Heat Shock Protein 70 Interacts with Aquaporin-2 and Regulates Its Trafficking
2007; Elsevier BV; Volume: 282; Issue: 39 Linguagem: Inglês
10.1074/jbc.m611101200
ISSN1083-351X
AutoresHua Lu, Tian‐Xiao Sun, Toshiyuki Matsuzaki, Xianhua Yi, Jairam R. Eswara, Richard Bouley, Mary McKee, Dennis Brown,
Tópico(s)Genetics, Aging, and Longevity in Model Organisms
ResumoThe trafficking of aquaporin-2 (AQP2) involves multiple complex pathways, including regulated, cAMP-, and cGMP-mediated pathways, as well as a constitutive recycling pathway. Although several accessory proteins have been indirectly implicated in AQP2 recycling, the direct protein-protein interactions that regulate this process remain largely unknown. Using yeast two-hybrid screening of a human kidney cDNA library, we have identified the 70-kDa heat shock proteins as AQP2-interacting proteins. Interaction was confirmed by mass spectrometry of proteins pulled down from rat kidney papilla extract using a GST-AQP2 C-terminal fusion protein (GST-A2C) as a bait, by co-immunoprecipitation (IP) assays, and by direct binding assays using purified hsc70 and the GST-A2C. The direct interaction of AQP2 with hsc70 is partially inhibited by ATP, and the Ser-256 residue in the AQP2 C terminus is important for this direct interaction. Vasopressin stimulation in cells enhances the interaction of hsc70 with AQP2 in IP assays, and vasopressin stimulation in vivo induces an increased co-localization of hsc70 and AQP2 on the apical membrane of principal cells in rat kidney collecting ducts. Functional knockdown of hsc70 activity in AQP2 expressing cells results in membrane accumulation of AQP2 and reduced endocytosis of rhodamine-transferrin. Our data also show that AQP2 interacts with hsp70 in multiple in vitro binding assays. Finally, in addition to hsc70 and hsp70, AQP2 interacts with several other key components of the endocytotic machinery in co-IP assays, including clathrin, dynamin, and AP2. To summarize, we have identified the 70-kDa heat shock proteins as a AQP2 interactors and have shown for hsc70 that this interaction is involved in AQP2 trafficking. The trafficking of aquaporin-2 (AQP2) involves multiple complex pathways, including regulated, cAMP-, and cGMP-mediated pathways, as well as a constitutive recycling pathway. Although several accessory proteins have been indirectly implicated in AQP2 recycling, the direct protein-protein interactions that regulate this process remain largely unknown. Using yeast two-hybrid screening of a human kidney cDNA library, we have identified the 70-kDa heat shock proteins as AQP2-interacting proteins. Interaction was confirmed by mass spectrometry of proteins pulled down from rat kidney papilla extract using a GST-AQP2 C-terminal fusion protein (GST-A2C) as a bait, by co-immunoprecipitation (IP) assays, and by direct binding assays using purified hsc70 and the GST-A2C. The direct interaction of AQP2 with hsc70 is partially inhibited by ATP, and the Ser-256 residue in the AQP2 C terminus is important for this direct interaction. Vasopressin stimulation in cells enhances the interaction of hsc70 with AQP2 in IP assays, and vasopressin stimulation in vivo induces an increased co-localization of hsc70 and AQP2 on the apical membrane of principal cells in rat kidney collecting ducts. Functional knockdown of hsc70 activity in AQP2 expressing cells results in membrane accumulation of AQP2 and reduced endocytosis of rhodamine-transferrin. Our data also show that AQP2 interacts with hsp70 in multiple in vitro binding assays. Finally, in addition to hsc70 and hsp70, AQP2 interacts with several other key components of the endocytotic machinery in co-IP assays, including clathrin, dynamin, and AP2. To summarize, we have identified the 70-kDa heat shock proteins as a AQP2 interactors and have shown for hsc70 that this interaction is involved in AQP2 trafficking. Aquaporin 2 is expressed in principal cells of the kidney collecting duct and mediates water absorption and urinary concentration in response to vasopressin (VP). 