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Inositol 1,4,5-Trisphosphate Receptor Ubiquitination Is Mediated by Mammalian Ubc7, a Component of the Endoplasmic Reticulum-associated Degradation Pathway, and Is Inhibited by Chelation of Intracellular Zn2+

2003; Elsevier BV; Volume: 278; Issue: 40 Linguagem: Inglês

10.1074/jbc.m305600200

ISSN

1083-351X

Autores

Jack M. Webster, Swati Tiwari, Allan M. Weissman, Richard J.H. Wojcikiewicz,

Tópico(s)

Autophagy in Disease and Therapy

Resumo

In response to activation of certain cell surface receptors, inositol 1,4,5-trisphosphate receptors (InsP3Rs), which are located in the endoplasmic reticulum, can be rapidly ubiquitinated and then degraded by the proteasome. Ubiquitination is mediated by the concerted action of ubiquitin-conjugating enzymes (Ubcs or E2s) and ubiquitin-protein ligases (E3s). In the present study we have examined the enzymology of ubiquitination of endogenous InsP3Rs in muscarinic agonist-stimulated SH-SY5Y human neuroblastoma cells, focusing our attention on two mammalian E2s, MmUbc6 and MmUbc7, that have been implicated in endoplasmic reticulum-associated degradation (ERAD) and are homologous to the yeast ERAD E2s, Ubc6p and Ubc7p. Analysis of SH-SY5Y cells stably expressing these enzymes and their dominant-negative mutants revealed that MmUbc7 mediates InsP3R ubiquitination and down-regulation, but that MmUbc6 does not. These data indicate that InsP3Rs are processed by a component of the ERAD pathway and suggest that MmUbc7 may be employed selectively to ubiquitinate proteins, like InsP3Rs, that are subject to regulated ERAD. Additional studies showed that the Zn2+ chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine blocked InsP3R ubiquitination, suggesting that a RING finger domain-containing E3 is also involved in this process. Finally, muscarinic agonist-induced InsP3R ubiquitination was seen in rat brain slices, indicating that the results obtained from SH-SY5Y cells reflect a physiological process. In response to activation of certain cell surface receptors, inositol 1,4,5-trisphosphate receptors (InsP3Rs), which are located in the endoplasmic reticulum, can be rapidly ubiquitinated and then degraded by the proteasome. Ubiquitination is mediated by the concerted action of ubiquitin-conjugating enzymes (Ubcs or E2s) and ubiquitin-protein ligases (E3s). In the present study we have examined the enzymology of ubiquitination of endogenous InsP3Rs in muscarinic agonist-stimulated SH-SY5Y human neuroblastoma cells, focusing our attention on two mammalian E2s, MmUbc6 and MmUbc7, that have been implicated in endoplasmic reticulum-associated degradation (ERAD) and are homologous to the yeast ERAD E2s, Ubc6p and Ubc7p. Analysis of SH-SY5Y cells stably expressing these enzymes and their dominant-negative mutants revealed that MmUbc7 mediates InsP3R ubiquitination and down-regulation, but that MmUbc6 does not. These data indicate that InsP3Rs are processed by a component of the ERAD pathway and suggest that MmUbc7 may be employed selectively to ubiquitinate proteins, like InsP3Rs, that are subject to regulated ERAD. Additional studies showed that the Zn2+ chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine blocked InsP3R ubiquitination, suggesting that a RING finger domain-containing E3 is also involved in this process. Finally, muscarinic agonist-induced InsP3R ubiquitination was seen in rat brain slices, indicating that the results obtained from SH-SY5Y cells reflect a physiological process. Inositol 1,4,5,-trisphosphate (InsP3) 1The abbreviations used are: InsP3, inositol 1,4,5-trisphosphate; ER, endoplasmic reticulum; InsP3R, inositol 1,4,5-trisphosphate receptor; [Ca2+]i, cytoplasmic free Ca2+ concentration; GPCR, G protein-coupled receptor; Ubc or