pmid: 21594802
references.citation: J Biol Chem. 1999 Feb 5;274(6):3602-9
references.pmid: 9920908
references.citation: Am J Physiol. 1997 Jul;273(1 Pt 1):L127-33
references.pmid: 9252549
references.citation: J Biol Chem. 2010 Aug 27;285(35):27008-27018
references.pmid: 20525683
references.citation: Commun Integr Biol. 2012 Sep 1;5(5):473-6
references.pmid: 23181163
references.citation: Curr Opin Cell Biol. 2000 Aug;12(4):483-90
references.pmid: 10873817
references.citation: J Neurochem. 1997 Aug;69(2):859-67
references.pmid: 9231748
references.citation: J Biol Chem. 2004 Sep 3;279(36):38025-31
references.pmid: 15247260
references.citation: Am J Physiol Cell Physiol. 2000 Aug;279(2):C461-79
references.pmid: 10913013
references.citation: J Cyst Fibros. 2014 Jan;13(1):29-36
references.pmid: 23891399
references.citation: Mol Cell Biol. 2011 Nov;31(22):4392-404
references.pmid: 21930781
references.citation: Am J Physiol Cell Physiol. 2013 Jan 15;304(2):C164-9
references.pmid: 23114966
references.citation: Am J Physiol Lung Cell Mol Physiol. 2007 Jun;292(6):L1352-60
references.pmid: 17307814
references.citation: Biochem J. 1997 Dec 1;328 ( Pt 2):353-61
references.pmid: 9371688
references.citation: Genes Cells. 2004 Mar;9(3):219-32
references.pmid: 15005709
references.citation: J Cell Sci. 2007 Dec 15;120(Pt 24):4278-88
references.pmid: 18029400
references.citation: Biochim Biophys Acta. 2009 Jun;1788(6):1341-9
references.pmid: 19328185
references.citation: Nat Neurosci. 2001 Apr;4(4):374-81
references.pmid: 11276227
references.citation: J Biol Chem. 2009 Feb 27;284(9):5645-53
references.pmid: 19095655
references.citation: J Biol Chem. 2010 May 28;285(22):17177-87
references.pmid: 20351096
references.citation: Am J Respir Crit Care Med. 2004 Oct 1;170(7):815-20
references.pmid: 15447951
references.citation: Biochem Biophys Res Commun. 1989 Mar 31;159(3):871-7
references.pmid: 2539153
references.citation: Neuroscience. 2007 Aug 24;148(2):510-21
references.pmid: 17651901
references.citation: Annu Rev Physiol. 2006;68:543-61
references.pmid: 16460283
references.citation: ACS Chem Biol. 2013 Feb 15;8(2):432-42
references.pmid: 23148778
references.citation: Genes Cells. 2008 May;13(5):483-95
references.pmid: 18429820
references.citation: Nature. 1994 Sep 29;371(6496):419-23
references.pmid: 8090221
references.citation: Chest. 2011 Jun;139(6):1480-1490
references.pmid: 21652558
references.citation: Oncogene. 2012 May 31;31(22):2773-82
references.pmid: 21996745
references.citation: J Biol Chem. 2005 Nov 4;280(44):36762-72
references.pmid: 16131493
references.citation: Commun Integr Biol. 2013 Mar 1;6(2):e23094
references.pmid: 23750297
references.citation: Science. 1989 Sep 8;245(4922):1059-65
references.pmid: 2772657
references.citation: Eur Respir J. 2004 Jan;23(1):146-58
references.pmid: 14738247
references.citation: Toxins (Basel). 2012 Oct;4(10):940-54
references.pmid: 23162706
references.citation: J Biol Chem. 2002 Oct 18;277(42):40099-105
references.pmid: 12167629
references.citation: BMC Cancer. 2010 Jun 22;10:315
references.pmid: 20569440
references.citation: Clin Biochem. 2012 Oct;45(15):1132-44
references.pmid: 22698459
references.citation: Mol Cell Biol. 2011 Oct;31(19):4076-86
references.pmid: 21807898
references.citation: Nature. 1994 Sep 29;371(6496):423-6
references.pmid: 8090222
references.citation: J Biol Chem. 2001 Mar 23;276(12):8942-50
references.pmid: 11124952
references.citation: J Biol Chem. 1998 Oct 9;273(41):26862-9
references.pmid: 9756932
references.citation: Nat Rev Mol Cell Biol. 2006 Jun;7(6):426-36
references.pmid: 16723978
references.citation: Mol Cell Proteomics. 2006 Nov;5(11):2124-30
references.pmid: 16887929
references.citation: Mol Med. 2012 May 09;18:685-96
references.pmid: 22396015
references.citation: J Vis Exp. 2013 Dec 13;(82):e50867
references.pmid: 24378656
references.citation: J Biol Chem. 1998 Aug 21;273(34):21759-68
references.pmid: 9705313
references.citation: Methods Mol Biol. 2011;741:39-54
references.pmid: 21594777
references.citation: Physiol Rev. 1997 Jul;77(3):759-803
references.pmid: 9234965
references.citation: Nucleic Acids Res. 2013 Jan;41(Database issue):D348-52
references.pmid: 23197659
references.citation: Thorax. 2012 Jan;67(1):12-8
references.pmid: 21825083
references.citation: Am J Physiol Cell Physiol. 2003 Jul;285(1):C1-18
references.pmid: 12777252
references.citation: Am J Respir Cell Mol Biol. 2004 Aug;31(2):140-6
references.pmid: 15039139
references.citation: J Biol Chem. 2012 Apr 27;287(18):15087-99
references.pmid: 22399289
references.citation: Am J Physiol Lung Cell Mol Physiol. 2013 Oct 15;305(8):L530-41
references.pmid: 23934925
references.citation: Front Pharmacol. 2012 Sep 26;3:160
references.pmid: 23055971
references.citation: PLoS One. 2013 May 09;8(5):e63167
references.pmid: 23671668
references.citation: Angew Chem Int Ed Engl. 2010 Dec 17;49(51):9907-11
references.pmid: 21105033
references.citation: Pflugers Arch. 2009 Mar;457(5):1071-8
references.pmid: 18752001
references.citation: J Neurochem. 2012 May;121(3):327-8
references.pmid: 22224437
references.citation: Annu Rev Biochem. 2008;77:701-26
references.pmid: 18304008
references.citation: Biochem J. 2012 Jan 15;441(2):633-43
references.pmid: 21995445