4The abbreviations used are: VPvasopressinAQP2aquaporin-2GSTglutathione S-transferaseco-IPco-immunoprecipitationPBSphosphate-buffered salineNBDnucleotide binding domainWTwild type. 4The abbreviations used are: VPvasopressinAQP2aquaporin-2GSTglutathione S-transferaseco-IPco-immunoprecipitationPBSphosphate-buffered salineNBDnucleotide binding domainWTwild type. Upon stimulation by VP, AQP2 accumulates on the plasma membrane of collecting duct principal cells (1Brown D. Am. J. Physiol. 2003; 284: F893-F901Crossref PubMed Scopus (219) Google Scholar, 2Knepper M.A. Wade J.B. Terris J. Ecelbarger C.A. Marples D. Mandon B. Chou C.L. Kishore B.K. Nielsen S. Kidney Int. 1996; 49: 1712-1717Abstract Full Text PDF PubMed Scopus (151) Google Scholar, 3Nielsen S. Kwon T.H. Christensen B.M. Promeneur D. Frokiaer J. Marples D. J. Am Soc. 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Many of the cytosolic proteins known to be involved in vesicular trafficking in general have also been implicated in AQP2 trafficking, including cytoskeletal components such as actin filaments, myosins, and microtubules, SNARE proteins, Rab proteins, and heterotrimeric G proteins, as well as protein kinases and their associated proteins (1Brown D. Am. J. Physiol. 2003; 284: F893-F901Crossref PubMed Scopus (219) Google Scholar, 11Klussmann E. Maric K. Rosenthal W. Rev. Physiol. Biochem. Pharmacol. 2000; 141: 33-95Crossref PubMed Google Scholar, 12Nielsen S. Marples D. Birn H. Mohtashami M. Dalby N.O. Trimble M. Knepper M. J. Clin. Investig. 1995; 96: 1834-1844Crossref PubMed Scopus (140) Google Scholar, 13Valenti G. Procino G. Tamma G. Carmosino M. Svelto M. Endocrinology. 2005; 146: 5063-5070Crossref PubMed Scopus (148) Google Scholar, 14Marples D. Schroer T.A. Ahrens N. Taylor A. Knepper M.A. Nielsen S. Am. J. Physiol. 1998; 274: F384-F394Crossref PubMed Google Scholar, 15Inoue T. 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Microarray analysis of the mouse kidney after water deprivation reveals an up-regulation of hsp70 expression, and a more recent proteomic analysis of the inner medullary collecting duct of Brattleboro rats after vasopressin treatment revealed that the level of heat shock protein 70 is increased in response to [deamino-Cys, d-Arg8] vasopressin (34van Balkom B.W. Hoffert J.D. Chou C.L. Knepper M.A. Am. J. Physiol. 2004; 286: F216-F224Crossref PubMed Scopus (29) Google Scholar, 35van Balkom B.W. van Raak M. Breton S. Pastor-Soler N. Bouley R. van der Sluijs P. Brown D. Deen P.M. J. Biol. Chem. 2003; 278: 1101-1107Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). All these data and the emerging appreciation of the role hsc70 in mediating channel trafficking raised the possibility that the 70-kDa heat shock proteins may also play a role in mediating AQP2 recycling. In this study, we use convergent techniques to show that both hsc70 and hsp70 interact with AQP2 directly and that the interaction with hsc70 is important for AQP2 trafficking/recycling. Chemicals and Antibodies—Lysine VP was obtained from Sigma. Tetramethylrhodamine transferrin conjugate was supplied by Molecular Probes (Eugene, OR). GST, glutathione S-transferase-agarose, and protein A-Sepharose were obtained from Amersham Biosciences. Purified, biotinylated hsc70 and purified hsp70 were purchased from StressGen (Victoria, British