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• W2033846769 abstract "An emerging theme in cell signaling is that membrane-bound channels and receptors are organized into supramolecular signaling complexes for optimum function and cross-talk. In this study, we determined how protein kinase C (PKC) phosphorylation influences the scaffolding protein Na+/H+ exchanger regulatory factor 1 (NHERF) to assemble protein complexes of cystic fibrosis transmembrane conductance regulator (CFTR), a chloride ion channel that controls fluid and electrolyte transport across cell membranes. NHERF directs polarized expression of receptors and ion transport proteins in epithelial cells, as well as organizes the homo- and hetero-association of these cell surface proteins. NHERF contains two modular PDZ domains that are modular protein-protein interaction motifs, and a C-terminal domain. Previous studies have shown that NHERF is a phosphoprotein, but how phosphorylation affects NHERF to assemble macromolecular complexes is unknown. We show that PKC phosphorylates two amino acid residues Ser-339 and Ser-340 in the C-terminal domain of NHERF, but a serine 162 of PDZ2 is specifically protected from being phosphorylated by the intact C-terminal domain. PKC phosphorylation-mimicking mutant S339D/S340D of NHERF has increased affinity and stoichiometry when binding to C-CFTR. Moreover, solution small angle x-ray scattering indicates that the PDZ2 and C-terminal domains contact each other in NHERF, but such intramolecular domain-domain interactions are released in the PKC phosphorylation-mimicking mutant indicating that PKC phosphorylation disrupts the autoinhibition interactions in NHERF. The results demonstrate that the C-terminal domain of NHERF functions as an intramolecular switch that regulates the binding capability of PDZ2, and thus controls the stoichiometry of NHERF to assemble protein complexes. An emerging theme in cell signaling is that membrane-bound channels and receptors are organized into supramolecular signaling complexes for optimum function and cross-talk. In this study, we determined how protein kinase C (PKC) phosphorylation influences the scaffolding protein Na+/H+ exchanger regulatory factor 1 (NHERF) to assemble protein complexes of cystic fibrosis transmembrane conductance regulator (CFTR), a chloride ion channel that controls fluid and electrolyte transport across cell membranes. NHERF directs polarized expression of receptors and ion transport proteins in epithelial cells, as well as organizes the homo- and hetero-association of these cell surface proteins. NHERF contains two modular PDZ domains that are modular protein-protein interaction motifs, and a C-terminal domain. Previous studies have shown that NHERF is a phosphoprotein, but how phosphorylation affects NHERF to assemble macromolecular complexes is unknown. We show that PKC phosphorylates two amino acid residues Ser-339 and Ser-340 in the C-terminal domain of NHERF, but a serine 162 of PDZ2 is specifically protected from being phosphorylated by the intact C-terminal domain. PKC phosphorylation-mimicking mutant S339D/S340D of NHERF has increased affinity and stoichiometry when binding to C-CFTR. Moreover, solution small angle x-ray scattering indicates that the PDZ2 and C-terminal domains contact each other in NHERF, but such intramolecular domain-domain interactions are released in the PKC phosphorylation-mimicking mutant indicating that PKC phosphorylation disrupts the autoinhibition interactions in NHERF. The results demonstrate that the C-terminal domain of NHERF functions as an intramolecular switch that regulates the binding capability of PDZ2, and thus controls the stoichiometry of NHERF to assemble protein complexes. It is becoming clear that cell surface receptors and ion transport proteins function as part of larger macromolecular complexes (1Guggino W.B. Stanton B.A. Nat. Rev. Mol. Cell Biol. 2006; 7: 426-436Crossref PubMed Scopus (347) Google Scholar, 2Bulenger S. Marullo S. Bouvier M. Trends Pharmacol. Sci. 2005; 26: 131-137Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar, 3Engelman D.M. Nature. 