Modulation of Mature Cystic Fibrosis Transmembrane Regulator Protein by the PDZ Domain Protein CAL
2004; Elsevier BV; Volume: 279; Issue: 3 Linguagem: Inglês
10.1074/jbc.m308640200
ISSN1083-351X
AutoresJie Cheng, Hua Wang, William B. Guggino,
Tópico(s)Genetic and Kidney Cyst Diseases
ResumoWe have previously identified the cystic fibrosis transmembrane regulator (CFTR)-interacting protein CAL and demonstrated that CAL modulates CFTR plasma membrane expression by retaining CFTR within the cell. Here, we report that in addition to regulating membrane expression, CAL also regulates the expression of mature CFTR. The co-expression of hemagglutinin-tagged or Myc-tagged CAL with green fluorescent protein (GFP)-CFTR in COS-7 cells causes a dose-dependent reduction in mature GFP-CFTR, independent of its tags. Bafilomycin A1, a lysosomal proton pump inhibitor, increases mature GFP-CFTR, confirming previous reports of lysosomal degradation of mature CFTR. Importantly, bafilomycin A1 reverses CAL-mediated CFTR degradation. The proteasome inhibitor, MG132, on the other hand, does not reverse the effect of CAL. CAL has no effect on CFTR maturation, suggesting that it exerts its effects on mature CFTR. Co-expression of CAL enhances the degradation of CFTR. We showed previously that CAL reduces the half-life of CFTR at the cell surface. Here we show that expression of dominant-negative dynamin 2 K44A, a large GTPase inhibitor that is known to inhibit clathrin-mediated endocytosis and vesicle formation in the Golgi, increases cell surface CFTR as measured by surface biotinylation. More importantly, dynamin 2 K44A also restores cell surface CFTR in CAL-overexpressing cells and partially blocks the CAL-mediated degradation of mature CFTR. These data suggest a model in which CAL retains CFTR in the cell and targets CFTR for degradation. We have previously identified the cystic fibrosis transmembrane regulator (CFTR)-interacting protein CAL and demonstrated that CAL modulates CFTR plasma membrane expression by retaining CFTR within the cell. Here, we report that in addition to regulating membrane expression, CAL also regulates the expression of mature CFTR. The co-expression of hemagglutinin-tagged or Myc-tagged CAL with green fluorescent protein (GFP)-CFTR in COS-7 cells causes a dose-dependent reduction in mature GFP-CFTR, independent of its tags. Bafilomycin A1, a lysosomal proton pump inhibitor, increases mature GFP-CFTR, confirming previous reports of lysosomal degradation of mature CFTR. Importantly, bafilomycin A1 reverses CAL-mediated CFTR degradation. The proteasome inhibitor, MG132, on the other hand, does not reverse the effect of CAL. CAL has no effect on CFTR maturation, suggesting that it exerts its effects on mature CFTR. Co-expression of CAL enhances the degradation of CFTR. We showed previously that CAL reduces the half-life of CFTR at the cell surface. Here we show that expression of dominant-negative dynamin 2 K44A, a large GTPase inhibitor that is known to inhibit clathrin-mediated endocytosis and vesicle formation in the Golgi, increases cell surface CFTR as measured by surface biotinylation. More importantly, dynamin 2 K44A also restores cell surface CFTR in CAL-overexpressing cells and partially blocks the CAL-mediated degradation of mature CFTR. These data suggest a model in which CAL retains CFTR in the cell and targets CFTR for degradation. Cystic fibrosis is a common lethal genetic disease caused by autosomal recessive mutations of cystic fibrosis transmembrane regulator (CFTR) 1The abbreviations used are: CFTR, cystic fibrosis transmembrane regulator; NHE-RF, Na+/H+ exchanger regulatory factor; Dyn2, dynamin 2; GFP, green fluorescent protein; HA, hemagglutinin; ER, endoplasmic reticulum; DMEM, Dulbecco's modified Eagle's medium; TGN, trans-Golgi network; SNARE, soluble NSF attachment protein receptors.1The abbreviations used are: CFTR, cystic fibrosis transmembrane regulator; NHE-RF, Na+/H+ exchanger regulatory factor; Dyn2, dynamin 2; GFP, green fluorescent protein; HA, hemagglutinin; ER, endoplasmic reticulum; DMEM, Dulbecco's modified Eagle's medium; TGN, trans-Golgi network; SNARE, soluble NSF attachment protein receptors. (1.Kerem B. Rommens J.M. Buchanan J.A. Markiewicz D. Cox T.K. Chakravarti A. Buchwald M. Tsui L.C. Science. 