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• W2046274277 abstract "The Menkes protein (MNK) is a copper-transporting P-type ATPase, which has six highly conserved metal-binding sites, GMTCXXC, at the N terminus. The metal-binding sites may be involved in MNK trafficking and/or copper-translocating activity. In this study, we report the detailed functional analysis in mammalian cells of recombinant human MNK and its mutants with various metal-binding sites altered by site-directed mutagenesis. The results of the study, both in vitro and in vivo, provide evidence that the metal-binding sites of MNK are not essential for the ATP-dependent copper-translocating activity of MNK. Moreover, metal-binding site mutations, which resulted in a loss of ability of MNK to traffick to the plasma membrane, produced a copper hyperaccumulating phenotype. Using an in vitro vesicle assay, we demonstrated that the apparent Km and Vmax values for the wild type MNK and its mutants were not significantly different. The results of this study suggest that copper-translocating activity of MNK and its copper-induced relocalization to the plasma membrane represent a well coordinated copper homeostasis system. It is proposed that mutations in MNK which alter either its catalytic activity or/and ability to traffick can be the cause of Menkes disease. The Menkes protein (MNK) is a copper-transporting P-type ATPase, which has six highly conserved metal-binding sites, GMTCXXC, at the N terminus. The metal-binding sites may be involved in MNK trafficking and/or copper-translocating activity. In this study, we report the detailed functional analysis in mammalian cells of recombinant human MNK and its mutants with various metal-binding sites altered by site-directed mutagenesis. The results of the study, both in vitro and in vivo, provide evidence that the metal-binding sites of MNK are not essential for the ATP-dependent copper-translocating activity of MNK. Moreover, metal-binding site mutations, which resulted in a loss of ability of MNK to traffick to the plasma membrane, produced a copper hyperaccumulating phenotype. Using an in vitro vesicle assay, we demonstrated that the apparent Km and Vmax values for the wild type MNK and its mutants were not significantly different. The results of this study suggest that copper-translocating activity of MNK and its copper-induced relocalization to the plasma membrane represent a well coordinated copper homeostasis system. It is proposed that mutations in MNK which alter either its catalytic activity or/and ability to traffick can be the cause of Menkes disease. The Menkes protein (MNK, ATP7A) 1The abbreviations used are: MNK, human Menkes protein; WND, Wilson protein; MBS, metal-binding site; PM, plasma membrane; TGN, trans-Golgi network; EV, empty expression vector; DTT, dithiothreitol; CHO cells, Chinese hamster ovary cells; ANOVA, analysis of variance.1The abbreviations used are: MNK, human Menkes protein; WND, Wilson protein; MBS, metal-binding site; PM, plasma membrane; TGN, trans-Golgi network; EV, empty expression vector; DTT, dithiothreitol; CHO cells, Chinese hamster ovary cells; ANOVA, analysis of variance. is a copper-transporting P-type ATPase (1Vulpe C. Levinson B. Whitney S. Packman S. Gitschier J. Nat. Genet. 1993; 3: 7-13Crossref PubMed Scopus (1208) Google Scholar, 2Chelly J. Tumer Z. Tonnesen T. Petterson A. Ishikawa Brush Y. Tommerup N. Horn N. Monaco A.P. Nat. Genet. 1993; 3: 14-19Crossref PubMed Scopus (623) Google Scholar, 3Mercer J.F. Livingston J. Hall B. Paynter J.A. Begy C. Chandrasekharappa S. Lockhart P. Grimes A. Bhave M. Siemieniak D. Glover T.W. Nat. Genet. 