2005; 438: 578-580Crossref PubMed Scopus (687) Google Scholar, 4Prinster S.C. Hague C. Hall R.A. Pharmacol. Rev. 2005; 57: 289-298Crossref PubMed Scopus (339) Google Scholar). The formation of membrane protein oligomers is necessary for the biosynthesis, signal transduction, and function of membrane receptors and ion transport proteins (5Shenolikar S. Voltz J.W. Minkoff C.M. Wade J.B. Weinman E.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11470-11475Crossref PubMed Scopus (283) Google Scholar, 6Guggino W.B. Banks-Schlegel S.P. Am. J. Respir. Crit. Care Med. 2004; 170: 815-820Crossref PubMed Scopus (44) Google Scholar, 7Scott K. Zuker C.S. Nature. 1998; 395: 805-808Crossref PubMed Scopus (136) Google Scholar). Understanding the mechanisms and functional significance of membrane protein assembly is essential for the identification of molecular targets for therapeutic purposes. The cystic fibrosis transmembrane conductance regulator (CFTR) 3The abbreviations used are:CFTRcystic fibrosis transmembrane conductance regulatorDLSdynamic light scatteringERMezrin-radixin-moesinFERMconserved domain shared by protein 4.1, ezrin, radixin, moesinIEFisoelectric focusingNHERF-1Na+/H+ exchanger regulatory factor 1NHERF(S339D/S340D)double mutants (S339D and S340D) of NHERFPDZpostsynaptic density 95/disk-large/zonula occluden-1PDZ2-CTtruncated construct of NHERF that includes PDZ2 and the C-terminal domain of NHERFPDZ2-CT(S339D/S340D)double mutant (S339D and S340D) of PDZ2-CTPKAprotein kinase APKCprotein kinase CSAXSsmall angle x-ray scatteringSLSstatic light scatteringSPRsurface plasmon resonancePDBProtein Data Bank is a chloride ion channel in several epithelial tissues, where CFTR is responsible for fluid and electrolyte transport across cell membranes (8Riordan J.R. Rommens J.M. Kerem B. Alon N. Rozmahel R. Grzelczak Z. Zielenski J. Lok S. Plavsic N. Chou J.L. Drumm M.L. Ianuzzi M.C. Collins F.S. Tsui L-C. Science. 1989; 245: 1066-1073Crossref PubMed Scopus (5946) Google Scholar, 9Pilewski J.M. Frizzell R.A. Physiol. Rev. 1999; 79: S215-S255Crossref PubMed Scopus (384) Google Scholar, 10Duan D.Y. Liu L.L. Bozeat N. Huang Z.M. Xiang S.Y. Wang G.L. Ye L. Hume J.R. Acta Pharmacol. Sin. 2005; 26: 265-278Crossref PubMed Scopus (66) Google Scholar). Malfunction in CFTR ion transport causes devastating human diseases, such as cystic fibrosis and secretory diarrhea (11Welsh M.J. Smith A.E. Sci. Am. 1995; 273: 52-59Crossref PubMed Scopus (104) Google Scholar, 12Barrett K.E. Keely S.J. Annu. Rev. Physiol. 2000; 62: 535-572Crossref PubMed Scopus (384) Google Scholar). CFTR is controlled by ATP hydrolysis and by protein kinase-dependent phosphorylation (13Bear C.E. Li C.H. Kartner N. Bridges R.J. Jensen T.J. Ramjeesingh M. Riordan J.R. Cell. 1992; 68: 809-818Abstract Full Text PDF PubMed Scopus (778) Google Scholar, 14Gadsby D.C. Nairn A.C. Physiol. Rev. 1999; 79: S77-S107Crossref PubMed Scopus (369) Google Scholar, 15Vergani P. Lockless S.W. Nairn A.C. Gadsby D.C. Nature. 2005; 433: 876-880Crossref PubMed Scopus (339) Google Scholar, 16Riordan J.R. Annu. Rev. Physiol. 2005; 67: 701-718Crossref PubMed Scopus (193) Google Scholar, 17Biemans-Oldehinkel E. Doeven M.K. Poolman B. FEBS Lett. 2006; 580: 1023-1035Crossref PubMed Scopus (181) Google Scholar), but recent studies suggest that CFTR is also regulated by a network of protein-protein interactions. The multiprotein complexes that interact with CFTR influence the intracellular trafficking of CFTR and coordinate the ion transport functions of CFTR to maintain cellular ion homeostasis, reviewed in Refs. 1Guggino W.B. Stanton B.A. Nat. Rev. Mol. Cell Biol. 2006; 7: 426-436Crossref PubMed Scopus (347) Google Scholar, 18Mazzochi C. Benos D.J. Smith P.R. Am. J. Physiol. 