Columbia, Canada). Purified hsp70 was biotinylated using a biotinylation kit from Pierce. Anti-c-Myc monoclonal antibody was obtained from the supernatant of the 9E10 hybridoma cell line, which was purchased from the ATCC. Polyclonal antibodies against clathrin heavy chain, AP2, and dynamin 2 were kindly provided by Dr. Sanja Sever (36Sever S. Damke H. Schmid S.L. J. Cell Biol. 2000; 150: 1137-1148Crossref PubMed Scopus (194) Google Scholar). Our affinity-purified rabbit polyclonal antibody against the AQP2 C terminus has been described previously (37Sabolic I. Katsura T. Verbavatz J.M. Brown D. J. Membr. Biol. 1995; 143: 165-175Crossref PubMed Scopus (214) Google Scholar). Antibodies against hsc70 (rat monoclonal, SPA815) and hsp70 (mouse monoclonal, SPA 810, and rabbit polyclonal, SPA 812) were purchased from StressGen. Secondary fluorescein isothiocyanate or CY3-conjugated antibodies were purchased from Jackson ImmunoResearch (West Grove, PA). Secondary horseradish peroxidase-conjugated antibodies were purchased from Santa Cruz Biotechnology. Stable Cell Lines, DNA Constructs, and Recombinant Adenovirus—The generation of LLC-PK1 cell lines stably expressing c-Myc-tagged wild type AQP2, referred to as W2 in Figs. 3 and 4 and LLC-AQP2 in the remainder of the text and figures, has been described previously (7Katsura T. Verbavatz J.M. Farinas J. Ma T. Ausiello D.A. Verkman A.S. Brown D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7212-7216Crossref PubMed Scopus (170) Google Scholar, 38Katsura T. Gustafson C.E. Ausiello D.A. Brown D. Am. J. Physiol. 1997; 272: F817-F822Crossref PubMed Google Scholar). All cell lines were routinely maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37 °C with 5% CO2.FIGURE 4AQP2 co-immunoprecipitates with hsc70 and hsp70. Panel A co-IP of AQP2 and hsc70 from AQP2 stably transfected LLC-AQP2 (W2) cells using an antibody against AQP2. Both cell lysate and kidney papilla extract were pre-cleared by protein A beads prior to co-IP experiments. hsc70 is detected in the co-IP complex pulled down with the anti-AQP2 antibody; AQP2 was also detected using anti-AQP2 antibody as expected (lower panel). Panel B shows co-IP of hsc70 and AQP2 using anti-hsc70 antibody. Both panels A and B show that antibodies against AQP2 or hsc70 are able to co-IP their binding counterparts from cell lysates, and this two way co-IP assay suggests an interaction between AQP2 and hsc70. Panel C shows that AQP2 is not only able to co-IP with hsc70 but also co-IP with hsp70 from rat kidney (Kid) papilla extract. Antibodies against hsc70 and hsp70 were used in this co-IP experiment. The molecular weight of AQP2 from LLC-AQP2 (W2) cells is greater than that from kidney papilla extract because of the presence of c-Myc tag in AQP2-transfected cells. Arrows indicate proteins being detected. WB, Western blot.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The rat aquaporin-2 coding sequence was cloned into a GAL4:DBD vector pAS2-1 (Clontech) as a bait construct for yeast two-hybrid screening. This bait protein was engineered as a single fusion peptide consisting of GAL4:DBD, plus the sequence coding for amino acids Met1-Glu16 from the N terminus of AQP2, Ala147-Ala161 from the second intracellular loop of AQP2, and Asn220-Ala271 from the C-tail of AQP2. The coding sequence of this C-terminal chimera construct was also subcloned into the bacterial expression vector pET41a (Novagen) as a fusion protein with a GST tag on its N terminus (39Bouley R. Breton S. Sun T. McLaughlin M. Nsumu N.N. Lin H.Y. Ausiello D.A. Brown D. J. Clin. Investig. 