1989; 245: 1073-1080Crossref PubMed Scopus (3161) Google Scholar, 2.Riordan J.R. 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Med. 2002; 70: 229-243PubMed Google Scholar). That which is folded efficiently traffics to the Golgi and acquires complex glycosylation characteristic of the mature CFTR. The most common mutation in CF, ΔF508CFTR, is a misfolded protein and is recognized as such by the ER, translocated to the cytosol, and degraded by the 26 S proteasome (9.Benharouga M. Sharma M. Lukacs G.L. Methods Mol. Med. 2002; 70: 229-243PubMed Google Scholar). Thus, the predominant majority of the ΔF508 never matures and traffics to the plasma membrane.The degradation of mature CFTR is less well understood. Recently, Benharouga et al. (10.Benharouga M. Haardt M. Kartner N. Lukacs G.L. J. Cell Biol. 2001; 153: 957-970Crossref PubMed Scopus (75) Google Scholar) found that both the lysosome and proteasome are involved. They found that lysosomal inhibitors increase the half-life of mature wild type CFTR in baby hamster kidney cells transfected with CFTR and in the endogenous CFTR in Caco-2 and T84 cells. Interestingly, C-terminal truncation mutants do not traffic to the lysosome. Instead, they are degraded in the proteasome (10.Benharouga M. Haardt M. Kartner N. Lukacs G.L. J. Cell Biol. 2001; 153: 957-970Crossref PubMed Scopus (75) Google Scholar). Thus, CFTR appears to enter different degradative pathways depending on signals within its C terminus.CFTR possesses a type I, C-terminal, PDZ (PSD-95/DLG/ZO-1)-binding motif. Several PDZ domain proteins known to interact with CFTR include NHE-RF/EBP-50, CAP70, and CAL (CFTR-associated ligand) (11.Cheng J. Moyer B.D. Milewski M. Loffing J. Ikeda M. Mickle J.E. Cutting G.R. Li M. Stanton B.A. Guggino W.B. J. Biol. Chem. 2002; 277: 3520-3529Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 12.Hall 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, 13.Short D.B. Trotter K.W. Reczek D. Kreda S.M. 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In addition, NHE-RF anchors CFTR to cytoskeleton stabilizing the cell surface CFTR (13.Short 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 (398) Google Scholar, 17.Swiatecka-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 (172) Google Scholar). Both NHE-RF and its related protein, E3KARP, link cAMP-dependent protein kinase to CFTR (13.Short 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 (398) Google Scholar, 18.Sun F. Hug M.J. Lewarchik C.M. Yun C.H. Bradbury N.A. Frizzell R.A. J. Biol. Chem. 2000; 275: 29539-29546Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). Moreover, NHE-RF was shown to link β2-adrenergic receptor to CFTR (19.Naren 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 (184) Google Scholar). Formation of such macromolecular complexes facilitates the efficiency of activation CFTR.CAL (also known as PIST (PDZ domain protein interacting specifically with TC10), GOPC (Golgi-associated PDZ and coiled-coil motif containing), and FIG (fused in glioblastoma)) (11.Cheng J. Moyer B.D. Milewski M. Loffing J. Ikeda M. Mickle J.E. Cutting G.R. Li M. Stanton B.A. Guggino W.B. J. Biol. Chem. 2002; 277: 3520-3529Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 20.Charest A. Lane K. McMahon K. Housman D.E. J. Biol. Chem. 2001; 276: 29456-29465Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 21.Yao R. Maeda T. Takada S. Noda T. Biochem. Biophys. Res. Commun. 