2006; 291: F1113-F1122Crossref PubMed Scopus (93) Google Scholar, 19Lamprecht G. Seidler U. Am. J. Physiol. 2006; 291: G766-G777Crossref PubMed Scopus (121) Google Scholar. cystic fibrosis transmembrane conductance regulator dynamic light scattering ezrin-radixin-moesin conserved domain shared by protein 4.1, ezrin, radixin, moesin isoelectric focusing Na+/H+ exchanger regulatory factor 1 double mutants (S339D and S340D) of NHERF postsynaptic density 95/disk-large/zonula occluden-1 truncated construct of NHERF that includes PDZ2 and the C-terminal domain of NHERF double mutant (S339D and S340D) of PDZ2-CT protein kinase A protein kinase C small angle x-ray scattering static light scattering surface plasmon resonance Protein Data Bank The macromolecular interactions of CFTR with other proteins are organized by scaffolding proteins. CFTR contains a DTRL motif in the cytoplasmic C terminus that interacts with a class of postsynaptic density 95/disk-large/zonula occluden-1 (PDZ) scaffolding proteins (20Wang S. Raab R.W. Schatz P.J. Guggino W.B. Li M. FEBS Lett. 1998; 427: 103-108Crossref PubMed Scopus (250) Google Scholar, 21Hall R.A. Ostedgaard L.S. Premont R.T. Blitzer J.T. Rahman N. Welsh M.J. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8496-8501Crossref PubMed Scopus (375) Google Scholar, 22Karthikeyan S. Leung T. Ladias J.A. J. Biol. Chem. 2001; 276: 19683-19686Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 23Wang S. Yue H. Derin R.B. Guggino W.B. Li M. Cell. 2000; 103: 169-179Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). PDZ is a protein-protein interaction modular domain that binds to the cytoplasmic domains of a number of transmembrane receptors and ion transport proteins (24Fanning A.S. Anderson J.M. Curr. Biol. 1996; 6: 1385-1388Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 25Harris B.Z. Lim W.A. J. Cell Sci. 2001; 114: 3219-3231Crossref PubMed Google Scholar). Scaffolding proteins containing multiple PDZ domains assemble and localize large signaling complexes at specific locations in cells (19Lamprecht G. Seidler U. Am. J. Physiol. 2006; 291: G766-G777Crossref PubMed Scopus (121) Google Scholar, 26Sheng M. Sala C. Annu. Rev. Neurosci. 2001; 24: 1-29Crossref PubMed Scopus (1044) Google Scholar, 27Swiatecka-Urban A. Duhaime M. Coutermarsh B. Karlson K.H. Collawn J. Milewski M. Cutting G.R. Guggino W.B. Langford G. Stanton B.A. J. Biol. Chem. 2002; 277: 40099-40105Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 28Moyer B.D. Duhaime M. Shaw C. Denton J. Reynolds D. Karlson K.H. Pfeiffer J. Wang S. Mickle J.E. Milewski M. Cutting G.R. Guggino W.B. Li M. Stanton B.A. J. Biol. Chem. 2000; 275: 27069-27074Abstract Full Text Full Text PDF PubMed Google Scholar, 29Haggie P.M. Stanton B.A. Verkman A.S. J. Biol. Chem. 2004; 279: 5494-5500Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). In particular, the cytoplasmic domain of CFTR binds to the scaffolding protein Na+/H+ exchanger regulatory factor-1 (NHERF) (20Wang S. Raab R.W. Schatz P.J. Guggino W.B. Li M. FEBS Lett. 1998; 427: 103-108Crossref PubMed Scopus (250) Google Scholar, 21Hall R.A. Ostedgaard L.S. Premont R.T. Blitzer J.T. Rahman N. Welsh M.J. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8496-8501Crossref PubMed Scopus (375) Google Scholar). NHERF contains two PDZ domains, PDZ1 and PDZ2 (shown in Fig. 1), and a C-terminal domain that binds to a class of membrane-cytoskeleton adapter proteins, ezrin-radixinmoesin (30Shenolikar S. Voltz J.W. Cunningham R. Weinman E.J. Physiology (Bethesda). 