Polycystin-1 C-terminal Cleavage Is Modulated by Polycystin-2 Expression
2009; Elsevier BV; Volume: 284; Issue: 31 Linguagem: Inglês
10.1074/jbc.m109.017756
ISSN1083-351X
AutoresClaudia A. Bertuccio, Hannah C. Chapin, Yiqiang Cai, Kavita Mistry, Véronique Chauvet, Stefan Somlo, Michael J. Caplan,
Tópico(s)Genetic Syndromes and Imprinting
ResumoAutosomal dominant polycystic kidney disease is caused by mutations in the genes encoding polycystin-1 (PC-1) and polycystin-2 (PC-2). PC-1 cleavage releases its cytoplasmic C-terminal tail (CTT), which enters the nucleus. To determine whether PC-1 CTT cleavage is influenced by PC-2, a quantitative cleavage assay was utilized, in which the DNA binding and activation domains of Gal4 and VP16, respectively, were appended to PC-1 downstream of its CTT domain (PKDgalvp). Cells cotransfected with the resultant PKDgalvp fusion protein and PC-2 showed an increase in luciferase activity and in CTT expression, indicating that the C-terminal tail of PC-1 is cleaved and enters the nucleus. To assess whether CTT cleavage depends upon Ca2+ signaling, cells transfected with PKDgalvp alone or together with PC-2 were incubated with several agents that alter intracellular Ca2+ concentrations. PC-2 enhancement of luciferase activity was not altered by any of these treatments. Using a series of PC-2 C-terminal truncated mutations, we identified a portion of the PC-2 protein that is required to stimulate PC-1 CTT accumulation. These data demonstrate that release of the CTT from PC-1 is influenced and stabilized by PC-2. This effect is independent of Ca2+ but is regulated by sequences contained within the PC-2 C-terminal tail, suggesting a mechanism through which PC-1 and PC-2 may modulate a novel signaling pathway. Autosomal dominant polycystic kidney disease is caused by mutations in the genes encoding polycystin-1 (PC-1) and polycystin-2 (PC-2). PC-1 cleavage releases its cytoplasmic C-terminal tail (CTT), which enters the nucleus. To determine whether PC-1 CTT cleavage is influenced by PC-2, a quantitative cleavage assay was utilized, in which the DNA binding and activation domains of Gal4 and VP16, respectively, were appended to PC-1 downstream of its CTT domain (PKDgalvp). Cells cotransfected with the resultant PKDgalvp fusion protein and PC-2 showed an increase in luciferase activity and in CTT expression, indicating that the C-terminal tail of PC-1 is cleaved and enters the nucleus. To assess whether CTT cleavage depends upon Ca2+ signaling, cells transfected with PKDgalvp alone or together with PC-2 were incubated with several agents that alter intracellular Ca2+ concentrations. PC-2 enhancement of luciferase activity was not altered by any of these treatments. Using a series of PC-2 C-terminal truncated mutations, we identified a portion of the PC-2 protein that is required to stimulate PC-1 CTT accumulation. These data demonstrate that release of the CTT from PC-1 is influenced and stabilized by PC-2. This effect is independent of Ca2+ but is regulated by sequences contained within the PC-2 C-terminal tail, suggesting a mechanism through which PC-1 and PC-2 may modulate a novel signaling pathway. Autosomal dominant polycystic kidney disease (ADPKD) 2The abbreviations used are: ADPKDautosomal dominant polycystic kidney diseasePC-1polycystin-1PC-2polycystin-2CTTcytoplasmic C-terminal tailPKDgalvpPC-1-Gal4VP16 fusion proteinFFirefly luciferaseRRenilla luciferaseGd3+gadoliniumBAPTA-AM1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetate-acetoxymethyl esterTGthapsigarginYCB9anti-PC-2 polyclonal antibodyGPSG protein-coupled receptor proteolytic siteHAhemagglutininRLUrelative light unitα-MEMα-minimum Eagle's mediumERendoplasmic reticulumJNKc-Jun N-terminal kinaseCTFC-terminal