2001; 286: 771-778Crossref PubMed Scopus (68) Google Scholar, 22.Neudauer C.L. Joberty G. Macara I.G. Biochem. Biophys. Res. Commun. 2001; 280: 541-547Crossref PubMed Scopus (72) Google Scholar) is a CFTR-interacting PDZ domain protein that associates predominantly with the Golgi apparatus. Overexpression of CAL reduces CFTR chloride currents in mammalian cells by decreasing the expression of CFTR in the plasma membrane (11.Cheng J. Moyer B.D. Milewski M. Loffing J. Ikeda M. Mickle J.E. Cutting G.R. Li M. Stanton B.A. Guggino W.B. J. Biol. Chem. 2002; 277: 3520-3529Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). This effect of CAL can be overcome by NHE-RF, which restores the plasma membrane expression of CFTR. CAL favors the retention of CFTR within the cell, whereas NHE-RF favors surface expression by competing with CAL for the binding of CFTR (11.Cheng J. Moyer B.D. Milewski M. Loffing J. Ikeda M. Mickle J.E. Cutting G.R. Li M. Stanton B.A. Guggino W.B. J. Biol. Chem. 2002; 277: 3520-3529Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). Interestingly, overexpression of CAL also reduces the amount of CFTR (23.Gentzsch M. Cui L. Mengos A. Chang X.B. Chen J.H. Riordan J.R. J. Biol. Chem. 2003; 278: 6440-6449Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). These findings point to a role for CAL in the intracellular trafficking of CFTR. Consistent with this notion, CAL interacts with proteins involved in membrane vesicle trafficking such as the SNARE protein, syntaxin 6, and the small GTPase, TC10 (20.Charest A. Lane K. McMahon K. Housman D.E. J. Biol. Chem. 2001; 276: 29456-29465Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 22.Neudauer C.L. Joberty G. Macara I.G. Biochem. Biophys. Res. Commun. 2001; 280: 541-547Crossref PubMed Scopus (72) Google Scholar). Furthermore, CAL also interacts with and potentially regulates the intracellular trafficking of the Wnt receptors, frizzled 5 and 8, neural members of the epidermal growth factor receptor family, CALEB/NGC (21.Yao R. Maeda T. Takada S. Noda T. Biochem. Biophys. Res. Commun. 2001; 286: 771-778Crossref PubMed Scopus (68) Google Scholar, 24.Hassel B. Schreff M. Stuebe E.M. Blaich U. Schumacher S. J. Biol. Chem. 2003; 278: 40136-40143Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar), and the chloride channel, CLC-3B (23.Gentzsch M. Cui L. Mengos A. Chang X.B. Chen J.H. Riordan J.R. J. Biol. Chem. 2003; 278: 6440-6449Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar).Recently, PDZ domain interactions were found to be involved in trafficking of other plasma membrane proteins both to and from the plasma membrane. For example, synapse-associated protein 102 is involved in the delivery of N-methyl-d-aspartate receptors to the cell surface (25.Sans N. Prybylowski K. Petralia R.S. Chang K. Wang Y.X. Racca C. Vicini S. Wenthold R.J. Nat. Cell Biol. 2003; 6: 520-530Crossref Scopus (258) Google Scholar). In addition, NHE-RF regulates β2-adrenergic receptor recycling to plasma membrane (26.Cao T.T. Deacon H.W. Reczek D. Bretscher A. von Zastrow M. Nature. 1999; 401: 286-290Crossref PubMed Scopus (562) Google Scholar).In addition to the PDZ domain, the C-tail of CFTR contains motifs for endocytosis. A tyrosine motif at the C terminus of CFTR binds directly with the μ light chain of the AP2 complex involved in sorting CFTR for endocytosis into clathrin-coated pits (27.Weixel K.M. Bradbury N.A. Pfleugers Arch. Eur. J. Physiol. 