E2, ubiquitin-conjugating enzyme; E3, ubiquitin-protein ligase; HECT-E3, ubiquitin-protein ligase containing a domain homologous to the E6-associated protein carboxyl terminus; RING-E3, RING finger domain-containing ubiquitin-protein ligase; U-box-E3, U-box domain-containing ubiquitin-protein ligase; ERAD, endoplasmic reticulum-associated degradation; HMGR, 3-hydroxy 3-methylglutarylcoenzyme A reductase; mamUbc6, mammalian homologue of yeast Ubc6p; mamUbc7, mammalian homologue of yeast Ubc7p; MmUbc6, murine homologue of yeast Ubc6p; MmUbc7, murine homologue of yeast Ubc7p; DN, dominant negative; HA, hemagglutinin; DTT, dithiothreitol; PBS, phosphate-buffered saline; TPEN, N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine; ALLN, N-acetyl-Leu-Leu-norleucinal; RERAD, regulated endoplasmic reticulum-associated degradation; GFP, green fluorescent protein. receptors are proteins that form tetrameric ion channels in endoplasmic reticulum (ER) membranes. Upon binding of the second messenger InsP3, the channels open and Ca2+ stored in the ER is released into the cytosol (1Patel S. Joseph S.K. Thomas A.P. Cell Calcium. 1999; 25: 247-264Crossref PubMed Scopus (373) Google Scholar, 2Berridge M.J. Lipp P. Bootman M.D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 11-21Crossref PubMed Scopus (4493) Google Scholar). In this way, InsP3 receptors (InsP3Rs) link cell-surface receptor-mediated InsP3 formation to increases in cytoplasmic free Ca2+ concentration ([Ca2+] i) (2Berridge M.J. Lipp P. Bootman M.D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 11-21Crossref PubMed Scopus (4493) Google Scholar). Upon activation of certain phosphoinositidase C-linked G protein-coupled receptors (GPCRs), InsP3Rs are rapidly degraded (3Wojcikiewicz R.J.H. Nahorski S.R. J. Biol. Chem. 1991; 266: 22234-22241Abstract Full Text PDF PubMed Google Scholar, 4Wojcikiewicz R.J.H. Furuichi T. Nakade S. Mikoshiba K. Nahorski S.R. J. Biol. 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Chem. 2003; 278: 940-947Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), in response to stimulation with muscarinic agonists, certain secretagogues (cholecystokinin, bombesin, and pituitary adenylate cyclase-activating polypeptide), certain peptide hormones (vasopressin and bradykinin), angiotensin II, and gonadotropin-releasing hormone, respectively. Persistent elevation of InsP3 concentration (4Wojcikiewicz R.J.H. Furuichi T. Nakade S. Mikoshiba K. Nahorski S.R. J. Biol. Chem. 1994; 269: 7963-7969Abstract Full Text PDF PubMed Google Scholar, 8Oberdorf J. Webster J.M. Zhu C.C. Luo S.G. Wojcikiewicz R.J.H. Biochem. J. 1999; 339: 453-461Crossref PubMed Scopus (74) Google Scholar, 10Willars G.B. Royall J.E. Nahorski S.R. El-Gehani F. Everest H. McArdle C.A. J. Biol. Chem. 2001; 276: 3123-3129Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 12Brind S. Swann K. Carroll J. Dev. Biol. 2000; 223: 251-265Crossref PubMed Scopus (111) Google Scholar) and InsP3 binding (7Zhu C.C. Furuichi T. Mikoshiba K. Wojcikiewicz R.J.H. J. Biol. Chem. 1999; 274: 3476-3484Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 11Wojcikiewicz R.J.H. Xu Q. Webster J.M. Alzayady K. Gao C. J. Biol. Chem. 2003; 278: 940-947Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 13Zhu C.C. Wojcikiewicz R.J.H. Biochem. J. 2000; 348: 551-556Crossref PubMed Scopus (32) Google Scholar) appear to be the signals that initiate InsP3R down-regulation, and this adaptive response allows chronically stimulated cells to reduce the sensitivity of their Ca2+ stores to InsP3 (3Wojcikiewicz R.J.H. Nahorski S.R. J. Biol. Chem. 1991; 266: 22234-22241Abstract Full Text PDF PubMed Google Scholar, 6Bokkala S. Joseph S.K. J. Biol. Chem. 1997; 272: 12454-12461Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 9Sipma H. Deelman L. De Smedt H. Missiaen L. Parys J.B. Vanlingen S. Henning R.H. Casteels R. Cell Calcium. 