fragmentPBSphosphate-buffered salineNTFN-terminal fragmentNSnot significant. is one of the most common genetic disorders, affecting 1 of every 1000 people (1Gabow P.A. N. Engl. J. Med. 1993; 329: 332-342Crossref PubMed Scopus (871) Google Scholar). It is characterized by the slow development of multiple bilateral cysts in the kidneys, resulting in progressive renal failure in 50% of patients by their 6th decade of life. ADPKD is a systemic disease with a number of extrarenal manifestations, including hepatic cysts, cardiac valvular anomalies, intracranial aneurysms, and colonic diverticulae (1Gabow P.A. N. Engl. J. Med. 1993; 329: 332-342Crossref PubMed Scopus (871) Google Scholar). More than 95% of cases of ADPKD are because of mutations in two recently identified genes, PKD1 and PKD2, which encode the polycystin-1 (PC-1) and polycystin-2 (PC-2) proteins, respectively (2Burn T.C. Connors T.D. Dackowski W.R. Petry L.R. Van Raay T.J. Millholland J.M. Venet M. Miller G. Hakim R.M. Landes G.M. KIinger K.W. Qian F. Onuchic L.F. Watnick T. Germino G.G. Doggett N.A. Hum. Mol. Genet. 1995; 4: 575-582Crossref PubMed Scopus (241) Google Scholar, 3Mochizuki T. Wu G. Hayashi T. Xenophontos S.L. Veldhuisen B. Saris J.J. Reynolds D.M. Cai Y. Gabow P.A. Pierides A. Kimberling W.J. Breuning M.H. Deltas C.C. Peters D.J. Somlo S. Science. 1996; 272: 1339-1342Crossref PubMed Scopus (1214) Google Scholar). autosomal dominant polycystic kidney disease polycystin-1 polycystin-2 cytoplasmic C-terminal tail PC-1-Gal4VP16 fusion protein Firefly luciferase Renilla luciferase gadolinium 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetate-acetoxymethyl ester thapsigargin anti-PC-2 polyclonal antibody G protein-coupled receptor proteolytic site hemagglutinin relative light unit α-minimum Eagle's medium endoplasmic reticulum c-Jun N-terminal kinase C-terminal fragment phosphate-buffered saline N-terminal fragment not significant. PC-1 is composed of a very large N-terminal extracellular region that incorporates a combination of functional motifs, followed by 11 transmembrane segments, and an ∼240-amino acid intracellular C terminus (4Hughes J. Ward C.J. Peral B. Aspinwall R. Clark K. San Millán J.L. Gamble V. Harris P.C. Nat. Genet. 1995; 10: 151-160Crossref PubMed Scopus (794) Google Scholar). PC-1 is believed to play a role in cell-cell or cell-matrix interactions, renal tubulogenesis, and intracellular signaling pathways (5Qian F. Boletta A. Bhunia A.K. Xu H. Liu L. Ahrabi A.K. Watnick T.J. Zhou F. Germino G.G. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 16981-16986Crossref PubMed Scopus (247) Google Scholar, 6Boletta A. Germino G.G. Trends Cell Biol. 2003; 13: 484-492Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 7Arnould T. Kim E. Tsiokas L. Jochimsen F. Grüning W. Chang J.D. Walz G. J. Biol. Chem. 1998; 273: 6013-6018Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 8Kim E. Arnould T. Sellin L.K. Benzing T. Fan M.J. Grüning W. Sokol S.Y. Drummond I. Walz G. J. Biol. Chem. 1999; 274: 4947-4953Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). Embedded within the N-terminal extracellular domain immediately proximal to the first transmembrane domain is a G protein-coupled receptor proteolytic site (GPS) (9Moy G.W. Mendoza L.M. Schulz J.R. Swanson W.J. Glabe C.G. Vacquier V.D. J. Cell Biol. 1996; 133: 809-817Crossref PubMed Scopus (220) Google Scholar). Qian et al. (5Qian F. Boletta A. Bhunia A.K. Xu H. Liu L. Ahrabi A.K. Watnick T.J. Zhou F. Germino G.G. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 16981-16986Crossref PubMed Scopus (247) Google Scholar) have demonstrated that PC-1 undergoes cleavage at the GPS domain after its synthesis in vivo, and a large proportion of the population of the ∼350-kDa N-terminal fragment generated through this cleavage remains tethered at the cell surface. The second fragment generated by this cleavage is a polypeptide of ∼150 kDa that is comprised of all of the transmembrane domains and the cytoplasmic tail (5Qian F. Boletta A. Bhunia A.K. Xu H. Liu L. Ahrabi A.K. Watnick T.J. Zhou F. Germino G.G. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 16981-16986Crossref PubMed Scopus (247) Google Scholar). Chauvet et al. (10Chauvet V. Tian X. Husson H. Grimm D.H. Wang T. Hiesberger T. Igarashi P. Bennett A.M. Ibraghimov-Beskrovnaya O. Somlo S. Caplan M.J. J. Clin. Invest. 2004; 114: 1433-1443Crossref PubMed Scopus (225) Google Scholar) have recently reported that the PC-1 protein is subject to an additional proteolytic cleavage. This proteolytic cleavage releases the polycystin-1 cytoplasmic C-terminal tail (CTT), which can enter the nucleus where it is able to regulate the expression of genes involved in cellular proliferation and differentiation. This cleavage appears to be stimulated, at least in part, by the absence of mechanical stimuli detected by the primary cilium (10Chauvet V. Tian X. Husson H. Grimm D.H. Wang T. Hiesberger T. Igarashi P. Bennett A.M. Ibraghimov-Beskrovnaya O. Somlo S. Caplan M.J. J. Clin. Invest. 2004; 114: 1433-1443Crossref PubMed Scopus (225) Google Scholar). Moreover, PC-1 is subjected to an additional cleavage that leads to the release of the C-terminal half of the cytoplasmic tail. This C-terminal half of the PC-1 tail interacts with the transcription factor STAT6 and the coactivator P100, and it stimulates STAT6-dependent gene expression (11Low S.H. Vasanth S. Larson C.H. Mukherjee S. Sharma N. Kinter M.T. Kane M.E. Obara T. Weimbs T. Dev. Cell. 2006; 10: 57-69Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). In summary, PC-1 undergoes several different cleavages, each of which generates fragments that may participate in specific signaling processes. Polycystin-2 is predicted to possess six membrane-spanning segments, with both the N and C termini exposed at the cytosolic surface of the membrane (3Mochizuki T. Wu G. Hayashi T. Xenophontos S.L. Veldhuisen B. Saris J.J. Reynolds D.M. Cai Y. Gabow P.A. Pierides A. Kimberling W.J. Breuning M.H. Deltas C.C. Peters D.J. Somlo S. Science. 1996; 272: 1339-1342Crossref PubMed Scopus (1214) Google Scholar, 12Hayashi T. Mochizuki T. Reynolds D.M. Wu G. Cai Y. Somlo S. Genomics. 1997; 44: 131-136Crossref PubMed Scopus (85) Google Scholar). The C-terminal tail of PC-2 contains a calcium-binding EF hand (3Mochizuki T. Wu G. Hayashi T. Xenophontos S.L. Veldhuisen B. Saris J.J. Reynolds D.M. Cai Y. Gabow P.A. Pierides A. Kimberling W.J. Breuning M.H. Deltas C.C. Peters D.J. Somlo S. Science. 1996; 272: 1339-1342Crossref PubMed Scopus (1214) Google Scholar, 13Li Q. Liu Y. Zhao W. Chen X.Z. FEBS Lett. 2002; 516: 270-278Crossref PubMed Scopus (41) Google Scholar) and several potential phosphorylation sites (3Mochizuki T. Wu G. Hayashi T. Xenophontos S.L. Veldhuisen B. Saris J.J. Reynolds D.M. Cai Y. Gabow P.A. Pierides A. Kimberling W.J. Breuning M.H. Deltas C.C. Peters D.J. Somlo S. Science. 1996; 272: 1339-1342Crossref PubMed Scopus (1214) Google Scholar). Polycystin-2 is predicted to form homomultimers as well as heteromultimers, particularly with PC-1 via coiled-coil domains that are predicted to reside in each of their cytoplasmic tails (14Qian F. Germino F.J. Cai Y. Zhang X. Somlo S. Germino G.G. Nat. Genet. 1997; 16: 179-183Crossref PubMed Scopus (566) Google Scholar, 15Tsiokas L. Kim E. Arnould T. Sukhatme V.P. Walz G. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 6965-6970Crossref PubMed Scopus (423) Google Scholar). These two proteins are thought to interact and to participate in common signaling pathways. Coexpression of PC-1 with PC-2 has been shown to promote the translocation of PC-2 to the plasma membrane, where PC-2 can function as a nonselective Ca2+-permeable cation channel (16Hanaoka K. Qian F. Boletta A. Bhunia A.K. Piontek K. Tsiokas L. Sukhatme V.P. Guggino W.B. Germino G.G. Nature. 2000; 408: 990-994Crossref PubMed Scopus (681) Google Scholar). It remains unclear, however, whether PC-1 is an obligate component of the channel complex or merely acts as a chaperone or anchoring partner. The mechanosensitive channel activity located on the primary cilia has been hypothesized to include the PC-1-PC-2 complex. It has been suggested that PC-1 senses flow and activates its binding partner PC-2, which then mediates Ca2+ entry into the cell (17Nauli S.M. Alenghat F.J. Luo Y. Williams E. Vassilev P. Li X. Elia A.E. Lu W. Brown E.M. Quinn S.J. Ingber D.E. Zhou J. Nat. Genet. 2003; 33: 129-137Crossref PubMed Scopus (1673) Google Scholar). Koulen et al. (18Koulen P. Cai Y. Geng L. Maeda Y. Nishimura S. Witzgall R. Ehrlich B.E. Somlo S. Nat. Cell Biol. 2002; 4: 191-197Crossref PubMed Scopus (577) Google Scholar) have demonstrated that PC-2 can also act as a Ca2+-activated Ca2+-release channel in the ER that is dependent upon the activation of the inositol trisphosphate receptor. This mechanism does not require coassembly with PC-1 (18Koulen P. Cai Y. Geng L. Maeda Y. Nishimura S. Witzgall R. Ehrlich B.E. Somlo S. Nat. Cell Biol. 2002; 4: 191-197Crossref PubMed Scopus (577) Google Scholar). We wondered whether PC-2 and the formation of the PC-1-PC-2 complex may regulate the PC-1 C-terminal tail cleavages. Toward this end, we took advantage of an assay that allows us to detect and quantify the cleavage and translocation of the PC-1 CTT to the nucleus. We demonstrate that the cleavage of polycystin-1 C-terminal tail is increased in the presence of polycystin-2. This mechanism, however, does not appear to require the coiled-coil domain interaction, and it is not modulated by Ca2+-signaling pathways. Full-length cDNA encoding mouse PC-1 was modified to include the N-terminal FLAG and C-terminal HA epitopes (19Grimm D.H. Cai Y. Chauvet V. Rajendran V. Zeltner R. Geng L. Avner E.D. Sweeney W. Somlo S. Caplan M.J. J. Biol. Chem. 2003; 278: 36786-36793Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar) and was subcloned into pcDNA3.1 (Invitrogen). The DNA sequence encoding the Gal4VP16 tag was PCR-amplified using CMV-Gal4VP16 (CMV-GV) as template, and BsiWI restriction enzyme sites were added at each end. Gal4VP16 was inserted at the BsiWI site located in the middle of one HA tag in the C terminus of PC-1, generating a PC-1-Gal4VP16 fusion protein (Fig. 1A, PKDgalvp). GL4.31[luc2P/GAL4UAS/Hygro] (F) (Promega, Madison, WI) was used as a Gal4 promoter-driven firefly luciferase reporter construct. This reporter vector contains five consensus binding sequences for the Gal4 DNA-binding protein upstream of an adenoviral promoter, followed by the firefly luciferase gene. In transient transfection experiments, a vector constitutively expressing Renilla luciferase (R), pRL-TK, was included as an internal control to normalize for transfection differences. pRL-TK and CMV-GV vectors were generous gifts from Dr. Yung-Feng Liao (Institute of Zoology, Academia Sinica, Taiwan). The p200galvp construct, corresponding to the last 200 amino acids of mouse PC-1 protein, was generated by PCR from PKDgalvp and subcloned into pcDNA 3.1. The generation of the human PC-2 construct has already been described (20Cai Y. Maeda Y. Cedzich A. Torres V.E. Wu G. Hayashi T. Mochizuki T. Park J.H. Witzgall R. Somlo S. J. Biol. Chem. 1999; 274: 28557-28565Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar). All PCR primer details are available upon request. The D511V