2000; 106: 1115-1126Crossref PubMed Scopus (189) Google Scholar). All the constructs were sequenced to confirm their reading frame and predicted composition. The GAL4 AD:human kidney cDNA library constructed in the yeast plasmid pACT2 was purchased from Clontech. Recombinant adenoviruses expressing wild type hsc70 (Ad-hsc70), an ATPase-deficient hsc70 mutant (Ad-hscT204V), and a GTPase-deficient dynamin mutant (Ad-Dyn/K44A) were kindly provided by Dr. Sandra Schmidt (27Newmyer S.L. Schmid S.L. J. Cell Biol. 2001; 152: 607-620Crossref PubMed Scopus (129) Google Scholar). Details of the infection of epithelial cells with adenovirus were described in our previous publication (6Sun T.X. Van Hoek A. Huang Y. Bouley R. McLaughlin M. Brown D. Am. J. Physiol. 2002; 282: F998-F1011Crossref PubMed Scopus (101) Google Scholar). Briefly, recombinant adenovirus of ∼5 multiplicity of infection was used to infect LLC-AQP2 stably transfected cells. After incubation for 36 h, cells were harvested and used for immunochemistry or endocytosis assays with rhodamine-transferrin. Yeast Two-hybrid Screening and Yeast Two-hybrid Assay—The yeast two-hybrid screening was performed in AH109 Saccharomyces cerevisiae (Clontech) that contains three reporters, ADE2, HIS3, and MEL1. The BD-expressing plasmid contains TRP1, and the AD-expressing plasmid contains LEU2. The medium stringency selection medium lacks tryptophan, leucine, and histidine; the high stringency selection medium lacks tryptophan, leucine, histidine, and adenine but contains X-β-gal: 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (20 mg/ml). 3-Aminotriazole (2.5 mm) was added to both medium and high stringency selection media to inhibit HIS3 leakiness. The AH109 containing the pAS2-1/ALCT plasmid was transformed with 0.2 mg of AD:cDNA plasmid library using a modified lithium acetate method. The transformed cells were plated onto medium stringency selection medium and incubated at 30 °C for 14 days. Positive colonies were patched onto high stringency selection medium plates and incubated at 30 °C until sufficient growth was achieved and colonies turned blue. Colonies that activated all of the reporter genes in the AH109 stain were further analyzed. The AD:cDNA plasmid encoding the interacting protein was isolated from yeast cells and transformed to KC8 Escherichia coli. The colonies containing AD:cDNA plasmid were rescued on M9/-Leu selection medium plates, and the plasmid DNA was isolated and sent for sequencing. Yeast two-hybrid assays were performed to further confirm the positive interaction of pAS2-1/ALCT and AD:cDNA plasmids as follows. AD:cDNA plasmids isolated from the primary screen were used to co-transform AH109 with pAS2-1/ALCT to confirm activation of reporters. The potential interacting clones were also co-transformed to AH109 with BD vector alone to test for false positivity. Transformed cells were plated on high stringency selection medium. AD:cDNA clones that were positive with pAS2-1/ALCT but negative with pAS2-1 were further characterized by DNA sequencing. Preparation of Cell Culture Lysates and Rat Kidney Papilla Extract—Cells were grown in 10-cm culture dishes in complete Dulbecco's modified Eagle's medium (with 10% fetal bovine serum) to 95% confluence. After various treatments, cells were washed three times with cold PBS and were scraped into lysis buffer (PBS buffer, pH 7.4, NaF 20 mm, Na3VO4 2mm, 0.5% Nonidet P-40, and 0.1%Triton X-100, and a mixture of protease inhibitors (Roche Applied Science)). Cell lysates were incubated on ice for 20 min and then passed 5-10 times through a 27-gauge syringe and centrifuged at 14,000 × g for 5 min at 4 °C to remove cell debris. The supernatants were used for pull down and co-immunoprecipitation assays. Kidney papilla extract