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W2123170497 abstract "The neuronal adaptor protein X11α participates in the formation of multiprotein complexes and intracellular trafficking. It contains a series of discrete protein-protein interaction domains including two contiguous C-terminal PDZ domains. We used the yeast two-hybrid system to screen for proteins that interact with the PDZ domains of human X11α, and we isolated a clone encoding domains II and III of the copper chaperone for Cu,Zn-superoxide dismutase-1 (CCS). The X11α/CCS interaction was confirmed in coimmunoprecipitation studies plus glutathioneS-transferase fusion protein pull-down assays and was shown to be mediated via PDZ2 of X11α and a sequence within the carboxyl terminus of domain III of CCS. CCS delivers the copper cofactor to the antioxidant superoxide dismutase-1 (SOD1) enzyme and is required for its activity. Overexpression of X11α inhibited SOD1 activity in transfected Chinese hamster ovary cells which suggests that X11α binding to CCS is inhibitory to SOD1 activation. X11α also interacts with another copper-binding protein found in neurons, the Alzheimer's disease amyloid precursor protein. Thus, X11α may participate in copper homeostasis within neurons. The neuronal adaptor protein X11α participates in the formation of multiprotein complexes and intracellular trafficking. It contains a series of discrete protein-protein interaction domains including two contiguous C-terminal PDZ domains. We used the yeast two-hybrid system to screen for proteins that interact with the PDZ domains of human X11α, and we isolated a clone encoding domains II and III of the copper chaperone for Cu,Zn-superoxide dismutase-1 (CCS). The X11α/CCS interaction was confirmed in coimmunoprecipitation studies plus glutathioneS-transferase fusion protein pull-down assays and was shown to be mediated via PDZ2 of X11α and a sequence within the carboxyl terminus of domain III of CCS. CCS delivers the copper cofactor to the antioxidant superoxide dismutase-1 (SOD1) enzyme and is required for its activity. Overexpression of X11α inhibited SOD1 activity in transfected Chinese hamster ovary cells which suggests that X11α binding to CCS is inhibitory to SOD1 activation. X11α also interacts with another copper-binding protein found in neurons, the Alzheimer's disease amyloid precursor protein. Thus, X11α may participate in copper homeostasis within neurons. amyloid precursor protein Chinese hamster ovary polyacrylamide gel electrophoresis glutathione S-transferase superoxide dismutase-1 amyotrophic lateral sclerosis The X11s, also known as mints (munc18interacting proteins 1–3), are a family of adaptor proteins with three members (α, β, and γ) encoded by separate genes on, respectively, human chromosomes 9, 15, and 19 (1–8). Expression of X11α and X11β is restricted to neurons, whereas X11γ is ubiquitously expressed (1Duclos F. Boschert U. Sirugo G. Mandel J.-L. Hen R. Koenig M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 109-113Crossref PubMed Scopus (66) Google Scholar, 4Okamoto M. Sudhof T.C. Eur. J. Cell Biol. 1998; 77: 161-165Crossref PubMed Scopus (68) Google Scholar, 6Tanahashi H. Tabira T. Biochem. Biophys. Res. Commun. 1999; 255: 663-667Crossref PubMed Scopus (72) Google Scholar, 7Tomita S. Ozaki T. Taru H. Oguchi S. Takeda S. Yagi Y. Sakiyama S. Kirino Y. Suzuki T. J. Biol. Chem. 1999; 274: 2243-2254Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 8McLoughlin D.M. Irving N.G. Brownlees J. Brion J.-P. Leroy K. Miller C.C.J. Eur. J. Neurosci. 1999; 11: 1988-1994Crossref PubMed Scopus (75) Google Scholar, 9Borg J.P. Lopez-Figueroa M.O. De Taddéo-Borg M. Kroon D.E. Turner R.S. Watson S.J. Margolis B. J. Neurosci. 1999; 19: 1307-1316Crossref PubMed Google Scholar). The X11s diverge substantially in their N-terminal regions, but they all contain a centrally located phosphotyrosine binding domain (10Margolis B. Borg J.P. Straight S. Meyer D. Kidney Int. 1999; 56: 1230-1237Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), through which all three X11s bind to the cytoplasmic domain of the Alzheimer's disease amyloid precursor protein (APP)1 (2McLoughlin D.M. Miller C.C.J. FEBS Lett. 1996; 397: 197-200Crossref PubMed Scopus (134) Google Scholar, 6Tanahashi H. Tabira T. Biochem. Biophys. Res. Commun. 