mutant cDNA construct was made by introducing GTT (Val) to replace GAT (Asp) at codon 511 of PC-2 by site-directed mutagenesis in pcDNA3.1 (18Koulen P. Cai Y. Geng L. Maeda Y. Nishimura S. Witzgall R. Ehrlich B.E. Somlo S. Nat. Cell Biol. 2002; 4: 191-197Crossref PubMed Scopus (577) Google Scholar) (Fig. 1B) using the QuickChange kit (Stratagene, La Jolla, CA). In addition, we produced a PC-2 clone truncated after leucine 703 (L703X) by introduction of an influenza virus hemagglutinin protein epitope tag and an in-frame stop codon as has been described previously (20Cai Y. Maeda Y. Cedzich A. Torres V.E. Wu G. Hayashi T. Mochizuki T. Park J.H. Witzgall R. Somlo S. J. Biol. Chem. 1999; 274: 28557-28565Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar) (Fig. 1B). We also introduced two naturally occurring human PC-2 mutations, R742X and R872X (3Mochizuki T. Wu G. Hayashi T. Xenophontos S.L. Veldhuisen B. Saris J.J. Reynolds D.M. Cai Y. Gabow P.A. Pierides A. Kimberling W.J. Breuning M.H. Deltas C.C. Peters D.J. Somlo S. Science. 1996; 272: 1339-1342Crossref PubMed Scopus (1214) Google Scholar), into PC-2 by site-directed mutagenesis using the QuickChange kit. Additional truncation constructs were produced by introducing stop codons at lysine 859 (K859X), aspartate 847 (D847X), glutamate 787 (E787X), and alanine 753 (A753X) (Fig. 1B). These mutagenized clones were constructed in pcDNA3.1 and were confirmed by direct sequencing. Amino acid substitution point mutations in predicted phosphorylation sites of the C terminus of PC-2 were introduced by site-directed mutagenesis using the QuickChange kit (21Cai Y. Anyatonwu G. Okuhara D. Lee K.B. Yu Z. Onoe T. Mei C.L. Qian Q. Geng L. Wiztgall R. Ehrlich B.E. Somlo S. J. Biol. Chem. 2004; 279: 19987-19995Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Briefly, we substituted alanine at Thr721 (T721A) or Ser812 (S812A) as single point substitutions and in combinations of three (Ser801, Ser812, and Ser829, clone PC-2 B5; Fig. 1C) or four sites (Thr721, Ser801, Ser812, and Ser829, clone PC-2-B10; Fig. 1C). PC-2 clones were generated using PC-2 subcloned into the pcDNA3.1 vector. COS-7 cells were cultured in a 5% CO2, 95% air humidified incubator at 37 °C in α-MEM (Invitrogen) supplemented with 10% fetal bovine serum, 2 mml-glutamine, 50 units/ml penicillin, and 50 μg/ml streptomycin. COS-7 cells were transfected using Lipofectamine 2000 (Invitrogen). Briefly, COS-7 cells were plated onto 6-well tissue culture plates and grown to about 60–80% confluency prior to transfection. GL4.31[luc2P/GAL4UAS/Hygro] (1 μg), pRL-TK (0.2 μg), PKDgalvp (1 μg), and PC2 (6 μg) were mixed with 4 μl of Lipofectamine 2000. Transfection mixtures were added dropwise into cell culture medium and incubated at 37 °C for 40 h. The amount of DNA in each well was equalized by the addition of a control plasmid, pcDNA3.1, which was also used for the mock transfection. Transfected cells were harvested with PBS and lysed with 250 μl of 1× passive lysis buffer (Promega, Madison, WI). Cell lysates were subjected to luciferase assay using the dual luciferase assay reagent kit (Promega, Madison, WI). Luciferase signals were determined in a GloMaxTM 20/20 luminometer (Promega, Madison, WI). All the experiments were compared with parallel experiments in which we cotransfected the CMV-GV vector, firefly and Renilla luciferases in the absence of PKDgalvp. Luciferase values in these experiments did not change in the presence of PC-2, its mutants, or when we added any of the chemical compounds tested (data not shown). Several series of experiments were performed to assess the effects of a variety of bioactive compounds on the PC-1 cleavage assay. Clasto-lactacystin β-lactone (15 μm) was added to the media 16 h