2004; 19: 362-369Crossref PubMed Scopus (135) Google Scholar, 31Weinman E.J. Hall R.A. Friedman P.A. Liu-Chen L.Y. Shenolikar S. Annu. Rev. Physiol. 2006; 68: 491-505Crossref PubMed Scopus (156) Google Scholar, 32Reczek D. Berryman M. Bretscher A. J. Cell Biol. 1997; 139: 169-179Crossref PubMed Scopus (518) Google Scholar, 33Bretscher A. Edwards K. Fehon R.G. Nat. Rev. Mol. Cell Biol. 2002; 3: 586-599Crossref PubMed Scopus (1136) Google Scholar, 34Li J. Dai Z. Jana D. Callaway D.J. Bu Z. J. Biol. Chem. 2005; 280: 37634-37643Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 35Weinman E.J. Steplock D. Donowitz M. Shenolikar S. Biochemistry. 2000; 39: 6123-6129Crossref PubMed Scopus (128) Google Scholar). NHERF is thus also called ezrin-binding protein 50 (EBP-50). As a member of a family of proteins that contains two or multiple copies of PDZ domains (36Donowitz M. Cha B. Zachos N.C. Brett C.L. Sharma A. Tse C.M. Li X. J. Physiol. (Lond.). 2005; 567: 3-11Crossref Scopus (182) Google Scholar, 37Thelin W.R. Hodson C.A. Milgram S.L. J. Physiol. (Lond.). 2005; 567: 13-19Crossref Scopus (35) Google Scholar), NHERF participates in directing polarized expression of ion channels and transporters within specific membrane domains of epithelial cells, as well as recruiting cell signaling macromolecular complexes (1Guggino W.B. Stanton B.A. Nat. Rev. Mol. Cell Biol. 2006; 7: 426-436Crossref PubMed Scopus (347) Google Scholar, 18Mazzochi C. Benos D.J. Smith P.R. Am. J. Physiol. 2006; 291: F1113-F1122Crossref PubMed Scopus (93) Google Scholar, 19Lamprecht G. Seidler U. Am. J. Physiol. 2006; 291: G766-G777Crossref PubMed Scopus (121) Google Scholar, 27Swiatecka-Urban A. Duhaime M. Coutermarsh B. Karlson K.H. Collawn J. Milewski M. Cutting G.R. Guggino W.B. Langford G. Stanton B.A. J. Biol. Chem. 2002; 277: 40099-40105Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 28Moyer B.D. Duhaime M. Shaw C. Denton J. Reynolds D. Karlson K.H. Pfeiffer J. Wang S. Mickle J.E. Milewski M. Cutting G.R. Guggino W.B. Li M. Stanton B.A. J. Biol. Chem. 2000; 275: 27069-27074Abstract Full Text Full Text PDF PubMed Google Scholar, 29Haggie P.M. Stanton B.A. Verkman A.S. J. Biol. Chem. 2004; 279: 5494-5500Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 30Shenolikar S. Voltz J.W. Cunningham R. Weinman E.J. Physiology (Bethesda). 2004; 19: 362-369Crossref PubMed Scopus (135) Google Scholar). Notably, recent studies find that NHERF increases the cell surface expression and rescues the function of ΔF508 CFTR (38Guerra L. Fanelli T. Favia M. Riccardi S.M. Busco G. Cardone R.A. Carrabino S. Weinman E.J. Reshkin S.J. Conese M. Casavola V. J. Biol. Chem. 2005; 280: 40925-40933Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 39Bossard F. Robay A. Toumaniantz G. Dahimene S. Becq F. Merot J. Gauthier C. Am. J. Physiol. 2007; 292: L1085-L1094Google Scholar), a mutant of CFTR that fails to reach the cell surface and causes cystic fibrosis (11Welsh M.J. Smith A.E. Sci. Am. 1995; 273: 52-59Crossref PubMed Scopus (104) Google Scholar). NHERF assembles the homotypic CFTR-CFTR association, which is thought to increase the channel activities of CFTR (40Raghuram V. Mak D.D. Foskett J.K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1300-1305Crossref PubMed Scopus (197) Google Scholar, 41Li C. Roy K. Dandridge K. Naren A.P. J. Biol. Chem. 2004; 279: 24673-24684Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). NHERF also organizes the heterotypic interactions of CFTR with other channels, receptors, and with intracellular signaling proteins (42Yoo D. Flagg T.P. Olsen O. Raghuram V. Foskett J.K. Welling P.A. J. Biol. Chem. 2004; 279: 6863-6873Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 43Guerra L. Favia M. Fanelli T. Calamita G. Svelto M. Bagorda A. Jacobson K.A. Reshkin