1999; 255: 663-667Crossref PubMed Scopus (72) Google Scholar, 7Tomita S. Ozaki T. Taru H. Oguchi S. Takeda S. Yagi Y. Sakiyama S. Kirino Y. Suzuki T. J. Biol. Chem. 1999; 274: 2243-2254Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 11Borg J.-P. Ooi J. Levy E. Margolis B. Mol. Cell. Biol. 1996; 16: 6229-6241Crossref PubMed Scopus (436) Google Scholar, 12Zhang Z. Lee C.-H. Mandiyan V. Borg J.-P. Margolis B. Schlessinger J. Kuriyan J. EMBO J. 1997; 16: 6141-6150Crossref PubMed Scopus (139) Google Scholar), and two contiguous C-terminal PDZ (PSD-95,Drosophila disks-large, ZO-1) domains (13Fanning A.S. Anderson J.M. J. Clin. Invest. 1999; 103: 767-772Crossref PubMed Scopus (401) Google Scholar). These, and a variety of other less well characterized protein-protein interaction regions (Fig. 1 A), mediate the binding of the X11s to a number of proteins. X11α binds to the pre-synaptic adaptor protein CASK via a sequence preceding the phosphotyrosine binding domain (9Borg J.P. Lopez-Figueroa M.O. De Taddéo-Borg M. Kroon D.E. Turner R.S. Watson S.J. Margolis B. J. Neurosci. 1999; 19: 1307-1316Crossref PubMed Google Scholar, 14Butz S. Okamoto M. Sudhof T.C. Cell. 1998; 94: 773-782Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar, 15Borg J.-P. Straight S.W. Kaech S.M. De Taddéo-Borg M. Kroon D.E. Karnak D. Turner R.S. Kim S.K. Margolis B. J. Biol. Chem. 1998; 273: 31633-31636Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). CASK also binds both Veli1 and members of the neurexin family of pre-synaptic membrane-spanning proteins (14Butz S. Okamoto M. Sudhof T.C. Cell. 1998; 94: 773-782Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar, 16Hata Y. Butz S. Sudhof T.C. J. Neurosci. 1996; 16: 2488-2494Crossref PubMed Google Scholar). The X11α-CASK-Veli pre-synaptic complex is highly evolutionarily conserved and is found in orthologous form in Caenorhabditis elegans as the LIN-10-LIN-2-LIN-7 complex (17Kaech S.M. Whitfield C.M. Kim S.K. Cell. 1998; 94: 761-771Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar) that regulates basolateral sorting of the epidermal growth factor receptor LET-23 and maintains cell polarity (18Rongo C. Whitfield C.W. Rodal A. Kim S.K. Kaplan J.M. Cell. 1998; 94: 751-759Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 19Whitfield C.W. Benard C. Barnes T. Hekimi S. Kim S.K. Mol. Biol. Cell. 1999; 10: 2087-2100Crossref PubMed Scopus (91) Google Scholar). X11α also binds to the synaptic vesicle docking protein Munc-18 through an N-terminal region (3Okamoto M. Sudhof T.C. J. Biol. Chem. 1997; 272: 31459-31464Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 4Okamoto M. Sudhof T.C. Eur. J. Cell Biol. 1998; 77: 161-165Crossref PubMed Scopus (68) Google Scholar, 14Butz S. Okamoto M. Sudhof T.C. Cell. 1998; 94: 773-782Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar). Via its first PDZ domain (PDZ1), X11α has been reported to bind both the kinesin superfamily motor protein KIF17 (20Setou M. Nakagawa T. Seog D.-H. Hirokawa N. Science. 2000; 288: 1796-1802Crossref PubMed Scopus (607) Google Scholar) and the C terminus of the N-type Ca2+ channel pore-forming α1B-subunit, which in turn can bind the SH3 region of CASK (21Maximov A. Sudhof T.C. Bezprozvanny I. J. Biol. Chem. 1999; 274: 24453-24456Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). Finally, we have shown that X11α binds, via both of its PDZ domains, to presenilin-1 and mediates interactions between presenilin-1 and APP (22Lau K.-F. McLoughlin D.M. Standen C. Miller C.C.J. Mol. Cell. Neurosci. 2000; 16: 555-563Crossref Scopus (81) Google Scholar). Thus, similar to several other PDZ-bearing proteins (for reviews see Refs. 13Fanning A.S. Anderson J.M. J. Clin. Invest. 1999; 103: 767-772Crossref PubMed Scopus (401) Google Scholar and23Garner C.C. Nash J. Huganir R.L. Trends Cell Biol. 2000; 10: 274-280Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar), X11α appears to provide a framework, or scaffolding, for the assembly of multimolecular complexes and functions in the trafficking and sorting of proteins to different neuronal compartments. The full complement of ligands that bind to the X11 proteins is not yet known. In this study, we demonstrate that, via its second PDZ domain (PDZ2), X11α interacts with the copper chaperone for copper/zinc superoxide dismutase-1 (CCS). CCS delivers and inserts the copper cofactor into the antioxidant enzyme copper/zinc superoxide dismutase-1 (SOD1) and is required for its activation (24Culotta V.C. Klomp L.W.J. Strain J. Casareno R.L.B. Krems B. Gitlin J.D. J. Biol. Chem. 1997; 272: 23469-23472Abstract Full Text Full Text PDF PubMed Scopus (686) Google Scholar, 25Corson L.B. Strain J.J. Culotta V.C. Cleveland D.