before luciferase assay. To assess the potential role of extracellular calcium, cells were incubated in calcium-free medium containing dialyzed calcium-free serum for 6 h prior to the luciferase assay. In addition, cells in calcium-containing medium were subjected to incubation with EGTA, gadolinium (Gd3+), A23187, ionomycin, [1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetate-acetoxymethyl ester] (BAPTA-AM), or thapsigargin at the indicated concentrations (Fig. 4) for 6 h before the luciferase assay. Extracellular ATP disodium salt, at the indicated concentration (Fig. 4), was added for 9 h prior to the luciferase assay. COS-7 cells from a 10-cm dish were lysed in 400 μl of cold lysis buffer containing non-ionic detergent (1% Nonidet P-40) and a mixture of protease inhibitors (Complete, Roche Applied Science). Cell lysates were exposed to brief probe sonication to thoroughly disrupt nuclei. The protein concentration of each sample was determined via a colorimetric protein concentration assay (Bio-Rad), and equal amounts of each sample were loaded and separated on an 8% SDS-polyacrylamide gel. The gel was then electrophoretically transferred to a nitrocellulose membrane (Bio-Rad), incubated in blocking buffer (150 mm NaCl, 20 mm Tris, 5% (w/v) powdered milk, 0.1% Tween) for 60 min, and then incubated with one of the following primary antibodies at 4 °C overnight: monoclonal anti-HA antibody (1:1000, Invitrogen), monoclonal anti-FLAG (1:3000, Sigma), or anti-PC-2 (YCB9) polyclonal antibody (20Cai Y. Maeda Y. Cedzich A. Torres V.E. Wu G. Hayashi T. Mochizuki T. Park J.H. Witzgall R. Somlo S. J. Biol. Chem. 1999; 274: 28557-28565Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar). Subsequently, primary antibody binding was detected with horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (1:10,000; Jackson ImmunoResearch Laboratories, West Grove, PA), and proteins were visualized with an enhanced chemiluminescence detection kit (ECL, Amersham Biosciences). The nuclear preparation protocol was modified from Lin et al. (22Lin S.Y. Makino K. Xia W. Matin A. Wen Y. Kwong K.Y. Bourguignon L. Hung M.C. Nat. Cell Biol. 2001; 3: 802-808Crossref PubMed Scopus (915) Google Scholar). COS-7 cells grown in 10-cm dishes were harvested in cold PBS, centrifuged for 5 min at 500 × g, and resuspended in hypotonic buffer (10 mm HEPES, 1.5 mm MgCl2, 10 mm KCl, and a standard mixture of protease inhibitors). Cells were homogenized in a tight-fitting Dounce homogenizer, chilled on ice for 10 min, and then shaken for 15 min with an Eppendorf mixer 5432. Nonidet P-40 was added (1% final concentration), and the preparations were shaken for an additional 15 min. The lysates were centrifuged at 1500 × g for 5 min. The resulting supernatant formed the non-nuclear fractions. The pellets (nuclear fractions) were washed in hypotonic buffer, resuspended in high salt buffer (20 mm HEPES, 420 mm NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, 25% glycerol, and protease inhibitors), shaken for 20 min, and finally centrifuged at 14,000 × g for 30 min. The pellets were sonicated and prepared for immunoblot analysis. All steps were performed at 4 °C. The protein concentration of each sample was determined using a Bio-Rad colorimetric protein concentration assay. COS-7 cells grown in 10-cm dishes were transfected as was described above. At 8 h after transfection, cell monolayers were washed twice with serum-free medium, after which the cultures were maintained in serum-free medium for 34 h. The culture medium was then collected, subjected to centrifugation to remove residual cells, and concentrated 200-fold by using a 100K Amicon membrane filter (Amicon-Ultra, Millipore, Tullagreen, Ireland). Each sample was stored at −80 °C. COS-7 cells