S.J. Casavola V. Pfluegers Arch. 2004; 449: 66-75Crossref PubMed Scopus (18) Google Scholar, 44Short D.B. Trotter K.W. Reczek D. Kreda S.M. Bretscher A. Boucher R.C. Stutts M.J. Milgram S.L. J. Biol. Chem. 1998; 273: 19797-19801Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar). For instance, NHERF forms multiprotein complexes that include CFTR, ezrin, and PKA, which implies that NHERF and ezrin bring PKA and CFTR into a macromolecular complex for PKA-mediated phosphorylation of CFTR (45Sun F. Hug M.J. Bradbury N.A. Frizzell R.A. J. Biol. Chem. 2000; 275: 14360-14366Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). In addition, NHERF recruits a multiprotein complex that includes CFTR as well as a β2-adrenergic receptor. This hetero-protein complex transmits signals from the agonists of the β2-adrenergic receptor to stimulate CFTR activities (46Naren A.P. Cobb B. Li C. Roy K. Nelson D. Heda G.D. Liao J. Kirk K.L. Sorscher E.J. Hanrahan J. Clancy J.P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 342-346Crossref PubMed Scopus (185) Google Scholar). Furthermore, NHERF mediates the macromolecular interactions of CFTR with an inwardly rectifying potassium channel, and such interactions may be responsible for coordinated ion transport activities of these channels (42Yoo D. Flagg T.P. Olsen O. Raghuram V. Foskett J.K. Welling P.A. J. Biol. Chem. 2004; 279: 6863-6873Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). NHERF is a phosphoprotein; it was originally isolated from cells in a phosphorylated state (32Reczek D. Berryman M. Bretscher A. J. Cell Biol. 1997; 139: 169-179Crossref PubMed Scopus (518) Google Scholar). Later studies have identified a number of kinases that phosphorylate NHERF. Hall et al. domain (47Hall R.A. Spurney R.F. Premont R.T. Rahman N. Blitzer J.T. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1999; 274: 24328-24334Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar) finds that a G protein-coupled receptor kinase phosphorylates NHERF at Ser-289 in the C terminus. NHERF is phosphorylated in a cell cycle-dependent manner by cdc2 kinase at Ser-279 and Ser-301 (48He J. Lau A.G. Yaffe M.B. Hall R.A. J. Biol. Chem. 2001; 276: 41559-41565Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). NHERF is also phosphorylated by PKC in response to extracellular signals such as by the stimulation of G protein-coupled receptors (49Deliot N. Hernando N. Horst-Liu Z. Gisler S.M. Capuano P. Wagner C.A. Bacic D. O'Brien S. Biber J. Murer H. Am. J. Physiol. 2005; 289: C159-C167Crossref PubMed Scopus (99) Google Scholar). However, attempts to identify the PKC phosphorylation sites in NHERF-1 have generated controversial results (50Raghuram V. Hormuth H. Foskett J.K. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 9620-9625Crossref PubMed Scopus (85) Google Scholar, 51Fouassier L. Nichols M.T. Gidey E. McWilliams R.R. Robin H. Finnigan C. Howell K.E. Housset C. Doctor R.B. Exp. Cell Res. 2005; 306: 264-273Crossref PubMed Scopus (34) Google Scholar). More importantly, the effects of phosphorylation on the ability of NHERF to assemble protein complexes, although essential, remain unknown. Phosphorylation is thought to induce oligomerization of NHERF that changes the ability of NHERF to assemble signaling complexes (51Fouassier L. Nichols M.T. Gidey E. McWilliams R.R. Robin H. Finnigan C. Howell K.E. Housset C. Doctor R.B. Exp. Cell Res. 2005; 306: 264-273Crossref PubMed Scopus (34) Google Scholar, 52Lau A.G. Hall R.A. Biochemistry. 