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6361-6366Crossref PubMed Scopus (143) Google Scholar, 26Casareno R.L.B. Waggoner D. Gitlin J.D. J. Biol. Chem. 1998; 273: 23625-23628Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 27Gamonet F. Lauquin G.J.M. Eur. J. Biochem. 1998; 251: 716-723Crossref PubMed Scopus (42) Google Scholar, 28Rae T.D. Schmidt P.J. Pufahl R.A. Culotta V.C. O'Halloran T.V. Science. 1999; 284: 805-808Crossref PubMed Scopus (1379) Google Scholar). We also demonstrate that overexpression of X11α inhibits SOD1 activity in transfected cells, consistent with a role for X11α in regulating CCS function and SOD1 activation. Interactive cloning experiments, including relevant controls, were performed as described previously (2McLoughlin D.M. Miller C.C.J. FEBS Lett. 1996; 397: 197-200Crossref PubMed Scopus (134) Google Scholar). In brief, the sequence encoding the two human X11α PDZ domains (PDZ1 and -2; X11α amino acid sequence 649–837) was amplified by polymerase chain reaction (22Lau K.-F. McLoughlin D.M. Standen C. Miller C.C.J. Mol. Cell. Neurosci. 2000; 16: 555-563Crossref Scopus (81) Google Scholar), subcloned into the EcoRI site of the yeast “bait” vector pY3 (29Sadowski I. Bell B. Broad P. Hollis M. Gene ( Amst. ). 1992; 118: 137-141Crossref PubMed Scopus (201) Google Scholar), and used to screen a human brain cDNA library (CLONTECH). Yeast colonies were grown using selective media lacking tryptophan, leucine, and histidine. Vigorously growing colonies were subjected to freeze-fracture β-galactosidase assays, and candidate library plasmids were rescued from positive colonies by transformation intoEscherichia coli HB101. Brain library cDNA inserts were sequenced using a Cyclist Exo− Pfu DNA sequencing kit (Stratagene). Chinese hamster ovary (CHO) cells and rat primary cortical neurones were cultured as described previously (8McLoughlin D.M. Irving N.G. Brownlees J. Brion J.-P. Leroy K. Miller C.C.J. Eur. J. Neurosci. 1999; 11: 1988-1994Crossref PubMed Scopus (75) Google Scholar, 30Brownlees J. Yates A. Bajaj N.P. Davis D. Anderton B.H. Leigh P.N. Shaw C.E. Miller C.C.J. J. Cell Sci. 2000; 113: 401-407Crossref PubMed Google Scholar). CHO cells were transfected using LipofectAMINE (Life Technologies, Inc.) as per the manufacturer's instructions. Full-length C-terminal Myc-tagged human X11α was as described previously (22Lau K.-F. McLoughlin D.M. Standen C. Miller C.C.J. Mol. Cell. Neurosci. 2000; 16: 555-563Crossref Scopus (81) Google Scholar). The cDNA encoding human CCS (24Culotta V.C. Klomp L.W.J. Strain J. Casareno R.L.B. Krems B. Gitlin J.D. J. Biol. Chem. 1997; 272: 23469-23472Abstract Full Text Full Text PDF PubMed Scopus (686) Google Scholar) was subcloned into pCDNA3.1 (Invitrogen), and human SOD1 was expressed in pCIneo (Promega). X11α was detected using rabbit polyclonal antibodies raised against X11α-(161–421) (22Lau K.-F. McLoughlin D.M. Standen C. Miller C.C.J. Mol. Cell. Neurosci. 2000; 16: 555-563Crossref Scopus (81) Google Scholar). The same immunogen was also used to generate mouse polyclonal antibodies. Antibody specificity was confirmed by competing out signals with immunogen (data not shown). Rabbit polyclonal antibodies raised against CCS residues 1–85 were used to detect CCS (26Casareno R.L.B. Waggoner D. Gitlin J.D. J. Biol. Chem. 1998; 273: 23625-23628Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). SOD1 sheep polyclonal antibody was purchased from Calbiochem, and the 9E10 anti-Myc monoclonal antibody was purchased from Sigma. CHO cells were doubly or singly transfected as indicated with Myc-tagged X11α, CCS, and SOD1. Transfected cells were harvested in ice-cold lysis buffer (50 mm Tris-HCl, 150 mm NaCl, 1 mmEDTA, 1% Triton X-100, 5 μg/ml leupeptin, 2 μg/ml aprotinin, 5 μg/ml pepstatin, and 0.25 mm phenylmethylsulfonyl fluoride) and incubated on ice for 20 min. In binding studies with transfected SOD1, the cell lysates also underwent one freeze-thaw cycle (26Casareno R.L.B. Waggoner D. Gitlin J.D. J. Biol. Chem. 1998; 273: 23625-23628Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). Lysates were then centrifuged at 14,000 rpm for 10 min at 4 °C. For immunoprecipitation studies the supernatant was precleared with protein A-Sepharose beads (Sigma). Myc-tagged X11α was immunoprecipitated from 500 μg of total protein lysate using antibody 9E10. The antibody was captured using protein A-Sepharose beads that were then washed four times with ice-cold lysis buffer. Immunoprecipitates were analyzed by SDS-PAGE and immunoblotting as described (8McLoughlin D.M. Irving N.G. Brownlees J. Brion J.