1993; 3: 20-25Crossref PubMed Scopus (624) Google Scholar) found in most tissues except the liver. Mutations in the MNK gene cause Menkes disease, a disorder associated with systemic copper deficiency, which is believed to be because of low copper absorption from the small intestine (4Danks D.M. Scriver C.R. Beaudet A.L. Sly W.V. Valle D. The Metabolic Basis of Inherited Disease. McGraw-Hill, New York1995: 2211-2235Google Scholar). Severe neurodegenerative and connective tissue disorders observed in Menkes patients are thought to be caused by partial or complete loss of catalytic activity of essential cuproenzymes (4Danks D.M. Scriver C.R. Beaudet A.L. Sly W.V. Valle D. The Metabolic Basis of Inherited Disease. McGraw-Hill, New York1995: 2211-2235Google Scholar, 5Mercer J.F.B. Camakaris J. Silver S. Walden W. Metal Ions in Gene Regulation. Chapman and Hall, New York1997: 250-276Google Scholar).The cDNA-derived amino acid sequence of the MNK protein reveals significant structural similarity with transmembrane P-type ATPases, a common class of cation-transporting transmembrane proteins (6Solioz M. Odermatt A. Krapf R. FEBS Lett. 1994; 346: 44-47Crossref PubMed Scopus (107) Google Scholar). Among these are heavy metal-transporting Cu+/2+ and Cd2+ ATPases (7Møller J.V. Juul B. le Maire M. Biochim. Biophys. Acta. 1996; 1286: 1-51Crossref PubMed Scopus (656) Google Scholar). A unique feature of copper-transporting P-type ATPases is the presence of putative metal-binding site(s) (MBS), GMCXXC, in the N-terminal region: one in bacteriaEnterococcus hirae (CopB), two in yeast Saccharomyces cerevisiae (Ccc2p), three in nematodes Caenorhabditis elegans (8Sambongi Y. Wakabayashi T. Yoshimizu T. Omote H. Oka T. Futai M. J. Biochem (Tokyo). 1997; 121: 1169-1175Crossref PubMed Scopus (50) Google Scholar), and six in mammals (MNK and Wilson protein, WND) (9Koch K.A.O. Pena M.M. Thiele D.J. Chem. Biol. 1997; 4: 549-560Abstract Full Text PDF PubMed Scopus (127) Google Scholar). The GMCXXC motif is also present in the putative copper chaperones Atox1 and Atx1, which have been proposed to deliver copper to MNK and Ccc2p, respectively, via a ligand exchange mechanism (10Pufahl R.A. Singer C.P. Peariso K.L. Lin S.-J. Schmidt P. Cizewski-Culotta V. Penner-Hahn J.E. O'Halloran T.V. Science. 1997; 278: 853-856Crossref PubMed Scopus (585) Google Scholar).Several studies have been conducted in an attempt to elucidate the role of MBSs. Lutsenko et al. (11Lutsenko S. Petrukhin K. Cooper M.J. Gilliam C.T. Kaplan J.H. J. Biol. Chem. 1997; 272: 18939-18944Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar) have demonstrated that the N-terminal domain of MNK binds six atoms of copper per molecule, suggesting each MBS binds one copper. By progressively mutating the MBS of MNK, Payne et al. (12Payne A.S. Gitlin J.D. J. Biol. Chem. 1998; 273: 3765-3770Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar) have recently demonstrated that MBSs 3–6 are required to complement the ΔCCC2 phenotype in S. cerevisiae. In contrast, Iida et al. (13Iida M. Terada K. Sambongi Y. Wakabayashi T. Miura N. Koyama K. Futai M. Sugiyama T. FEBS Lett. 1998; 428: 281-285Crossref PubMed Scopus (96) Google Scholar) have shown that only MBS 6 in WND was required to rescue the ΔCCC2 phenotype. Vulpe, et al. (14Vulpe C. Yuan D. Ibom V. Gitschier J. Copper and Zinc Receptors in Signalling, Trafficking and Disease. ASBMB, Granlibakken, Lake Tahoe, CA1997: 35Google Scholar) have provided evidence that the ΔCCC2 phenotype can be complemented by mutant forms of Ccc2p, but at least one MBS was required. In contrast, it has been shown that the mutation of the only MBS in the cadmium-transporting P-type ATPase in Staphylococcus aureus (CadA) reduced but did not abolish the catalytic activity of the protein, suggesting that the single MBS was not essential for the translocation of cadmium (15Konings W.N. Kaback H.R. Lolkema J.S. Transport Processes in Eukaryotic and Prokaryotic Organisms. Elsevier Science Publishers B.V., Amsterdam1996: 3-4Google Scholar).An important aspect of MNK physiology is that copper regulates the intracellular location of the protein (16Petris M.J. Mercer J.F. Culvenor J.G. Lockhart P. Gleeson P.A. Camakaris J. EMBO J. 1996; 15: 6084-6095Crossref PubMed Scopus (528) Google Scholar). MNK normally resides in the trans-Golgi network (TGN) (16Petris M.J. Mercer J.F. Culvenor J.G. Lockhart P. Gleeson P.A. Camakaris J. EMBO J. 1996; 15: 6084-6095Crossref PubMed Scopus (528) Google Scholar, 17Yamaguchi Y. Heiny M.E. Suzuki M. Gitlin J.