2001; 443: S70-S74Crossref PubMed Scopus (13) Google Scholar). Surface CFTR is endocytosed rapidly. For example, within 5 min, 50% CFTR at the plasma membrane is endocytosed via AP2 clathrin-coated pits. CFTR contained within endosomes can be either recycled back to the plasma membrane or degraded in the lysosome. Because surface CFTR has a long half-life (24–48h), the majority of CFTR must be recycled back to the cell surface instead of targeted to the lysosome for degradation. It is not clear how this process is regulated. In this study, we examined the role of CAL on the expression of mature CFTR protein. We found that CAL regulates the expression of mature CFTR through mechanisms that are both bafilomycin A1- and dynamin 2-sensitive.EXPERIMENTAL PROCEDURESCell Culture and Transfection—African green monkey kidney cells (COS-7) (obtained from American Type Tissue Culture) were maintained in DMEM, 20 mm l-glutamine, 100 unit/ml penicillin, 100 μg/ml streptomycin, and 10% fetal calf serum. Media and other components were purchased from (Invitrogen). The COS-7 cells were transfected using LipofectAMINE 2000 (Invitrogen) according to the manufacturer's instructions.Immunoblotting—The cells were harvested and processed as described previously (11.Cheng J. Moyer B.D. Milewski M. Loffing J. Ikeda M. Mickle J.E. Cutting G.R. Li M. Stanton B.A. Guggino W.B. J. Biol. Chem. 2002; 277: 3520-3529Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). Briefly, the cells were solubilized in lysis buffer (50 mm NaCl, 150 mm Tris-HCl, pH 7.4, 1% Nonidet P-40, and complete protease inhibitor; Roche Applied Sciences). The cell lysates were spun at 14,000 × g for 15 min at 4 °C to pellet insoluble material. The supernatants were subjected to SDS-PAGE and Western blotting followed by ECL (Amersham Biosciences). The chemiluminescence signal on the polyvinylidene difluoride membrane was directly captured by FujiFilm LAS-1000 plus system with 1,300,000-pixel cooled CCD camera that has 3.7-order of magnitude linearity. Quantification was carried out within the linear range using the Image Gauge version 3.2 software (FujiFilm). GFP-CFTR and Dyn2-GFP were detected with polyclonal GFP antibody (1:1000; BD Biosciences, Boston, MA). HA-CAL was detected with monoclonal HA antibody (1:2000; Roche Applied Sciences). Tubulin was detected with monoclonal tubulin antibody (1: 1000; Sigma).Surface Biotinylation—Surface biotinylation of CFTR at the plasma membrane was described previously (11.Cheng J. Moyer B.D. Milewski M. Loffing J. Ikeda M. Mickle J.E. Cutting G.R. Li M. Stanton B.A. Guggino W.B. J. Biol. Chem. 2002; 277: 3520-3529Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). Briefly, the cell surface proteins were labeled with cell-impermeable EZ-Link™ Sulfo-NHS-SS-Biotin (sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate; Pierce) at 4 °C for 15 min. The cell surface proteins were isolated by incubating with immobilized NeutrAvidin beads at 4 °C for 2 h (Pierce; catalog number 53151). The bound proteins were eluted with 2× Laemmli sample buffer supplemented with 100 mm dithiothreitol at 42 °C for 30 min. The eluted proteins were subjected to SDS-PAGE and Western blotting followed by ECL (Amersham Biosciences). GFP-CFTR was detected with polyclonal GFP antibody (1:1000; BD Biosciences).Pulse-Chase—COS-7 cells were washed twice in methionine/cysteine-free DMEM and then incubated for 30 min in methionine/cysteine free-DMEM. The cells were incubated in methionine/cysteine free DMEM containing TRAN-35S-label (250 μCi/ml) for 30 min. Subsequently, the cells were washed extensively with DMEM containing 10 mm of unlabeled methionine and 10 mm of unlabeled cysteine and chased in this solution for the indicated times. Radiolabeled CFTR was immunoprecipitated with M3A7 and L12B14 monoclonal antibodies (Upstate, Waltham, MA), separated on 7.5% SDS-PAGE, dried, and