-P. Leroy K. Miller C.C.J. Eur. J. Neurosci. 1999; 11: 1988-1994Crossref PubMed Scopus (75) Google Scholar). GlutathioneS-transferase (GST) fusion proteins with X11α PDZ1-(649–746), PDZ2-(742–837), and PDZ1 + 2-(649–837) were as described (22Lau K.-F. McLoughlin D.M. Standen C. Miller C.C.J. Mol. Cell. Neurosci. 2000; 16: 555-563Crossref Scopus (81) Google Scholar). The partial CCS clone (CCS-(48–274)) isolated in the library screen encoded the C-terminal 227 amino acids of CCS, incorporating the C-terminal 39 residues of domain I and all of domains II and III of CCS (Fig. 1 B). Digestion of this clone withBglII released cDNAs encoding CCS-(48–241) and CCS-(243–274) that were subcloned into the BamHI site of pGEX-5X-1 (Amersham Pharmacia Biotech). CCS-(48–241) encompasses all of domain II of CCS (amino acids 87–234), whereas CCS-(243–274) encodes the C-terminal 32 amino acids of CCS including the CXC copper-binding motif (31Schmidt P.J. Rae T.D. Pufahl R.A. Hamma T. Strain J. O'Halloran T.V. Culotta V.C. J. Biol. Chem. 1999; 274: 23719-23725Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 32Lamb A.L. Wernimont A.K. Pufahl R.A. Culotta V.C. O'Halloran T.V. Rosenzweig A.C. Nat. Struct. Biol. 1999; 6: 724-729Crossref PubMed Scopus (176) Google Scholar, 33Lamb A.L. Wernimont A.K. Pufahl R.A. O'Halloran T.V. Rosenzweig A.C. Biochemistry. 2000; 39: 1589-1595Crossref PubMed Scopus (87) Google Scholar). Digestion of the library clone with EcoRI released the CCS-(48–274) insert, which was subcloned into pGEX-5X-1. Fusion proteins containing full-length CCS or the cytoplasmic domain of APP were as described previously (26Casareno R.L.B. Waggoner D. Gitlin J.D. J. Biol. Chem. 1998; 273: 23625-23628Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar,34Lau K.-F. McLoughlin D.M. Standen C.L. Irving N.G. Miller C.C.J. Neuroreports. 2000; 11: 3607-3610Crossref PubMed Scopus (43) Google Scholar). Expression and purification of GST fusion proteins, plus their use as ligands in pull-down binding assays, were essentially as described (22Lau K.-F. McLoughlin D.M. Standen C. Miller C.C.J. Mol. Cell. Neurosci. 2000; 16: 555-563Crossref Scopus (81) Google Scholar). The immunoprecipitation and GST fusion assays to confirm the X11α and CCS interaction were performed three times with similar results. Transfected CHO cells and rat neuronal cultures were fixed and prepared for immunofluorescence as described (8McLoughlin D.M. Irving N.G. Brownlees J. Brion J.-P. Leroy K. Miller C.C.J. Eur. J. Neurosci. 1999; 11: 1988-1994Crossref PubMed Scopus (75) Google Scholar, 22Lau K.-F. McLoughlin D.M. Standen C. Miller C.C.J. Mol. Cell. Neurosci. 2000; 16: 555-563Crossref Scopus (81) Google Scholar, 34Lau K.-F. McLoughlin D.M. Standen C.L. Irving N.G. Miller C.C.J. Neuroreports. 2000; 11: 3607-3610Crossref PubMed Scopus (43) Google Scholar). X11α was detected in transfected CHO cells using the 9E10 antibody to the Myc tag; endogenous X11α in primary rat cortical neurons was detected with the mouse X11α antiserum. The rabbit antiserum was used to detect CCS in both transfected CHO cells and in neurons. Antibodies were visualized by goat anti-mouse Igs coupled to Texas Red and goat anti-rabbit Igs coupled to Oregon Green (Molecular Probes). CHO cells were transfected with 2.5 μg of SOD1, X11α, and CCS DNA as indicated; transfections receiving only one or two plasmids were balanced with empty vector such that all received the same total amount of DNA. Transfected cells were washed twice with PBS, harvested by scraping into cold PBS, and then centrifuged at 10,000 × g for 5 min at 4 °C. The pelleted cells were resuspended and lysed in water by a freeze-thaw cycle as described (35Borchelt D.R. Lee M.K. Slunt H.S. Guarnieri M. Xu Z.