1998; 23: 11-21Crossref PubMed Scopus (45) Google Scholar, 10Willars G.B. Royall J.E. Nahorski S.R. El-Gehani F. Everest H. McArdle C.A. J. Biol. Chem. 2001; 276: 3123-3129Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 14Sharma K. McGowan T.A. Wang L. Madesh M. Kaspar V. Szalai G. Thomas A.P. Hajnoczky G. Am. J. Physiol. 2000; 278: F1022-F1029PubMed Google Scholar, 15Tovey S.C. de Smet P. Lipp P. Thomas D. Young K.W. Missiaen L. De Smedt H. Parys J.B. Berridge M.J. Thuring J. Holmes A. Bootman M.D. J. Cell Sci. 2001; 114: 3979-3989Crossref PubMed Google Scholar). InsP3R down-regulation is mediated by the ubiquitin/proteasome pathway (6Bokkala S. Joseph S.K. J. Biol. Chem. 1997; 272: 12454-12461Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 8Oberdorf J. Webster J.M. Zhu C.C. Luo S.G. Wojcikiewicz R.J.H. Biochem. J. 1999; 339: 453-461Crossref PubMed Scopus (74) Google Scholar, 9Sipma H. Deelman L. De Smedt H. Missiaen L. Parys J.B. Vanlingen S. Henning R.H. Casteels R. Cell Calcium. 1998; 23: 11-21Crossref PubMed Scopus (45) Google Scholar, 11Wojcikiewicz R.J.H. Xu Q. Webster J.M. Alzayady K. Gao C. J. Biol. Chem. 2003; 278: 940-947Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) and is a specific response, because the levels of other ER and signaling proteins remain unaffected (4Wojcikiewicz R.J.H. Furuichi T. Nakade S. Mikoshiba K. Nahorski S.R. J. Biol. Chem. 1994; 269: 7963-7969Abstract Full Text PDF PubMed Google Scholar, 5Wojcikiewicz R.J.H. J. Biol. Chem. 1995; 270: 11678-11683Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar, 6Bokkala S. Joseph S.K. J. Biol. Chem. 1997; 272: 12454-12461Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 9Sipma H. Deelman L. De Smedt H. Missiaen L. Parys J.B. Vanlingen S. Henning R.H. Casteels R. Cell Calcium. 1998; 23: 11-21Crossref PubMed Scopus (45) Google Scholar, 16Wojcikiewicz R.J.H. Oberdorf J.A. J. Biol. Chem. 1996; 271: 16652-16655Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). To be degraded by the ubiquitin/proteasome pathway (17Glickman M.H. Ciechanover A. Physiol. Rev. 2002; 82: 373-428Crossref PubMed Scopus (3380) Google Scholar), the substrate destined for destruction is covalently modified by the attachment of a polyubiquitin chain in a multistep process. First, in an ATP-dependent reaction, ubiquitin is covalently bound to a cysteine residue of the ubiquitin-activating enzyme, via a thiol-ester bond. Next, ubiquitin is transferred to the "active site" cysteine residue of a ubiquitin-conjugating enzyme (Ubc or E2), again employing a thiol-ester bond. The Ubc then conjugates ubiquitin to a lysine residue of either the substrate or a previously linked ubiquitin moiety via an isopeptide bond, forming a polyubiquitin chain. The specific recognition of substrates to be degraded as well as the transfer of ubiquitin from Ubc to substrate is facilitated by a ubiquitin-protein ligase (E3), of which there are three major families. The HECT ligases (HECT-E3s), which contain a domain homologous to the E6-AP carboxyl terminus, form an intermediate thiol-ester bond with ubiquitin (17Glickman M.H. Ciechanover A. Physiol. Rev. 2002; 82: 373-428Crossref PubMed Scopus (3380) Google Scholar). In contrast, the RING ligases (RING-E3s), whose activity is dependent upon a Zn2+-binding RING finger domain (17Glickman M.H. Ciechanover A. Physiol. Rev. 2002; 82: 373-428Crossref PubMed Scopus (3380) Google Scholar, 18Jackson P.K. Eldridge A.G. Freed E. Furstenthal L. Hsu J.Y. Kaiser B.K. Reimann J.D.R. Trends Cell Biol. 2000; 10: 429-439Abstract Full Text Full Text PDF PubMed Scopus (553) Google Scholar), and the U-box ligases (U-box-E3s) (19Hatakeyama S. Yada M. Matsumoto M. Ishida N. Nakayama K. J. Biol. 