visualized by autoradiography. The radioactive signal on the dried SDS-PAGE was scanned with a FujiFilm BAS-100 system (FujiFilm). Quantification was carried out within the linear range using the Image Gauge version 3.2 software (FujiFilm).Confocal Microscopy—COS-7 cells were plated on glass coverslips 1 day before transfection. The cells were fixed in 4% paraformaldehyde and permeabilized in 0.2% Nonidet P-40 1 day post-transfection. Nonspecific binding sites were blocked with 5% normal goat serum. The cells were stained with anti-HA monoclonal antibody (1:2000; Roche Applied Sciences) in 5% normal goat serum, washed with 1% bovine serum albumin, and incubated with goat anti-mouse Cy3 secondary antibodies in 1% normal goat serum (1:200; Jackson ImmunoResearch, West Grove, PA). The GFP signal was detected by its fluorescence. The specimens were mounted and viewed on the UltraVIEW spinning disk confocal microscope (PerkinElmer Life Sciences).Calculation of Degradation—The percentage of bafilomycin A1-sensitive CFTR degradation was calculated as follows: [CFTRbafilomycin A1-treated – CFTRuntreated]/CFTRbafilomycin A1-treated × 100%.Statistical Analysis—The data are presented as the means ± S.E. Statistical significance was determined by Student's t test. We assigned significance at p < 0.05.RESULTSIs Expression of Mature CFTR Protein Influenced by CAL?— Co-expression of CAL reduces the total cellular CFTR (23.Gentzsch M. Cui L. Mengos A. Chang X.B. Chen J.H. Riordan J.R. J. Biol. Chem. 2003; 278: 6440-6449Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). To confirm these observations, we co-transfected GFP-CFTR into COS-7 cells with different concentrations of either HA- or Myc-tagged CAL. GFP-CFTR was previously shown to have the single channel properties and intracellular trafficking patterns similar to that of wild type CFTR (28.Moyer B.D. Loffing J. Schwiebert E.M. Loffing-Cueni D. Halpin P.A. Karlson K.H. Ismailov I.I. Guggino W.B. Langford G.M. Stanton B.A. J. Biol. Chem. 1998; 273: 21759-21768Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Fig. 1 depicts the steady state levels of CFTR, HA-CAL, and tubulin, which is used to assess protein loading. Note that CFTR resolves into two bands that are well known to correspond to the upper mature C-band (fully glycosylated) and the lower immature B band (core glycosylated) of CFTR (29.Cheng S.H. Gregory R.J. Marshall J. Paul S. Souza D.W. White G.A. O'Riordan C.R. Smith A.E. Cell. 1990; 63: 827-834Abstract Full Text PDF PubMed Scopus (1407) Google Scholar). Note that expression of either type of tagged CAL construct significantly reduced steady state levels of the mature band of CFTR assayed at 48 h post-transfection. Consistent with previous results (23.Gentzsch M. Cui L. Mengos A. Chang X.B. Chen J.H. Riordan J.R. J. Biol. Chem. 2003; 278: 6440-6449Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar), the effects of CAL on mature CFTR were dose-dependent and independent of the epitope tags. Both results argue that CAL down-regulates CFTR mature protein in a specific fashion. For example, the abundance of tubulin, a cytoskeleton protein, was not affected by either HA-CAL (Fig. 1, A and C) or Myc-CAL (Fig. 1, B and C). Likewise, co-expression of HA-CAL or Myc-CAL did not affect the expression of GFP, a cytosolic protein, or GFP-frizzled 4, a membrane protein (data not shown). Neither GFP nor frizzled 4 binds to CAL (11.Cheng J. Moyer B.D. Milewski M. Loffing J. Ikeda M. Mickle J.E. Cutting G.R. Li M. Stanton B.A. Guggino W.B. J. Biol. Chem. 2002; 277: 3520-3529Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). All of the GFP-tagged proteins used have the same cytomegalovirus promoters, eliminating a nonspecific effect on promoter activity. In