-S. Wong P.C. Brown Jr., R.H. Price D.L. Sisodia S.S. Cleveland D.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8292-8296Crossref PubMed Scopus (533) Google Scholar), centrifuged at 10,000 × g for 5 min, and the supernatant collected. A sample of each supernatant was removed for SDS-PAGE and immunoblot analyses; the remainder was adjusted to 0.125 m Tris chloride, pH 6.8, 20% (v/v) glycerol, 0.025% bromphenol blue, and 0.1% Nonidet P-40 (35Borchelt D.R. Lee M.K. Slunt H.S. Guarnieri M. Xu Z.-S. Wong P.C. Brown Jr., R.H. Price D.L. Sisodia S.S. Cleveland D.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8292-8296Crossref PubMed Scopus (533) Google Scholar). 30 μg of protein from each sample was run on 10% nondenaturing polyacrylamide gels, and SOD1 activities were determined by nitro blue tetrazolium in-gel staining assays as described (36Beauchamp C. Fridovich I. Anal. Biochem. 1971; 44: 276-287Crossref PubMed Scopus (10014) Google Scholar). 1 μg of protein from each supernatant sample was analyzed by immunoblotting to determine the expression of SOD1, X11α, and CCS. Protein quantities were assayed using Bradford reagent (Sigma) as per the manufacturer's instructions. SOD1 activity assay gels and immunoblots were further analyzed by pixel densitometry using a Bio-Rad GS710 scanner and Quantity 1 software to obtain the ratio of SOD1 activity to expressed protein level for each sample (35Borchelt D.R. Lee M.K. Slunt H.S. Guarnieri M. Xu Z.-S. Wong P.C. Brown Jr., R.H. Price D.L. Sisodia S.S. Cleveland D.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8292-8296Crossref PubMed Scopus (533) Google Scholar). Within each set of transfections, this ratio was set at 100% for the SOD1-only transfected cells, and the remaining activities were expressed relative to this. Data from three separate sets of transfections were analyzed together using the Kruskal-Wallis one-way analysis of variance. To identify binding partners for the X11α PDZ1 + 2 region, we used the yeast two-hybrid system to screen a human brain cDNA library. A cDNA encoding the C-terminal 227 amino acids of CCS (CCS-(48–274)) was isolated (Fig.1 B). To investigate if the full-length proteins could interact in mammalian cells, immunoprecipitation experiments were performed from X11α, CCS, and X11α/CCS cotransfected CHO cells. X11α was immunoprecipitated using the 9E10 antibody to the Myc tag. CCS was present only in immunoprecipitates obtained from X11α/CCS-cotransfected cells but not X11α or CCS alone transfected cells (Fig.2 A). Thus CCS coimmunoprecipitates with X11α from transfected CHO cells. Human CCS is comprised of three functional domains (31Schmidt P.J. Rae T.D. Pufahl R.A. Hamma T. Strain J. O'Halloran T.V. Culotta V.C. J. Biol. Chem. 1999; 274: 23719-23725Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 33Lamb A.L. Wernimont A.K. Pufahl R.A. O'Halloran T.V. Rosenzweig A.C. Biochemistry. 2000; 39: 1589-1595Crossref PubMed Scopus (87) Google Scholar). The N-terminal domain I (amino acids 1–86) is homologous to the secretory pathway copper chaperone HAH1 and its yeast ortholog Atx1p (37Klomp L.W.J. Lin S.-J. Yuan D.S. Klausner R.D. Culotta V.C. Gitlin J.D. J. Biol. Chem. 1997; 272: 9221-9226Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar); the central domain II is ∼50% identical to SOD1 (24Culotta V.C. Klomp L.W.J. Strain J. Casareno R.L.B. Krems B. Gitlin J.D. J. Biol. Chem. 1997; 272: 23469-23472Abstract Full Text Full Text PDF PubMed Scopus (686) Google Scholar); and the C-terminal domain III is unique to CCS (Fig. 1 B). As all of domain II of CCS was encoded in the CCS cDNA isolated in the yeast two-hybrid screen, this raised the possibility that X11α might also interact with SOD1. However, under similar experimental conditions as above, SOD1 did not coimmunoprecipitate with X11α (Fig. 2 B). To map further the regions required for binding of X11α with CCS, we used GST fusion protein binding assays. GST fusion proteins containing X11α PDZ1, PDZ2, and PDZ1 + 2 were used as baits in pull-down assays from CCS-transfected CHO cells. CCS strongly interacted with PDZ2 and PDZ1 + 2, whereas no interaction could be detected between CCS and PDZ1 or GST alone (Fig.3 A). Since domain II of CCS is homologous to SOD1, we also tested whether SOD1 could bind to