was prepared from Sprague-Dawley rat kidney. The papillae from two kidneys were washed three times with cold PBS and homogenized in a Teflon pestle homogenizer (Thomas Scientific). After passing through a syringe with a 25-gauge needle, the homogenates were resuspended in cold lysis buffer containing PBS, pH 7.4, 20 mm NaF, 2mm Na3VO4, 0.5% Nonidet P-40, 0.1% Triton X-100, and protease inhibitors. This rat kidney papilla extract was then used for pulldown and co-IP experiments. Expression and Purification of GST AQP2 Fusion Protein (GST-A2C)—Escherichia coli, BL21 (DE3) (Novagen), was used for GST fusion protein expression. DNA constructs encoding various GST-AQP2 C-terminal fusion proteins were transformed into bacteria using electroporation. Expression of GST-A2C in E. coli was induced by isopropyl 1-thio-β-d-galactopyranoside (1 mm) at 37 °C for 4 h. Bacteria were lysed by lysozyme (1 mg/ml) and 0.5% Nonidet P-40 in STE buffer (150 mm NaCl, 50 mm Tris-HCl, pH 7.4, EDTA 1 mm). Bacterial supernatant was allowed to bind to the glutathione-agarose column at 4 °C for 40 min with gentle rocking. After washing twice with STE buffer with 500 mm NaCl, 0.5% Nonidet P-40, and 0.1% Triton X-100 and three times with normal STE buffer, the GST fusion protein was eluted with elution buffer (5 mm reduced glutathione, 50 mm Tris-HCl, pH 8.0) and further dialyzed against PBS buffer at 4 °C. Purified GST and GST-AQP2 fusion proteins were subjected to SDS-PAGE and immunoblot analysis. Pulldown Experiments—Cell lysates and papilla extracts were pre-cleared on a glutathione column at 4 °C for 40 min. Approximately 20 μg of the GST or GST-A2C protein was incubated with 40 μl of glutathione beads and 500 μg to 1mg of pre-cleared cell lysate or rat kidney papilla extract at 4 °C with gentle rocking for 2 h. Then the glutathione beads were washed four times with PBS containing Nonidet P-40 (0.5%), Triton X-100 (0.5%), NaF (20 mm), and protease inhibitors to remove nonspecifically bound proteins. Finally, the pulled down material was analyzed by SDS-PAGE and immunoblot analysis. For mass spectrometry, the pulled down proteins from rat kidney papilla extracts were further processed with thrombin digestion (Novagen) and gel purification and finally sent for mass spectrometry analysis by MDS proteomics (Toronto, Canada). Meanwhile, purified GST-A2C fusion proteins were also used to pull down purified biotinylated hsc70 (StressGen) as mentioned above. Briefly, 20 μg of GST-A2C (wild type, S256A, and S256D constructs) was incubated with 40 μl of glutathione beads and 1 μg of biotinylated hsc70 at 4 °C for 2 h. After washing, the pulled down material was subjected to SDS-PAGE and Western blot analysis using horseradish peroxidase-conjugated streptavidin as a probe to detect the biotinylated hsc70 (Pierce). The immunoblots from five independent experiments were scanned, and mean signal intensity was calculated using IPLab Spectrum software. The percentages of the reduction of the ratio of biotinylation signal over AQP2 signal were calculated. Similar pulldown experiments were performed using purified wild type GST-A2C with purified, biotinylated hsc70 or hsp70, respectively, in the presence or absence of ADP (10 mm) or ATP (10 mm). Data were obtained from at least three independent experiments, analyzed using IPLab Spectrum software. As mentioned above, the percentages of the reduction of the signal intensity ratio of hsc70 (or hsp70) over AQP2 were calculated. Co-immunoprecipitation Procedure—Lysates from LLC-PK1, LLC-AQP2 (W2) cells, and rat kidney papilla extract were pre-cleared on protein A beads at 4 °C for 40 min prior to co-IP studies. Protein A beads (40 μl) were