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The process by which these substrates are destroyed is known as ER-associated degradation (ERAD), which is primarily considered to be a "quality control" system that functions to specifically degrade misfolded proteins and unassembled subunits of multimeric complexes (24Jarosch E. Lenk U. Sommer T. Int. Rev. Cytol. 2003; 223: 39-81Crossref PubMed Scopus (66) Google Scholar). It is possible, however, that certain functional, native ER proteins can also be degraded by ERAD, but in a regulated manner, the prototype for this being 3-hydroxy 3-methylglutaryl-coenzyme A reductase (HMGR) (26Hampton R.Y. Bhakta H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12944-12948Crossref PubMed Scopus (121) Google Scholar, 27Hampton R.Y. Curr. Opin. Cell Biol. 2002; 14: 476-482Crossref PubMed Scopus (404) Google Scholar, 28Hampton R.Y. Annu. Rev. Cell Dev. Biol. 2002; 18: 345-378Crossref PubMed Scopus (92) Google Scholar). Recent studies indicate that ubiquitination and degradation of HMGR is initiated by metabolites of the mevalonate pathway (27Hampton R.Y. Curr. Opin. Cell Biol. 2002; 14: 476-482Crossref PubMed Scopus (404) Google Scholar, 28Hampton R.Y. Annu. Rev. Cell Dev. Biol. 2002; 18: 345-378Crossref PubMed Scopus (92) Google Scholar, 29Ravid T. Avner R. Polak-Charcon S. Faust J.R. Roitelman J. J. Biol. Chem. 1999; 274: 29341-29351Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 30Ravid T. Doolman R. Avner R. Harats D. Roitelman J. J. Biol. Chem. 2000; 275: 35840-35847Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar), perhaps after a "structural transition" that gives HMGR characteristics of a misfolded protein (27Hampton R.Y. Curr. Opin. Cell Biol. 2002; 14: 476-482Crossref PubMed Scopus (404) Google Scholar, 28Hampton R.Y. Annu. Rev. Cell Dev. Biol. 2002; 18: 345-378Crossref PubMed Scopus (92) Google Scholar). With regard to the Ubcs that mediate ERAD, the enzymes Ubc6p and Ubc7p have often been implicated in this process in yeast, with Ubc7p playing the dominant role in regulated degradation of HMGR (24Jarosch E. Lenk U. Sommer T. Int. Rev. Cytol. 2003; 223: 39-81Crossref PubMed Scopus (66) Google Scholar, 26Hampton R.Y. Bhakta H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12944-12948Crossref PubMed Scopus (121) Google Scholar, 31Hiller M.M. Finger A. Schweiger M. Wolf D.H. Science. 1996; 273: 1725-1728Crossref PubMed Scopus (619) Google Scholar, 32Plemper R.K. Wolf D.H. Mol. Biol. Rep. 1999; 26: 125-130Crossref PubMed Google Scholar, 33Bays N.W. Gardner R.G. Seelig L.P. Joazeiro C.A. Hampton R.Y. Nat. Cell Biol. 2001; 3: 24-29Crossref PubMed Scopus (381) Google Scholar, 34Biederer T. Volkwein C. Sommer T. Science. 1997; 278: 1806-1809Crossref PubMed Scopus (331) Google Scholar, 35Plemper R.K. Wolf D.H. Trends Biochem. Sci. 1999; 24: 266-270Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). Likewise, in studies with exogenously expressed substrates in mammalian cells, a mammalian homologue of Ubc6p, hereafter termed mamUbc6, has been implicated in the ERAD of cystic fibrosis transmembrane conductance regulator and TCRα (23Lenk U. Yu H. Walter J. Gelman M.S. Hartmann E. Kopito R.R. Sommer T. J. Cell Sci. 2002; 115: 3007-3014Crossref PubMed Google Scholar), and a mammalian homologue of Ubc7p, hereafter termed mamUbc7, has been implicated in the ERAD of TCRα and CD3-δ (22Tiwari S. Weissman A.M. J. Biol. Chem. 2001; 276: 16193-16200Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). With regard to the endogenous expression of these Ubcs, mamUbc7 is expressed ubiquitously in all human tissues examined, including brain (36Katsanis N. Fisher E.M.C. Genomics. 