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14030-14035Crossref PubMed Scopus (190) Google Scholar, 18Dierick H.A. Adam A.N. Escara-Wilke J.F. Glover T.W. Hum. Mol. Genet. 1997; 6: 409-416Crossref PubMed Scopus (98) Google Scholar), but the elevation of extracellular concentrations of copper results in translocation of MNK from the TGN to the plasma membrane (PM), thus presumably facilitating copper efflux (16Petris M.J. Mercer J.F. Culvenor J.G. Lockhart P. Gleeson P.A. Camakaris J. EMBO J. 1996; 15: 6084-6095Crossref PubMed Scopus (528) Google Scholar). We have proposed, therefore, that MNK can maintain copper homeostasis by means of vesicular trafficking and ATP-dependent copper-translocating activity (16Petris M.J. Mercer J.F. Culvenor J.G. Lockhart P. Gleeson P.A. Camakaris J. EMBO J. 1996; 15: 6084-6095Crossref PubMed Scopus (528) Google Scholar). Strausaket al. (19Strausak D. La Fontaine S. Hill J. Firth S.D. Lockhart P.J. Mercer J.F. J. Biol. Chem. 1999; 274: 11170-11177Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar) demonstrated recently that CXXC to SXXS mutations in MBSs 4–6 or 1–6 abolished the copper-stimulated trafficking of MNK to the PM, whereas mutations in MBSs 1–3 had no effect on the trafficking of MNK compared with the wild-type protein. An important yet unanswered question is whether the processes of MNK trafficking and copper-translocating activity are co-dependent or independent events.The studies presented in this paper provide the first direct evidence of catalytic activity of human MNK and its variants with mutated MBSs expressed in mammalian cells. Recently we demonstrated that mammalian MNK translocates copper across membranes in vitro, and this is ATP-dependent (20Voskoboinik I. Brooks H. Smith S. Shen P. Camakaris J. FEBS Lett. 1998; 435: 178-182Crossref PubMed Scopus (62) Google Scholar). In this paper we used a similarin vitro system to demonstrate that MBSs are not essential for copper-translocating activity of MNK. Moreover, studies using whole cells demonstrated that mutations in MBSs 4–6 or all six MBSs resulted in a copper-accumulating phenotype. This phenomenon coincided with the inability of the same mutant proteins to traffick to the PM in response to copper (19Strausak D. La Fontaine S. Hill J. Firth S.D. Lockhart P.J. Mercer J.F. J. Biol. Chem. 1999; 274: 11170-11177Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Taken together, our results indicate that MNK trafficking and copper-translocating activity are integral components of intracellular copper homeostasis.DISCUSSIONThe present study provides the first detailed functional analysis of N-terminal putative copper-binding motifs (MBS) of MNK in mammalian cells by studying mutants where these motifs have been altered by site-directed mutagenesis. The overexpression of the normal and mutant MNKs enabled us to analyze the kinetics of MNK-mediated copper translocation using an in vitro vesicle assay. The results have shown that putative MBSs in the N-terminal domain of MNK were not essential for copper-translocating activity of MNK, as all the mutated proteins investigated retained their ATP-dependent copper-translocating activity (Table I, Fig. 4).The in vivo results (Fig. 5 and 6) are particularly noteworthy and are consistent with the retention of copper transport activity of the mutant MNKs in vitro, but those mutants which have lost the ability to traffick in response to copper (115 and 116) actually lead to enhanced copper