the X11α PDZ domain fusion proteins. No interaction between any of the X11α PDZ domains and SOD1 was detected (Fig. 3 A), which is consistent with the immunoprecipitation experiments (Fig.2 B). In a complementary series of experiments, we used GST fusion proteins containing different domains of CCS in pull-down experiments from X11α-transfected CHO cells. X11α bound to full-length CCS, CCS-(48–274), which includes both domains II and III and replicates the results of the yeast two-hybrid screen, and also to CCS-(243–274) (i.e. the C-terminal 32 amino acids of domain III) but not to CCS-(48–241). GST alone did not bind X11α (Fig. 3 B). Thus sequences within domain III of CCS mediate the interaction with X11α. The strength of the signals obtained using these CCS baits was generally weaker than those with the X11α baits. However, it is notable that in these experiments the strength of signals obtained with the CCS baits was similar to that obtained with a GST bait containing the C-terminal 47-amino acid cytoplasmic domain of APP (Fig. 3 B), which is known to bind to all X11 proteins (2McLoughlin D.M. Miller C.C.J. FEBS Lett. 1996; 397: 197-200Crossref PubMed Scopus (134) Google Scholar, 6Tanahashi H. Tabira T. Biochem. Biophys. Res. Commun. 1999; 255: 663-667Crossref PubMed Scopus (72) Google Scholar, 7Tomita S. Ozaki T. Taru H. Oguchi S. Takeda S. Yagi Y. Sakiyama S. Kirino Y. Suzuki T. J. Biol. Chem. 1999; 274: 2243-2254Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 11Borg J.-P. Ooi J. Levy E. Margolis B. Mol. Cell. Biol. 1996; 16: 6229-6241Crossref PubMed Scopus (436) Google Scholar, 12Zhang Z. Lee C.-H. Mandiyan V. Borg J.-P. Margolis B. Schlessinger J. Kuriyan J. EMBO J. 1997; 16: 6141-6150Crossref PubMed Scopus (139) Google Scholar). We also tested whether the CCS baits would interact with SOD1 in these pull-down assays. Only sequences that included domain II bound SOD1, a finding consistent with previous reports (26Casareno R.L.B. Waggoner D. Gitlin J.D. J. Biol. Chem. 1998; 273: 23625-23628Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar) (Fig. 3 B). Taken together, these binding studies indicate that the interaction between human X11α and CCS is mediated by the second PDZ domain of X11α and sequences within domain III of CCS. Immunocytochemical labeling of X11α/CCS cotransfected CHO cells revealed that both proteins had overlapping distribution patterns, particularly in the perinuclear region (Fig. 4 A). X11α and CCS also displayed an overlapping distribution pattern in rat primary cortical neurons where staining for both proteins was particularly pronounced in cell bodies with weaker labeling of neurites (Fig.4 B). These observations are consistent with previous reports that X11α is present in cell bodies, possibly including the Golgi apparatus and to a lesser extent in axons, dendrites, and synapses (9Borg J.P. Lopez-Figueroa M.O. De Taddéo-Borg M. Kroon D.E. Turner R.S. Watson S.J. Margolis B. J. Neurosci. 1999; 19: 1307-1316Crossref PubMed Google Scholar,38Okamoto M. Matsuyama T. Sugita M. Eur. J. Neurosci. 2000; 12: 3067-3072Crossref PubMed Scopus (27) Google Scholar), and that CCS is enriched in neuronal cell bodies (39Rothstein J.D. Dykes-Hoberg M. Corson L.B. Becker M. Cleveland D.W. Price D.L. Culotta V.C. Wong P.C. J. Neurochem. 1999; 72: 422-429Crossref PubMed Scopus (107) Google Scholar). CCS delivers the metal ion copper cofactor to SOD1 (24Culotta V.C. Klomp L.W.J. Strain J. Casareno R.L.B. Krems B. Gitlin J.D. J. Biol. Chem. 1997; 272: 23469-23472Abstract Full Text Full Text PDF PubMed Scopus (686) Google Scholar, 25Corson L.B. Strain J.J. Culotta V.C. Cleveland D.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6361-6366Crossref PubMed Scopus (143) Google Scholar, 26Casareno R.L.B. Waggoner D. Gitlin J.D. J. Biol. Chem. 1998; 273: 23625-23628Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 27Gamonet F. Lauquin G.J.M. Eur. J. Biochem. 1998; 251: 716-723Crossref PubMed Scopus (42) Google Scholar, 28Rae T.D. Schmidt P.J. Pufahl R.A. Culotta V.C. O'Halloran T.V. Science. 1999; 284: 805-808Crossref PubMed Scopus (1379) Google Scholar), and copper is required for SOD1 activity (40Fridovich I. Annu. Rev. Biochem. 