were grown on glass coverslips, fixed in 2% paraformaldehyde dissolved in PBS, permeabilized in PBS, 0.3% Triton, 0.1% BSA blocked in GSDB (16% goat serum, 120 mm sodium phosphate, 0.3% Triton X-100, and 450 mm NaCl), and incubated with primary antibody at room temperature. In clasto-lactacystin β-lactone experiments, full-length PKDgalvp construct was detected with a polyclonal antibody against HA (1:200 dilution; Invitrogen) and a monoclonal antibody against FLAG (1:200 dilution; Sigma). Fluorescein isothiocyanate-conjugated anti-rabbit IgG (1:100 dilution; Sigma) and rhodamine-conjugated anti-mouse IgG (1:100 dilution; Chemicon International, Billerica, MA) were used as secondary antibodies. In experiments involving full-length PC-2 or its mutants, HA and FLAG epitope tags in PKDgalvp were detected with monoclonal antibodies against HA and FLAG, respectively (1:200; Sigma), and fluorescein isothiocyanate-conjugated anti-mouse IgG (1:100, Sigma) was used as the secondary antibody. PC-2 or PC-2 mutants were detected with rabbit polyclonal YCB9 (N terminus) anti-PC-2 antibody (1:10,000) (20Cai Y. Maeda Y. Cedzich A. Torres V.E. Wu G. Hayashi T. Mochizuki T. Park J.H. Witzgall R. Somlo S. J. Biol. Chem. 1999; 274: 28557-28565Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar) and detected with rhodamine-conjugated anti-rabbit IgG (1:100; Chemicon International, Billerica, MA). Nuclei were stained with Hoechst 33342 (Molecular Probes, Invitrogen). Coverslips were mounted in Vectashield (Vector Laboratories, Burlingame, CA) and were examined using either an Olympus BX51 epifluorescent microscope equipped with a ×40 objective and a Magnafire digital camera or a Leica confocal microscope, using a ×40 oil-immersion objective. To quantify the nuclear localization of PC-1, a line was traced following the major axis of each nucleus, and the mean intensity along this line was determined for either PKDgalvp-HA or PKDgalvp-FLAG and for Hoechst staining. No differences were observed in nuclear Hoechst staining in each of the different treatments. Thus, Hoechst staining intensity was used to normalize nuclear CTT signals measured in different studies. Results are expressed as means ± S.E. Differences between means were evaluated using Student's t test or analysis of variance as appropriate. Values of p < 0.05 were considered to be significant. The following reagents were obtained from commercial sources: clasto-lactacystin β-lactone (Calbiochem), BAPTA-AM (Calbiochem), thapsigargin (Calbiochem), ATP disodium salt (Sigma), ionomycin calcium salt (Sigma), calcimycin A23187 (Sigma), and SMEM medium (Invitrogen). All the other reagents were at least molecular biology grade and obtained from standard suppliers. The goal of this study was to define the nature of the mechanisms responsible for PC-1 CTT cleavage. Our strategy made use of a sensitive and quantitative reporter gene assay. We generated a fusion protein in which a chimera of Gal4 and VP16 was inserted into full-length PC-1 downstream of the intracellular CTT domain (PKDgalvp). Gal4 is a yeast transcription factor, and VP16 is the transcriptional activating domain of the viral VP16 protein. The PKDgalvp construct also has a FLAG epitope tag at its N terminus and two HA epitope tags at its C terminus, one on either side of Gal4VP16 (Fig. 1A). The resultant cDNA encoding PKDgalvp was cotransfected with a Gal4-driven firefly luciferase reporter gene in COS-7 cells. Proteolytic cleavage of the PKDgalvp fusion protein will release the CTT, which may then translocate to the nucleus and activate expression of a firefly luciferase reporter gene under the control of the Gal4 promoter. Therefore, luciferase expression is a function of PC-1 CTT cleavage, and luciferase activity could