accumulation in whole cells relative to the EV control. We suggest that this is a result of these mutant MNK proteins transporting copper into an intracellular compartment, presumably the TGN, from where there is little or no copper-regulated trafficking of MNK to the PM (19Strausak D. La Fontaine S. Hill J. Firth S.D. Lockhart P.J. Mercer J.F. J. Biol. Chem. 1999; 274: 11170-11177Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar), and thus is substantially reducing copper efflux from these cells. The small reduction in 64Cu accumulation in mutant 115 (6 h, 189 μm copper; Fig. 5) may be because of copper efflux occurring as a consequence of a constitutive (copper-independent) recycling pool of catalytically active MNK (16Petris M.J. Mercer J.F. Culvenor J.G. Lockhart P. Gleeson P.A. Camakaris J. EMBO J. 1996; 15: 6084-6095Crossref PubMed Scopus (528) Google Scholar). Given the low expression of MNK in 115 cells (compared with 116), such a pool would be proportionately higher relative to the pool of catalytically active MNK that does not traffick in response to copper.The role of putative MBSs in the N terminus of MNK and other copper-transporting ATPases and in particular “the reason” for the six MBSs in the mammalian copper ATPases is not understood. Previous studies utilizing yeast complementation assays have suggested, in contrast to our results, that the MBSs are needed for the copper-transporting activity of MNK, WND, and Ccc2p (Refs. 12Payne A.S. Gitlin J.D. J. Biol. Chem. 1998; 273: 3765-3770Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 13Iida M. Terada K. Sambongi Y. Wakabayashi T. Miura N. Koyama K. Futai M. Sugiyama T. FEBS Lett. 1998; 428: 281-285Crossref PubMed Scopus (96) Google Scholar, 14Vulpe C. Yuan D. Ibom V. Gitschier J. Copper and Zinc Receptors in Signalling, Trafficking and Disease. ASBMB, Granlibakken, Lake Tahoe, CA1997: 35Google Scholar; see the Introduction). The yeast system involves an indirect measurement of the activity of MNK, WND, and Ccc2p through ability to form the [Fet 3-Cu] complex (12Payne A.S. Gitlin J.D. J. Biol. Chem. 1998; 273: 3765-3770Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 13Iida M. Terada K. Sambongi Y. Wakabayashi T. Miura N. Koyama K. Futai M. Sugiyama T. FEBS Lett. 1998; 428: 281-285Crossref PubMed Scopus (96) Google Scholar, 14Vulpe C. Yuan D. Ibom V. Gitschier J. Copper and Zinc Receptors in Signalling, Trafficking and Disease. ASBMB, Granlibakken, Lake Tahoe, CA1997: 35Google Scholar) in copper-deficient medium. Mutations of MNK or WND could reduce, but not abolish, their catalytic activities (as reported in this paper), but the reduced activity may be insufficient to complement the ΔCCC2 phenotype. In addition, MBSs may be required to scavenge and concentrate the low amounts of copper present used in the yeast ΔCCC2 complementation assay. It is noteworthy that, using a direct assay on the Cd2+-transporting P-type ATPase CadA in S. aureus, Nucifora et al. (26Nucifora G. Chu L. Misra T.K. Silver S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 3544-3548Crossref PubMed Scopus (288) Google Scholar) demonstrated that this protein contains only one MBS, and the mutations of cysteines in the sequence GFTCANC reduced but did not abolish the rates of Cd2+ transport (15Konings W.N. Kaback H.R. Lolkema J.S. Transport Processes in Eukaryotic and Prokaryotic Organisms. Elsevier Science Publishers B.V., Amsterdam1996: 3-4Google Scholar). Therefore it is clear that the metal-binding sites are required for some of the functions carried out by these enzymes, but our in vitro results show that copper translocation is possible without the N-terminal CXXC motifs. It is not possible to reconcile the results from the different groups at this stage. However, one should also consider the possibility that there may be a number of modes of delivery of copper to the ion channel, one