1998; 51: 128-131Crossref PubMed Scopus (28) Google Scholar), and although mamUbc6 distribution has not been studied, a chick homologue of Ubc6p is expressed in all avian tissues, again including brain (37Lester D. Farquharson C. Russell G. Houston B. Biochem. Biophys. Res. Commun. 2000; 269: 474-480Crossref PubMed Scopus (24) Google Scholar). In the present study we have examined whether the ERAD-linked Ubcs, mamUbc6 and mamUbc7, mediate GPCR-initiated ubiquitination and down-regulation of endogenous InsP3Rs in SH-SY5Y human neuroblastoma cells. We accomplished this by creating SH-SY5Y cell lines stably expressing murine homologues of Ubc6p and Ubc7p (MmUbc6 and MmUbc7), and their respective "dominant-negative" (DN) mutants, in which the active site cysteine residues have been mutated to serine (22Tiwari S. Weissman A.M. J. Biol. Chem. 2001; 276: 16193-16200Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 38Gonen H. Bercovich B. Orian A. Carrano A. Takizawa C. Yamanaka K. Pagano M. Iwai K. Ciechanover A. J. Biol. Chem. 1999; 274: 14823-14830Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Our results indicate that mamUbc7 mediates InsP3R ubiquitination and down-regulation and thus suggests that some of the same machinery that is responsible for the ERAD of misfolded and misassembled proteins also degrades InsP3Rs. Additionally, we provide evidence for the involvement of a RING-E3 in InsP3R ubiquitination and down-regulation, and demonstrate that muscarinic receptor activation can induce InsP3R ubiquitination in rat cerebral cortex. Plasmids—cDNAs encoding HA-MmUbc6, HA-MmUbc6DN(C94S), myc-MmUbc7, and myc-MmUbc7DN(C89S) (GenBank™ accession nos. AF296656 and AF296657), with the hemagglutinin (HA) epitope (YPYDVPDYA) and the myc epitope (EQKLISEEDLN) tags at the NH2 termini (22Tiwari S. Weissman A.M. J. Biol. Chem. 2001; 276: 16193-16200Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), were ligated into the EcoR1/SalI sites of the multiple cloning region of the pIRES2-EGFP plasmid (Clontech). The amino acid sequences of the MmUbcs used in this study are each ∼60% identical to yeast Ubc6p and Ubc7p (22Tiwari S. Weissman A.M. J. Biol. Chem. 2001; 276: 16193-16200Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). MmUbc6 is ∼95% identical to its human homologue (22Tiwari S. Weissman A.M. J. Biol. Chem. 2001; 276: 16193-16200Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), and MmUbc7 is 100% identical to its human homologue (accession no. NM_003343). Cell Culture and Transfection—SH-SY5Y human neuroblastoma cells were grown in monolayers as described (7Zhu C.C. Furuichi T. Mikoshiba K. Wojcikiewicz R.J.H. J. Biol. Chem. 1999; 274: 3476-3484Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Cells were subcultured with 10 mm Hepes, 155 mm NaCl, 1 mm EDTA, pH 7.4 (HBS-E), and medium was routinely changed every other day. Cells (∼75% confluent) were transfected with 1 μg of DNA and 8 μl of FuGENE 6 (Roche Applied Science) according to the instructions from the manufacturer. Cells were then subcultured and grown in the presence of 500 μg/ml G418. GFP-expressing colonies were identified by fluorescence microscopy of living cells and selected using sterile cloning cylinders. A second round of clonal selection followed to ensure that each clonal cell line was independently derived from a single transfected cell. Stock cultures were maintained in 250 μg/ml G418, whereas cells to be used in experiments were cultured in the absence of G418. Antibodies—CT1 was produced from a peptide corresponding to the COOH-terminal 19 residues of the rat type I InsP3R and is specific for type I InsP3Rs (5Wojcikiewicz R.J.H. J. Biol. Chem. 1995; 270: 11678-11683Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar). Anti-c-myc epitope clone 9E10 (α-myc) and anti-ubiquitin clone Ubi-1 (α-Ub) were from Zymed Laboratories Inc.; anti-HA epitope clone HA.11 (α-HA) was from Babco; rabbit