be measured using a quantitative assay. When the PKDgalvp protein was expressed, we observed the maximum luciferase activity after 36–42 h post-transfection. All data in Fig. 2 and in subsequent figures are normalized to the values obtained with triple transfection of Renilla luciferase + Gal4 promoter-driven firefly luciferase reporter construct + PKDgalvp, which is set to 100%. No increases in luciferase activity were observed in COS-7 cells transfected with R alone or with R + PKDgalvp or R + F (1.54 ± 0.45, 2.18 ± 0.72, and 2.56 ± 0.62% of the RLU/s value obtained with R + F + PKDgalvp, respectively; Fig. 2A, p, NS). Transfection with R + F + PKDgalvp produced a dramatic (∼100-fold) increase in luciferase activity (Fig. 2A). It should be noted that a very similar assay was developed and successfully employed to study the C-terminal tail cleavage of fibrocystin, the product of the gene mutated in autosomal recessive polycystic kidney disease. These investigators performed a control for nonspecific cleavage that made use of the human low density lipoprotein receptor, a single pass membrane protein, with Gal4-VP16 appended at its C terminus. They observed that this construct is not subjected to proteolysis, demonstrating the specificity of the assay (23Hiesberger T. Gourley E. Erickson A. Koulen P. Ward C.J. Masyuk T.V. Larusso N.F. Harris P.C. Igarashi P. J. Biol. Chem. 2006; 281: 34357-34364Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Chauvet et al. (10Chauvet V. Tian X. Husson H. Grimm D.H. Wang T. Hiesberger T. Igarashi P. Bennett A.M. Ibraghimov-Beskrovnaya O. Somlo S. Caplan M.J. J. Clin. Invest. 2004; 114: 1433-1443Crossref PubMed Scopus (225) Google Scholar) have demonstrated that PC-1 is subject to a proteolytic cleavage in response to alterations in mechanical stimuli, which releases the cytoplasmic CTT of the protein. The CTT can enter the nucleus, where it regulates the expression of genes involved in cellular proliferation and differentiation. It has also been shown that Siah-1, a protein that functions to mediate the ubiquitin-dependent degradation of target proteins, regulates the ubiquitination and degradation of PC-1 C-terminal fragments by the proteasome pathway (24Kim H. Jeong W. Ahn K. Ahn C. Kang S. J. Am. Soc. Nephrol. 2004; 15: 2042-2049Crossref PubMed Scopus (32) Google Scholar). To test whether the signaling capacity of the PC-1 CTT is decreased by proteasome degradation, we used COS-7 cells transiently transfected with PKDgalvp with or without the addition of clasto-lactacystin β-lactone, an irreversible inhibitor of the proteasome. In the presence of clasto-lactacystin β-lactone, luciferase activity increased significantly (∼4-fold) compared with R + F + PKDgalvp (438.22 ± 36.20% of the RLU/s value obtained with R + F + PKDgalvp, p < 0.001; Fig. 2A). Furthermore, Western blotting with an anti-HA epitope tag antibody that recognizes the C terminus of PKDgalvp revealed a band corresponding to the molecular weight of the CTT of PC-1 plus gal4/vp16 in total lysates when clasto-lactacystin β-lactone was added (Fig. 2B) but not in its absence. This band was also present when we performed Western blotting with the anti-HA antibody on nuclear fractions prepared from COS-7 cells transiently transfected with PKDgalvp and treated with clasto-lactacystin β-lactone (Fig. 2B). When an antibody against FLAG, which recognizes the tag located at the N terminus of the PKDgalvp protein, was used, a band corresponding to the size of the PC-1 N-terminal fragment (NTF) produced by autocatalytic cleavage at the GPS site was observed in cell lysates with or without clasto-lactacystin β-lactone. This band was not detected in the nuclear extract preparatio
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