involving the MBSs and another MBS independent, and the various assays are measuring these distinct modes of presentation.Related to this point is the involvement of copper chaperones,e.g. Atx1 in yeast and Atox1 in humans, in the delivery of copper to the enzymes. The inability of MBS mutants 115 and 116 to traffick, while being catalytically active suggests that one of the roles of MBSs is copper binding, possibly via the [Atox 1-Cu+] complex, followed by the recognition of copper-loaded MNK by the vesicle-assembling system. In the in vitro system described here, it is possible DTT functioned not only as a reducing agent for copper, but also as a copper chaperone as it can bind copper (27Kachur A.V. Held K.D. Koch C.J. Biaglow J.E. Radiat. Res. 1997; 147: 409-415Crossref PubMed Scopus (57) Google Scholar) in a complex similar to the CXXC motif (the structure of DTT is HS-CH2(CHOH)2CH2SH). One can argue that there is a potential for copper to bypass MBS(s) and be delivered to the channel directly, especially in MBS mutants and this may be the mechanism in cells exposed to high copper. As copper translocation for all the MNK proteins tested was stimulated by ATP and inhibited by orthovanadate, this direct delivery still has the properties of a P-type ATPase transport system.In conclusion, our studies provide biochemical and physiological evidence that MBSs of human MNK are not important for its catalytic activity, but the presence of at least MBS 4, 5, and 6, which may function as sensors for copper, appears to be essential for copper-regulated trafficking (19Strausak D. La Fontaine S. Hill J. Firth S.D. Lockhart P.J. Mercer J.F. J. Biol. Chem. 1999; 274: 11170-11177Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Copper-translocating activity and copper-stimulated trafficking of MNK together represent a finely tuned regulated intracellular copper homeostasis system. The current findings suggest that MNK can perform its physiological role in detoxification of copper and in the absorption of copper from small intestine into the blood stream only when the both functions are intact. It can be predicted that mutations, which result in the loss of catalytic activity and/or copper-stimulated trafficking ability of MNK, would result in copper accumulation and, potentially, Menkes disease. The Menkes protein (MNK, ATP7A) 1The abbreviations used are: MNK, human Menkes protein; WND, Wilson protein; MBS, metal-binding site; PM, plasma membrane; TGN, trans-Golgi network; EV, empty expression vector; DTT, dithiothreitol; CHO cells, Chinese hamster ovary cells; ANOVA, analysis of variance.1The abbreviations used are: MNK, human Menkes protein; WND, Wilson protein; MBS, metal-binding site; PM, plasma membrane; TGN, trans-Golgi network; EV, empty expression vector; DTT, dithiothreitol; CHO cells, Chinese hamster ovary cells; ANOVA, analysis of variance. is a copper-transporting P-type ATPase (1Vulpe C. Levinson B. Whitney S. Packman S. Gitschier J. Nat. Genet. 1993; 3: 7-13Crossref PubMed Scopus (1208) Google Scholar, 2Chelly J. Tumer Z. Tonnesen T. Petterson A. Ishikawa Brush Y. Tommerup N. Horn N. Monaco A.P. Nat. Genet. 1993; 3: 14-19Crossref PubMed Scopus (623) Google Scholar, 3Mercer J.F. Livingston J. Hall B. Paynter J.A. Begy C. Chandrasekharappa S. Lockhart P. Grimes A. Bhave M. Siemieniak D. Glover T.W. Nat. Genet. 