polyclonal anti-transaldolase (α-tal) was a kind gift from Drs. Andras Perl and Katalin Banki (39Banki K. Hutter E. Colombo E. Gonchoroff N.J. Perl A. J. Biol. Chem. 1996; 271: 32994-33001Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar); and anti-human p53 clone DO-1 (α-p53) was from Santa Cruz. Cell Fractionation—Cells were harvested in HBS-E, centrifuged at ∼700 × g for 3 min, and resuspended in 1 ml of 10 mm Tris base, 1 mm EGTA, pH 7.4 (hypotonic buffer) containing protease inhibitors (0.2 mm phenylmethylsulfonyl fluoride, 10 μm leupeptin, 10 μm pepstatin, 0.2 μm soybean trypsin inhibitor) and 1 mm dithiothreitol (DTT). Cells were then disrupted by sonication and centrifuged at 100,000 × g for 1 h. Supernatant was collected and represented the cytosolic fraction. The pellet was resuspended in 1 ml of hypotonic buffer and represented the membrane fraction. Membrane fraction protein (10 μg) and an equivalent volume of the cytosolic fraction were then electrophoresed and processed in immunoblots. Confocal Immunofluorescence Microscopy—Cells were grown on 8-chamber glass slides, fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min, and then washed three times in PBS. Fixed cells were then incubated in blocking solution (10% goat serum, 4% bovine serum albumin in PBS) containing 0.01% digitonin for 1 h, and then incubated overnight in blocking solution containing primary antibody (α-myc or α-HA) at 4 °C. Next, cells were washed six times in PBS and incubated for 2 h in blocking solution containing rhodamine-conjugated goat anti-mouse secondary antibody. Cells were then washed six times, and coverslips were mounted with anti-fade mount (Molecular Probes). Images were obtained with a Bio-Rad MRC-1024 confocal microscope using a 60× oil immersion objective. The 568-nm line and the 488-nm line of the krypton/argon laser were used to excite rhodamine and GFP, respectively. Emission was collected between 589 and 621 nm for rhodamine and between 506 and 538 nm for GFP. Preparation of Lysates for Immunoblots—Cells were grown to confluence and were stimulated, harvested in HBS-E, centrifuged (700 × g for 3 min), and lysed in 150 mm NaCl, 50 mm Tris base, 1 mm EDTA, 1% Triton X-100, protease inhibitors, 1 mm DTT, pH 8.0 (lysis buffer), for 20 min at 4 °C. Lysates were then centrifuged (16,000 × g for 10 min), and supernatants were collected, electrophoresed, and processed in immunoblots with CT1, α-HA, α-myc, α-Ub, or α-p53. For ubiquitin thiol-ester analysis, cells were lysed in non-reducing lysis buffer and electrophoresed under non-reducing conditions (in the absence of DTT). InsP3 Receptor Immunoprecipitation and Detection of Ubiquitination—Supernatants obtained from centrifuged lysates were collected, protein concentrations were equalized, and equal volumes of these supernatants were incubated for 4 h at 4 °C with CT1 and protein A-Sepharose CL-4B beads. Immunoprecipitated InsP3Rs were then electrophoresed and processed in immunoblots, first with α-Ub to assess polyubiquitin content, and then reprobed with CT1 to determine InsP3 receptor levels. When comparing between different clonal cell lines, the amount of polyubiquitin immunoreactivity associated with InsP3Rs was defined as the ratio of α-Ub immunoreactivity to CT1 immunoreactivity (α-Ub/CT1). This accounted for minor differences in InsP3R content between samples. This procedure accurately reflected ubiquitination, because the α-Ub/CT1 ratio remained constant when a range of amounts of ubiquitinated InsP3Rs from untransfected SH-SY5Y cells were analyzed (data not shown). Ubiquitination in Rat Cerebral Cortex—Rat cerebral cortex slices (350 × 350 μm) were prepared and incubated as described (40Challis R.A.J. Mistry R. Gray D.W. Nahorski S.R. Neuropharmacology. 