2001; 40: 8572-8580Crossref PubMed Scopus (102) Google Scholar). In this study, we identified the effects of PKC phosphorylation on NHERF in assembling CFTR complexes, using biochemical and biophysical methods. Our results demonstrate that there are intramolecular domain-domain interactions between PDZ2 and the C-terminal domain of NHERF. PKC phosphorylation abolishes the autoinhibitory-like interactions and increases the binding affinity of PDZ2 for its peptide ligand. PKC phosphorylation thus regulates the affinity and stoichiometry of NHERF to assemble macromolecular complexes. Protein Expression, Site-directed Mutagenesis, and Protein Purification—The human NHERF cDNA encoding PDZ1 (residues 11–99), PDZ2-(150–240), the C-terminal domain termed CT-(242–358), PDZ2 plus the C-terminal domain termed PDZ2-CT-(150–358), and the full-length NHERF-(11–358) were subcloned into the pET151/D-TOPO vectors (Invitrogen), respectively. The pET151/D-TOPO vector encodes a V5 epitope plus hexahistidine fusion tag at the N terminus of an expressed protein. Using the pET151/D-TOPO vector, we have also expressed and purified the last 70-residue peptide of human CFTR, termed C-CFTR-(1411–1480), which includes the PDZ-binding motif DTRL at the very C terminus. Fig. 1 summarizes the different constructs of NHERF used in this study. Site-directed mutants in the full-length NHERF and the differently truncated NHERF domains were generated with the QuikChange site-directed mutagenesis kit (Stratagene). All of the expression vectors were subjected to sequence analysis at the DNA Sequencing Facility at Fox Chase Cancer Center. Cell growth and protein purification have been described previously (34Li J. Dai Z. Jana D. Callaway D.J. Bu Z. J. Biol. Chem. 2005; 280: 37634-37643Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). PKC Phosphorylation Assays and Isoelectric Focusing Two-dimensional Electrophoresis—PKC phosphorylation assays were performed by adding 25 milliunits of PKC (1 μl) to a 20-μl solution containing 1.0 μg of purified protein in 20 mm HEPES, pH 7.4, 10 mm MgCl2, 1.0 mm dithiothreitol, 1.7 mm CaCl2, 0.020 mm ATP, and 14 μCi of [γ-32P]ATP. The catalytic domain of PKC from rat brain was purchased from EMD Biosciences, Inc. The solution was incubated at 30 °C for 30 min. The phosphorylation reaction was terminated by adding 20 μl of protein sample buffer. The samples were resolved by SDS-PAGE on 10% Nu/PAGE gels (Invitrogen). The gels were stained with Coomassie Blue, destained, dried, and exposed to chemiluminescence film (Eastman Kodak Co.) to detect γ-32P incorporation. The chemiluminescence image was quantified by densitometry, using an Epson Perfection V750 PRO digital scanner and a software UN-SCAN-IT Gel from the Silk Scientific. Isoelectric focusing (IEF) two-dimensional electrophoresis was also used to identify PKC-induced phosphorylation in the full-length NHERF. Before electrophoresis, the PKC phosphorylation reaction was performed by adding 25 milliunits of PKC to a 20-μl solution containing 1.0 μg of purified protein, 0.020 mm ATP, 20 mm HEPES, pH 7.4, 10 mm MgCl2, 1.0 mm dithiothreitol, and 1.7 mm CaCl2. The phosphorylation reaction was terminated by adding SDS sample buffer. The phosphorylated protein was resolved by IEF electrophoresis, using unphosphorylated NHERF as control. The isoelectric points (pI) of unphosphorylated and phosphorylated NHERF were estimated based on the amino acid sequence of the protein and the number of phosphorylated Ser/Thr sites. The pI of unphosphorylated NHERF was calculated using an isoelectric point calculator, assuming pKa = 4.3 for Glu, pKa = 3.7 for Asp, pKa = 10.5 for Lys, and pKa = 12.5 for Asn. The pI values of NHERF with different number of Ser/Thr phosphorylation sites were calculated, assuming pKa1 = 2.12 for the first ionization and the pKa2 = 7.21 for the second ionization of a phosphorylation group. Cell Culture and Delivering Proteins in Mammalian Cells—The BioTrek protein delivery reagent (Stratagene, La Jolla, CA) was used to deliver wild-type NHERF, NHERF(S339D), NHERF(S340D), and