1993; 3: 20-25Crossref PubMed Scopus (624) Google Scholar) found in most tissues except the liver. Mutations in the MNK gene cause Menkes disease, a disorder associated with systemic copper deficiency, which is believed to be because of low copper absorption from the small intestine (4Danks D.M. Scriver C.R. Beaudet A.L. Sly W.V. Valle D. The Metabolic Basis of Inherited Disease. McGraw-Hill, New York1995: 2211-2235Google Scholar). Severe neurodegenerative and connective tissue disorders observed in Menkes patients are thought to be caused by partial or complete loss of catalytic activity of essential cuproenzymes (4Danks D.M. Scriver C.R. Beaudet A.L. Sly W.V. Valle D. The Metabolic Basis of Inherited Disease. McGraw-Hill, New York1995: 2211-2235Google Scholar, 5Mercer J.F.B. Camakaris J. Silver S. Walden W. Metal Ions in Gene Regulation. Chapman and Hall, New York1997: 250-276Google Scholar). The cDNA-derived amino acid sequence of the MNK protein reveals significant structural similarity with transmembrane P-type ATPases, a common class of cation-transporting transmembrane proteins (6Solioz M. Odermatt A. Krapf R. FEBS Lett. 1994; 346: 44-47Crossref PubMed Scopus (107) Google Scholar). Among these are heavy metal-transporting Cu+/2+ and Cd2+ ATPases (7Møller J.V. Juul B. le Maire M. Biochim. Biophys. Acta. 1996; 1286: 1-51Crossref PubMed Scopus (656) Google Scholar). A unique feature of copper-transporting P-type ATPases is the presence of putative metal-binding site(s) (MBS), GMCXXC, in the N-terminal region: one in bacteriaEnterococcus hirae (CopB), two in yeast Saccharomyces cerevisiae (Ccc2p), three in nematodes Caenorhabditis elegans (8Sambongi Y. Wakabayashi T. Yoshimizu T. Omote H. Oka T. Futai M. J. Biochem (Tokyo). 1997; 121: 1169-1175Crossref PubMed Scopus (50) Google Scholar), and six in mammals (MNK and Wilson protein, WND) (9Koch K.A.O. Pena M.M. Thiele D.J. Chem. Biol. 1997; 4: 549-560Abstract Full Text PDF PubMed Scopus (127) Google Scholar). The GMCXXC motif is also present in the putative copper chaperones Atox1 and Atx1, which have been proposed to deliver copper to MNK and Ccc2p, respectively, via a ligand exchange mechanism (10Pufahl R.A. Singer C.P. Peariso K.L. Lin S.-J. Schmidt P. Cizewski-Culotta V. Penner-Hahn J.E. O'Halloran T.V. Science. 1997; 278: 853-856Crossref PubMed Scopus (585) Google Scholar). Several studies have been conducted in an attempt to elucidate the role of MBSs. Lutsenko et al. (11Lutsenko S. Petrukhin K. Cooper M.J. Gilliam C.T. Kaplan J.H. J. Biol. Chem. 1997; 272: 18939-18944Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar) have demonstrated that the N-terminal domain of MNK binds six atoms of copper per molecule, suggesting each MBS binds one copper. By progressively mutating the MBS of MNK, Payne et al. (12Payne A.S. Gitlin J.D. J. Biol. Chem. 1998; 273: 3765-3770Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar) have recently demonstrated that MBSs 3–6 are required to complement the ΔCCC2 phenotype in S. cerevisiae. In contrast, Iida et al. (13Iida M. Terada K. Sambongi Y. Wakabayashi T. Miura N. Koyama K. Futai M. Sugiyama T. FEBS Lett. 1998; 428: 281-285Crossref PubMed Scopus (96) Google Scholar) have shown that only MBS 6 in WND was required to rescue the ΔCCC2 phenotype. Vulpe, et al. (14Vulpe C. Yuan D. Ibom V. Gitschier J. Copper and Zinc Receptors in Signalling, Trafficking and Disease. ASBMB, Granlibakken, Lake Tahoe, CA1997: 35Google Scholar) have provided evidence that the ΔCCC2 phenotype can be complemented by mutant forms of Ccc2p, but at least one MBS was required. In contrast, it has been shown that the mutation of the only MBS in the cadmium-transporting P-type ATPase in Staphylococcus aureus (CadA) reduced but did not abolish the catalytic activity of the protein, suggesting that the single MBS was not essential for the translocation of cadmium (15Konings W.N. Kaback H.R. Lolkema J.S. Transport Processes in Eukaryotic and Prokaryotic Organisms. Elsevier Science Publishers B.V., Amsterdam1996: 3-4Google Scholar). An important aspect of MNK physiology is that copper regulates the intracellular location of the protein (16Petris M.J. Mercer J.F. Culvenor J.G. Lockhart P. Gleeson P.A. Camakaris J. EMBO J. 1996; 15: 6084-6095Crossref PubMed Scopus (528) Google Scholar). MNK normally resides in the trans-Golgi network (TGN) (16Petris M.J. Mercer J.F. Culvenor J.G. Lockhart P. Gleeson P.A. Camakaris J. EMBO J. 1996; 15: 6084-6095Crossref PubMed Scopus (528) Google Scholar, 17Yamaguchi Y. Heiny M.E. Suzuki M. Gitlin J.