addition, NHE-RF has no effect on the abundance of mature CFTR (11.Cheng J. Moyer B.D. Milewski M. Loffing J. Ikeda M. Mickle J.E. Cutting G.R. Li M. Stanton B.A. Guggino W.B. J. Biol. Chem. 2002; 277: 3520-3529Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 23.Gentzsch M. Cui L. Mengos A. Chang X.B. Chen J.H. Riordan J.R. J. Biol. Chem. 2003; 278: 6440-6449Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). These data suggest that CAL does not down-regulate GFP-CFTR by reducing its transcription (see more on pulse-chase experiments later) or by acting on the GFP moiety of the fusion proteins.To verify these results further, the effect of CAL on CFTR was investigated in cells stably expressing a low level of CFTR. This cell line was created by transfecting COS-7 cells with GFP-CFTR, followed by selection and expansion of isolated G418-resistant colonies. One resultant clone, 4F2, was obtained that expressed one-tenth of the GFP-CFTR cells transiently transfected with 3 μg of GFP-CFTR/100-mm dish (data not shown). In these cells, HA-CAL also reduced the steady state levels of CFTR (Fig. 2C, third lane is 65.6 ± 6.8% of first lane; p < 0.05, n = 3).Fig. 2Bafilomycin A1 reverses CAL-mediated reduction of CFTR. A, COS-7 Cells were co-transfected with GFP-CFTR and HA-CAL. Either the lysosome inhibitor bafilomycin A1 (1 μm) or the proteasome inhibitor MG132 (10 μm) was added 24 h post-transfection. The cells were lysed, and the proteins were detected by Western blotting as described in the legend to Fig. 1. B, the percentage of bafilomycin A1-sensitive CFTR degradation was calculated as the portion of CFTR protected by bafilomycin A1 (see "Experimental Procedures"). The data are the means of three experiments. The error bars are standard deviations. *, p < 0.01 versus the control. C, the COS-7 cell line, 4F2, stably expressing a low amount of GFP-CFTR was transfected with HA-CAL (6 μg/dish). Bafilomycin A1 (1 μm) was added 24 h post-transfection. The cells were lysed, and the proteins were detected as described above. D, the percentage of bafilomycin A1-sensitive CFTR degradation in 4F2. The data are the means of three experiments. The error bars are the standard deviations. *, p < 0.01 versus the control.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Is the Degradation of CFTR Influenced by CAL?—To investigate the role of the lysosome and the proteasome in CAL-mediated down-regulation of CFTR, we examined the effect of their respective inhibitors. As shown in Fig. 2A, treatment with bafilomycin A1, a lysosomal proton pump inhibitor (30.Bowman E.J. Siebers A. Altendorf K. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7972-7976Crossref PubMed Scopus (1586) Google Scholar), increases steady state protein levels of CFTR. Our data combined with previous findings obtained with other lysosome blocking agents (10.Benharouga M. Haardt M. Kartner N. Lukacs G.L. J. Cell Biol. 2001; 153: 957-970Crossref PubMed Scopus (75) Google Scholar, 11.Cheng J. Moyer B.D. Milewski M. Loffing J. Ikeda M. Mickle J.E. Cutting G.R. Li M. Stanton B.A. Guggino W.B. J. Biol. Chem. 2002; 277: 3520-3529Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar) suggest that lysosomal degradation is an important process in regulating mature CFTR abundance. Important for this study is that bafilomycin A1 reverses CAL-mediated reduction in mature CFTR (Fig. 2A). Fig 2A (first and third lanes) shows the typical effect of CAL on mature CFTR. In the absence of bafilomycin A1, CAL-transfected cells have significantly less mature CFTR than in cells not transfected with CAL (Fig. 2A, third lane is 57.1 ± 4.2% of first lane; p < 0.05, n = 3). Importantly, after 24–48 h of bafilomycin A1 treatment, the effect of CAL is significantly reduced (Fig. 2A, fourth