1995; 64: 97-112Crossref PubMed Scopus (2755) Google Scholar). We therefore tested whether expression of X11α could influence SOD1 activity. SOD1 activity assays were performed on mock-transfected CHO cells, and CHO cells transfected with SOD1, SOD1 + X11α, SOD + CCS, and SOD1 + CCS + X11α (Fig.5 A). Immunoblot analyses demonstrated that similar levels of transfected SOD1 were expressed in the different transfections (Fig. 5 B). However, SOD1 activity was significantly reduced in CHO cells expressing X11α (Kruskal-Wallis χ2 = 8.74, D.F. = 3,p = 0.03; Fig. 5, A and C). This included cells that were not cotransfected with CCS (Fig. 5 A, track 3) which demonstrates inhibition of the action of endogenous CCS by X11α. In the present study we have demonstrated that human X11α interacts with CCS in a variety of biochemical assays and that X11α and CCS display overlapping subcellular distribution patterns in both neurons and transfected CHO cells. The interaction between X11α and CCS is mediated via the second PDZ domain of X11α and sequences within the C-terminal 32 amino acids of CCS. CCS is the first reported ligand specific for PDZ2 of X11α. The prototypical PDZ domain binding sequence is the C-terminal motif X(S/T)X(V/I/L). It is now apparent, however, that PDZ domains bind to a much broader range of C-terminal sequences (for example see Ref. 41Songyang Z. Fanning A.S. Fu C. Xu J. Marfatia S.M. Chishti A.H. Crompton A. Chan A.C. Anderson J.M. Cantley L.C. Science. 1997; 275: 73-77Crossref PubMed Scopus (1224) Google Scholar) and can also bind to internal sequences as well as to other PDZ domains (13Fanning A.S. Anderson J.M. J. Clin. Invest. 1999; 103: 767-772Crossref PubMed Scopus (401) Google Scholar, 23Garner C.C. Nash J. Huganir R.L. Trends Cell Biol. 2000; 10: 274-280Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar,42Oschkinat H. Nat. Struct. Biol. 1999; 6: 408-410Crossref PubMed Scopus (17) Google Scholar). It is thus possible that PDZ2 of X11α binds to the C terminus of CCS (i.e. PAHL) or alternatively to an internal sequence within the C-terminal 32 amino acids of domain III. The anti-oxidant enzyme SOD1 catalyzes the disproportionation of superoxide anions through redox cycling of the bound copper ion in each monomer (40Fridovich I. Annu. Rev. Biochem. 1995; 64: 97-112Crossref PubMed Scopus (2755) Google Scholar). Copper is delivered to SOD1 via CCS, and CCS is thus required for SOD1 activity (24Culotta V.C. Klomp L.W.J. Strain J. Casareno R.L.B. Krems B. Gitlin J.D. J. Biol. Chem. 1997; 272: 23469-23472Abstract Full Text Full Text PDF PubMed Scopus (686) Google Scholar, 25Corson L.B. Strain J.J. Culotta V.C. Cleveland D.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6361-6366Crossref PubMed Scopus (143) Google Scholar, 27Gamonet F. Lauquin G.J.M. Eur. J. Biochem. 1998; 251: 716-723Crossref PubMed Scopus (42) Google Scholar, 28Rae T.D. Schmidt P.J. Pufahl R.A. Culotta V.C. O'Halloran T.V. Science. 1999; 284: 805-808Crossref PubMed Scopus (1379) Google Scholar, 43Wong P.C. Waggoner D. Subramaniam J.R. Tessarollo L. Bartnikas T.B. Culotta V.C. Price D.L. Rothstein J. Gitlin J.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2886-2891Crossref PubMed Scopus (265) Google Scholar). CCS comprises three domains as follows: an N-terminal domain I that is homologous to the Atx1p metallochaperone and that is involved in the recruitment of copper; a central domain II that exhibits homology to SOD1 and that facilitates the interaction of CCS with SOD1; and a C-terminal domain III that also binds copper but that additionally is involved in the interaction with SOD1 (26Casareno R.L.B. Waggoner D. Gitlin J.D. J. Biol. Chem. 1998; 273: 23625-23628Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 31Schmidt P.J. Rae T.D. Pufahl R.A. Hamma T. Strain J. O'Halloran T.V. Culotta V.C. J. Biol. Chem. 1999; 274: 23719-23725Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 44Schmidt P.J. Kunst C. Culotta V.C. J. Biol. Chem. 2000; 275: 33771-33776Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 45Rae T.D. Torres A.T. Pufahl R.A. O'Halloran T.V. J. Biol. Chem. 2001; 276: 5166-5176Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). We mapped the X11α-binding site to the C-terminal 32 amino acids of CCS, i.e. within domain III. Overexpression of X11α led to a down-regulation of SOD1 activity in transfected CHO cells, and this was not due to an effect of X11α on expression of either SOD1 or CCS (Fig. 5 