1994; 33: 15-25Crossref PubMed Scopus (23) Google Scholar). Slices were collected by centrifugation (700 × g for 2 min) and homogenized in lysis buffer, lysates were centrifuged (16,000 × g for 10 min), and InsP3Rs were immunoprecipitated with CT1, probed in immunoblots with α-Ub, and reprobed with CT1. Measurement of InsP3 Formation and Ca2 + Mobilization—SH-SY5Y cells were grown to confluence, harvested in HBS-E, washed, and resuspended in Krebs-Hepes buffer as described (7Zhu C.C. Furuichi T. Mikoshiba K. Wojcikiewicz R.J.H. J. Biol. Chem. 1999; 274: 3476-3484Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). For InsP3 measurements, triplicate 100-μl aliquots of cell suspensions were incubated without or with carbachol and incubations were terminated by the addition of 100 μl of 1 m trichloroacetic acid. InsP3 mass was then determined using a radioreceptor assay as previously described (41Wojcikiewicz R.J.H. Tobin A.B. Nahorski S.R. J. Neurochem. 1994; 63: 177-185Crossref PubMed Scopus (80) Google Scholar). For [Ca2+] i measurements, cell suspensions were incubated with 10 μm Fura2-AM for 60 min at 37 °C and then excited at 340 and 380 nm, and emission intensity at 510 nm was recorded with a computerized LS-50B luminescence spectrometer (PerkinElmer Life Sciences). [Ca2+] i was calculated as described (42Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar) using 0.1% Triton X-100 and 10 mm EGTA as calibrating agents. Materials—Cell culture materials were obtained from Invitrogen; G418 was from CellGro; T4 DNA ligase, EcoRI, and XhoI were from New England Biolabs; peroxidase-conjugated antibodies, Triton X-100, protease inhibitors, N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), carbachol, and atropine were from Sigma; protein A-Sepharose CL-4B was from Amersham Life Sciences; DTT and SDS-PAGE gel supplies were from Bio-Rad; and N-acetyl-Leu-Leu-norleucinal (ALLN) was from Alexis. Miscellaneous—Data shown are representative of at least two independent experiments. Quantitation of immunoreactivity was accomplished using a GeneGnome Imager and GeneTools software from Syngene. Combined quantitated data from immunoblots are mean ± S.E. of results from ≥3 independent experiments. Establishment of SH-SY5Y Cell Lines Stably Expressing Exogenous MmUbcs—The cDNAs encoding epitope-tagged MmUbc6 and MmUbc7 and their DN mutants (22Tiwari S. Weissman A.M. J. Biol. Chem. 2001; 276: 16193-16200Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar) were ligated into the pIRES2-EGFP vector, SH-SY5Y cells were transfected with the resulting plasmids, and G418-resistant, GFP-expressing clones were isolated. Expression of exogenous MmUbc6 and MmUbc6DN (HA-tagged), and MmUbc7 and MmUbc7DN (myc-tagged) was confirmed in immunoblots with α-HA and α-myc, which identified, respectively, ∼31-kDa proteins (Fig. 1A, upper panel, lanes 4 and 6) and ∼19-kDa proteins (Fig. 1B, upper panel, lanes 3–6). The clonal cell lines depicted in Fig. 1 were those that expressed the highest levels of MmUbcs; these and other independently derived cell lines expressing equivalent amounts of MmUbcs were used throughout the course of this study. GFP and exogenous MmUbc expression remained stable throughout, as determined by periodic fluorescence microscopy and immunoblot analysis, respectively (data not shown). Localization of Exogenous MmUbcs—To show that the exogenous MmUbcs were appropriately localized in SH-SY5Y cells, membrane and cytosol fractions were prepared from control and transfected cells. The efficiency of fractionation was established using the type I InsP3R as a marker for membranes, and transaldolase (an entirely cytosolic enzyme of the pentose-phosphate pathway) (39Banki K. Hutter E. Colombo E. Gonchoroff N.J. Perl A. J. Biol. Chem. 1996;

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