lane, is 75.7 ± 5.9% of second lane; p > 0.05, n = 3). The effect of bafilomycin is likely underestimated. Some CAL-induced reduction in mature CFTR was expected to take place prior to the addition of bafilomycin midway, at 24 h posttransfection. Thus, some CAL-induced reduction in mature CFTR was expected to take place prior to the addition of bafilomycin. Fig. 2B depicts the extent of CFTR bafilomycin A1 sensitivity (see "Experimental Procedures"). At 24–48 h posttransfection, ∼27.93 ± 2.85% of CFTR is bafilomycin-sensitive. In contrast, in cells transfected with 6 μg of HA-CAL/100-mm dish, ∼49.55 ± 7.62% of CFTR is bafilomycin-sensitive (p < 0.01). The reversal of CAL-mediated reduction of mature CFTR by bafilomycin A1 is also reproduced in the stable cell line 4F2 (Fig. 2C, fourth lane is 77.8 ± 9.6% of second lane; p > 0.05, n = 3; whereas Fig. 2C, third lane is 65.6 ± 6.8% of first lane, p < 0.05, n = 3). More importantly, transfection of HA-CAL leads to an increase in the extent of bafilomycin A1-sensitive degradation of CFTR (Fig. 2D). These results strongly suggested that CAL plays a key role in regulating the bafilomycin A1-sensitive degradation of CFTR. On the other hand, treating CAL-transfected cells with the proteasome inhibitor MG132 did not rescue mature GFP-CFTR (Fig. 2A) back to the control levels observed in the absence of CAL. In contrast to bafilomycin A1 treatment, in the presence of CAL, MG132 actually reduced levels of mature CFTR. It has been observed by several investigators that proteasome inhibition does not increase the maturation of CFTR (31.Jensen T.J. Loo M.A. Pind S. Williams D.B. Goldberg A.L. Riordan J.R. Cell. 1995; 83: 129-135Abstract Full Text PDF PubMed Scopus (767) Google Scholar, 32.Ward C.L. Omura S. Kopito R.R. Cell. 1995; 83: 121-127Abstract Full Text PDF PubMed Scopus (1127) Google Scholar). MG132 did not increase immature CFTR in the Nonidet P-40 lysates probably because blocking of proteosomal degradation leads to the accumulation of CFTR into a detergent-insoluble polyubiquitinated form of immature CFTR (32.Ward C.L. Omura S. Kopito R.R. Cell. 1995; 83: 121-127Abstract Full Text PDF PubMed Scopus (1127) Google Scholar). The failure of MG132 to inhibit CAL reduction of mature CFTR is further evidence that CAL can function downstream of the proteosome and influence the degradation of mature CFTR.What Is the Effect of CAL on CFTR Maturation?—The steady state level of mature CFTR is determined by new protein synthesis and the trafficking and maturation of CFTR from ER to Golgi as well as post-Golgi degradation. To eliminate the possibility that, in addition to enhancing the degradation of mature CFTR in the lysosome, CAL decreases CFTR synthesis and/or maturation, we examined the synthesis and maturation of CFTR by metabolic labeling and pulse-chase experiments. In Fig. 3A, COS-7 cells transfected with GFP-CFTR with or without HA-CAL were metabolically pulse-labeled with [35S]methionine and cysteine for 30 min at 16 h post-transfection. The cells were then chased in medium supplemented with unlabeled methionine and cysteine for 1–4 h to follow the maturation of CFTR. The lower band (Fig. 3A, band B) is the newly synthesized CFTR in ER that appears first. Not all newly synthesized CFTR is converted to mature CFTR (band C, upper band); because CFTR folds inefficiently, most of band B is degraded by ER-associated degradation (8.Kopito R.R. Physiol. Rev. 1999; 79: S167-S173Crossref PubMed Scopus (373) Google Scholar). Fig. 3 shows that GFP-CFTR matures similarly whether transfected or not transfected with CAL. Therefore, CAL exerts its effects on mature CFTR and does not affect the synthesis and trafficking to Golgi. To te
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