NHERF(S339D/S340D) in Calu-3 cells. Purified proteins or protein complexes were diluted to 150 μg/ml using phosphate-buffered saline. About 100 μl of the diluted protein solution was transferred to a tube containing the lyophilized BioTrek reagent. The mixture was resuspended and incubated at room temperature for about 5 min. Serum-free medium was then added to the tube to a final volume of 500 μl. The Calu-3 cell line was purchased from ATCC (Manassas, VA). Calu-3 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum at 37 °C in a humidified 5% CO2 atmosphere. The cells were grown in a 6-well plate with 30-mm-diameter wells. When the cell density reached 50–60% confluence, the culture medium was removed by aspiration. The cells were washed once with serum-free medium. After 500 μl of fresh serum-free medium was added to each well, 500 μl of the BioTrek/protein mixture was added dropwise to the cells. The cell culture plate was then incubated at 37 °C and 5% CO2 in a humidified incubator for 4–5 h before the immunoprecipitation experiments. Immunoprecipitation and Immunoblotting Experiments—The Calu-3 cell monolayer in a 30-mm-diameter well was washed with phosphate-buffered saline followed by lysis in radioimmunoprecipitation assay (RIPA) buffer at 4 °C. A mouse anti-V5 antibody (Invitrogen) was used to pull down the protein complex. About 1.0 μg of anti-V5 was added to 500 μl of cell lysate and shaken in a cold room for 4 h. About 20 μl of agarose beads (G PLUS; Santa Cruz Biotechnology) were added to the lysate, and the mixture was shaken in a cold room overnight. The beads were then spun and washed three times with 750 μl of RIPA buffer and then resuspended in 50 μl of SDS-PAGE sample buffer. A mouse anti-hemagglutinin antibody (Invitrogen) was used as a mock primary antibody during immunoprecipitation experiments for negative control to verify the specificity of the antibody used to pull down the protein complex. After electrophoresis, proteins in the SDS-polyacrylamide gel were transferred to nitrocellulose membranes by semi-dry blotting. The membranes were then blocked in 5% fat-free milk in TBS-T (10 mm Tris-HCl, pH 7.5, 150 mm NaCl, 0.05% Tween 20) for 3 h at room temperature. Primary rabbit anti-CFTR antibody (Alomone Labs, Jerusalem, Israel) was used to detect CFTR, and primary mouse anti-V5 antibody was used to detect NHERF. The primary antibody was added to the blots and incubated overnight in blocking solution. The following day the blots were washed three times with TBS-T buffer and then incubated with the secondary antibody (anti-mouse horseradish peroxidase for mouse anti-V5 antibody or anti-rabbit horseradish peroxidase for rabbit anti-CFTR primary antibody) for 1 h at room temperature and washed three times with changes of TBS-T buffer. The blots were then developed by enhanced chemiluminescence detection (Pierce) for analysis. The chemiluminescence image was quantified by densitometry, using an Epson Perfection V750 PRO digital scanner and the software UN-SCAN-IT gel. Light Scattering Experiments—Light scattering experiments were performed using a DynaPro Molecular Sizing Instrument (Wyatt Technology Corp., Santa Barbara, CA) with a laser of wavelength 824.7 nm at a fixed 90° scattering angle. The DynaPro was used to perform both dynamic light scattering (DLS) and static light scattering (SLS). DLS measures the hydrodynamic radius of the proteins. SLS determines the absolute molecular mass of a protein, which is independent of the size and shape of a protein. Before light scattering experiments, the sample was centrifuged at 10,000 rpm for 5–10 min to remove dust. Protein concentrations were varied from 0.5 to 3 mg/ml during light scattering measurements. The scattering intensity of the buffer background was subtracted from that of the sample