B). These observations suggest that X11α binding to CCS is inhibitory to CCS function. Recent studies have highlighted the crucial importance of domain III in the delivery of copper from CCS to SOD1 (31Schmidt P.J. Rae T.D. Pufahl R.A. Hamma T. Strain J. O'Halloran T.V. Culotta V.C. J. Biol. Chem. 1999; 274: 23719-23725Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 44Schmidt P.J. Kunst C. Culotta V.C. J. Biol. Chem. 2000; 275: 33771-33776Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 45Rae T.D. Torres A.T. Pufahl R.A. O'Halloran T.V. J. Biol. Chem. 2001; 276: 5166-5176Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). One suggestion is that domain III extends into the active site of SOD1 to facilitate insertion of copper into the enzyme (31Schmidt P.J. Rae T.D. Pufahl R.A. Hamma T. Strain J. O'Halloran T.V. Culotta V.C. J. Biol. Chem. 1999; 274: 23719-23725Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 44Schmidt P.J. Kunst C. Culotta V.C. J. Biol. Chem. 2000; 275: 33771-33776Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 45Rae T.D. Torres A.T. Pufahl R.A. O'Halloran T.V. J. Biol. Chem. 2001; 276: 5166-5176Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Thus, X11α may inhibit SOD1 activity by binding to CCS domain III and disrupting this process in some way. Altered copper homeostasis may be at least part of the pathogenic process in several neurodegenerative diseases (for reviews see Refs. 46Waggoner D.J. Bartnikas T.B. Gitlin J.D. Neurobiol. Dis. 1999; 6: 221-230Crossref PubMed Scopus (771) Google Scholarand 47Bush A.I. Curr. Opin. Chem. Biol. 2000; 4: 184-191Crossref PubMed Scopus (702) Google Scholar). In particular, mutations in SOD1 are the causative genetic defect in some familial forms of amyotrophic lateral sclerosis (ALS) (48Rosen D.R. Siddique T. Patterson D. Figlewicz D.A. Sapp P. Hentati A. Donaldson D. Goto J. O'Regan J.P. Deng H.X. Brown R. Nature. 1993; 362: 59-62Crossref PubMed Scopus (5568) Google Scholar, 49Deng H.X. Hentati A. Tainer J.A. Iqbal Z. Cayabyab A. Hung W.Y. Getzoff E.D. Hu P. Herzfeldt B. Roos R.P. Warner C. Deng G. Soriano E. Smyth C. Parge H.E. Ahmed A. Roses A.D. Hallewell R.A. Pericakvance M.A. Siddique T. Science. 1993; 261: 1047-1051Crossref PubMed Scopus (1360) Google Scholar). ALS mutant SOD1 proteins can bind CCS, and it has been suggested that mutant SOD1 may exert a toxic effect via altered cellular copper chemistry (25Corson L.B. Strain J.J. Culotta V.C. Cleveland D.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6361-6366Crossref PubMed Scopus (143) Google Scholar). In addition to binding CCS, X11α interacts with another copper-binding protein, APP, mutations in which can cause Alzheimer's disease (see for review Ref. 50Selkoe D.J. Nature. 1999; 399: 23-31Crossref PubMed Scopus (1534) Google Scholar). APP has a conserved copper binding region in its ectodomain (51Hesse L. Beher D. Masters C.L. Multhaup G. FEBS Lett. 1994; 349: 109-116Crossref PubMed Scopus (223) Google Scholar) and may play a role in the transport of copper to different cellular compartments (46Waggoner D.J. Bartnikas T.B. Gitlin J.D. Neurobiol. Dis. 1999; 6: 221-230Crossref PubMed Scopus (771) Google Scholar). APP has been shown to reduce Cu(II) to Cu(I) upon binding Cu(II) and may thereby mediate copper-induced toxicity and oxidative stress in neurons (52Multhaup G. Schlicksupp A. Hesse L. Beher D. Ruppert T. Masters C.L. Beyreuther K. Science. 1996; 271: 1406-1409Crossref PubMed Scopus (591) Google Scholar, 53Multhaup G. Ruppert T. Schlicksupp A. Hesse L. Bill E. Pipkorn R. Masters C.L. Beyreuther K. Biochemistry. 1998; 37: 7224-7230Crossref PubMed Scopus (135) Google Scholar, 54White A.R. Multhaup G. Maher F. Bellingham S. Camakaris J. Zheng H. Bush A.I. Beyreuther K. Masters C.L. Cappai R. J. Neurosci. 1999; 19: 9170-9179Crossref PubMed Google Scholar). Thus, through its interactions with both CCS and APP, X11α may play a general role in copper homeostasis within neurons. Defective copper metabolism, perhaps involving X11α, may be mechanistic in both ALS and Alzheimer's disease." @default.
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W2123170497 title "The Neuronal Adaptor Protein X11α Interacts with the Copper Chaperone for SOD1 and Regulates SOD1 Activity" @default.
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