incubated with 0.5 μg of antibody and 500 μg of cell lysate or rat kidney papilla extract with gentle rocking for 2 h at 4 °C. After washing five times with washing buffer (PBS buffer, protease inhibitors, 0.5% Nonidet P-40, and 0.1% Triton X-100), the co-IP samples were subjected to SDS-PAGE and immunoblot analysis using a variety of antibodies. Cell lysates incubated with protein A beads alone were routinely used as a negative control. Co-IP using lysates from nontransfected cells was also used as a negative control in each experiment. Studies were repeated at least three times. Immunocytochemistry and Electron Microscopy of Tissue and Cultured Cells—Rat kidney was fixed by cardiac perfusion with paraformaldehyde lysine periodate as described previously (39Bouley R. Breton S. Sun T. McLaughlin M. Nsumu N.N. Lin H.Y. Ausiello D.A. Brown D. J. Clin. Investig. 2000; 106: 1115-1126Crossref PubMed Scopus (189) Google Scholar) with or without pre-exposure to [deamino-Cys, d-Arg8] vasopressin administered for 3 days via Alzet osmotic minipumps implanted subcutaneously in the nape of the neck, and delivering about 1.2 μg or DDAVP per day. Tissues were rinsed three times for 5 min in PBS, cryo-protected in 30% sucrose in PBS, frozen, and sectioned at 5 μm for immunostaining. The kidney sections were treated with 1% SDS for 4 min to improve antigenicity (40Brown D. Lydon J. McLaughlin M. Stuart-Tilley A. Tyszkowski R. Alper S. Histochem. Cell Biol. 1996; 105: 261-267Crossref PubMed Scopus (278) Google Scholar) and then blocked with 1% bovine serum albumin in PBS buffer before immunostaining. Primary antibodies against AQP2 (1:100) or hsc70 (or hsp70) (1:100) were added to kidney sections and incubated at room temperature for 90 min. After washing with PBS, fluorescein isothiocyanate- and CY3-conjugated secondary antibodies were applied for 60 min at room temperature. After final washes with PBS, sections were mounted in Vectashield (Vector Laboratories, Burlingame CA) and examined by conventional immunofluorescence microscopy (Nikon Eclipse 800) and confocal microscopy (Bio-Rad Radiance 2000). LLC-PK1 or LLC-AQP2 cells were seeded onto coverslips and treated as described below. Cells grown on coverslips were fixed with 4% paraformaldehyde in PBS for 20 min, washed three times for 5 min with PBS, permeabilized with 1% Triton X-100 for 4 min, and then subjected to routine immunostaining for AQP2 or hsc70 as described previously (8Lu H. Sun T.X. Bouley R. Blackburn K. McLaughlin M. Brown D. Am. J. Physiol. 2004; 286: F233-F243Crossref PubMed Scopus (113) Google Scholar). An estimate of translocation of AQP2 to the plasma membrane was obtained by quantitative analysis of AQP2 labeling in three independent experiments from cells infected with empty adenovirus (control), and cells infected with adenovirus expressing wild type hsc70, ATPase-deficient hsc70 mutant, and the GTPase-deficient K44A dynamin mutant. The percentage area occupied by AQP2 staining in a region of interest of the cytoplasm in each of at least 20 cells per experimental condition was quantified using IPLab Spectrum software, as described previously (39Bouley R. Breton S. Sun T. McLaughlin M. Nsumu N.N. Lin H.Y. Ausiello D.A. Brown D. J. Clin. Investig. 2000; 106: 1115-1126Crossref PubMed Scopus (189) Google Scholar), and was compared with the control cells. The loss of cytoplasmic staining was shown previously to parallel an increase in membrane staining during the AQP2 trafficking process. For electron microscopy, kidneys were fixed by perfusion with paraformaldehyde lysine periodate. Small blocks of tissue were dehydrated through a
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