solution. Light scattering experiments were performed at 10 °C. The data were analyzed with the software Dynamics version 6. Surface Plasmon Resonance Binding Experiments and Data Analysis—Surface plasmon resonance (SPR) experiments were performed on a Biacore 1000 instrument (Biacore Life Sciences, NJ) at 25.0 °C. Before the binding experiments, the hydrogel matrix of the BIAcore CM5 Biosensor chips was activated by N-hydroxysuccinimide and N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide (Biacore Life Sciences, NJ). The ligand, which is 3 μl of 5 μg/ml C-CFTR dissolved in 10 mm sodium acetate, pH 5.2, was injected to coat the activated surface. Uncross-linked ligand was washed away, and uncoated sites were blocked by 1 m ethanolamine, pH 8.5. The analytes (NHERF or NHERF mutants) were prepared in HBS-EP buffer containing 10 mm HEPES buffer, pH 7.4, 150 mm NaCl, 3 mm EDTA, and 0.005% surfactant polysorbate 20. The analyte was injected over the C-CFTR-coated surfaces at a series of concentrations at 50 μl/min for 3 min. At the end of each injection, the sensor chip was regenerated with 4.0 m MgCl2, 50 mm triethylamine, pH 9.15, and HBS-EP buffer. The SPR response curves were obtained by subtracting the background signal generated by injecting the analyte over a control cell without ligand coating to remove the bulk refractive index effects. The nonspecific binding was corrected by subtracting the signal generated by HBS-EP buffer alone. The response curve reached an equilibrium plateau in all cases (shown in Fig. 5A), and the average of the plateau region was taken and plotted as a function of analyte concentrations to obtain the binding curve (shown in Fig. 5B). For monovalent analyte binding, the dissociation constant (Kd) was obtained by fitting the binding curve to a monovalent binding model as shown in Equation 1, RU=CA×maxCA+Kd(Eq. 1) where RU is the response unit when the analyte flows over the ligand-coated sensor chip; CA is the concentration of the analyte, and max is the maximum response unit. The Kd and max values were obtained by nonlinear curve fitting to Equation 1 using the commercial software Origin 6.1 (OriginLab Corp., Northhampton, MA). For bivalent analytes, the three flow cells of a sensor chip were coated with C-CFTR at three different densities by injecting the C-CFTR solution twice to each flow cell at 3 μl of 3 μg/ml, 1 μl of 10 μg/ml, and 3 μl of 20 μg/ml. This operation gave three values of response units of 50, 80, and 400 in the three flow cells, respectively, corresponding to three different coating densities. The analyte was then injected at various concentrations to flow over the C-CFTR-coated chip. After background subtraction, the response unit of the plateau region of each response curve was taken and plotted against the analyte concentrations. The bivalent binding model can be expressed as shown in Equation (see Supplemental Material for the derivation of Equation 2), RU=Am1LKd1+m2L2Kd1Kd2(Eq. 2) where Kd1 and Kd2 are the dissociation constants of the two" @default.
• W2033846769 created "2016-06-24" @default.
• W2033846769 creator A5008915529 @default.
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• W2033846769 date "2007-09-01" @default.
• W2033846769 modified "2023-10-14" @default.
• W2033846769 title "Protein Kinase C Phosphorylation Disrupts Na+/H+ Exchanger Regulatory Factor 1 Autoinhibition and Promotes Cystic Fibrosis Transmembrane Conductance Regulator Macromolecular Assembly" @default.
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• W2033846769 doi "https://doi.org/10.1074/jbc.m702019200" @default.
• W2033846769 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/17613530" @default.
• W2033846769 hasPublicationYear "2007" @default.
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