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14030-14035Crossref PubMed Scopus (190) Google Scholar, 18Dierick H.A. Adam A.N. Escara-Wilke J.F. Glover T.W. Hum. Mol. Genet. 1997; 6: 409-416Crossref PubMed Scopus (98) Google Scholar), but the elevation of extracellular concentrations of copper results in translocation of MNK from the TGN to the plasma membrane (PM), thus presumably facilitating copper efflux (16Petris M.J. Mercer J.F. Culvenor J.G. Lockhart P. Gleeson P.A. Camakaris J. EMBO J. 1996; 15: 6084-6095Crossref PubMed Scopus (528) Google Scholar). We have proposed, therefore, that MNK can maintain copper homeostasis by means of vesicular trafficking and ATP-dependent copper-translocating activity (16Petris M.J. Mercer J.F. Culvenor J.G. Lockhart P. Gleeson P.A. Camakaris J. EMBO J. 1996; 15: 6084-6095Crossref PubMed Scopus (528) Google Scholar). Strausaket al. (19Strausak D. La Fontaine S. Hill J. Firth S.D. Lockhart P.J. Mercer J.F. J. Biol. Chem. 1999; 274: 11170-11177Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar) demonstrated recently that CXXC to SXXS mutations in MBSs 4–6 or 1–6 abolished the copper-stimulated trafficking of MNK to the PM, whereas mutations in MBSs 1–3 had no effect on the trafficking of MNK compared with the wild-type protein. An important yet unanswered question is whether the processes of MNK trafficking and copper-translocating activity are co-dependent or independent events. The studies presented in this paper provide the first direct evidence of catalytic activity of human MNK and its variants with mutated MBSs expressed in mammalian cells. Recently we demonstrated that mammalian MNK translocates copper across membranes in vitro, and this is ATP-dependent (20Voskoboinik I. Brooks H. Smith S. Shen P. Camakaris J. FEBS Lett. 1998; 435: 178-182Crossref PubMed Scopus (62) Google Scholar). In this paper we used a similarin vitro system to demonstrate that MBSs are not essential for copper-translocating activity of MNK. Moreover, studies using whole cells demonstrated that mutations in MBSs 4–6 or all six MBSs resulted in a copper-accumulating phenotype. This phenomenon coincided with the inability of the same mutant proteins to traffick to the PM in response to copper (19Strausak D. La Fontaine S. Hill J. Firth S.D. Lockhart P.J. Mercer J.F. J. Biol. Chem. 1999; 274: 11170-11177Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Taken together, our results indicate that MNK trafficking and copper-translocating activity are integral components of intracellular copper homeostasis. DISCUSSIONThe present study provides the first detailed functional analysis of N-terminal putative copper-binding motifs (MBS) of MNK in mammalian cells by studying mutants where these motifs have been altered by site-directed mutagenesis. The overexpression of the normal and mutant MNKs enabled us to analyze the kinetics of MNK-mediated copper translocation using an in vitro vesicle assay. The results have shown that putative MBSs in the N-terminal domain of MNK were not essential for copper-translocating activity of MNK, as all the mutated proteins investigated retained their ATP-dependent copper-translocating activity (Table I, Fig. 4).The in vivo results (Fig. 5 and 6) are particularly noteworthy and are consistent with the retention of copper transport activity of the mutant MNKs in vitro, but those mutants which have lost the ability to traffick in response to copper (115 and 116) actually lead to enhanced copper accumulation in whole cells relative to the EV control. We suggest that this is a result of these mutant MNK proteins transporting copper into an intracellular compartment, presumably the TGN, from where there is little or no copper-regulated trafficking of MNK to the PM (19Strausak D. La Fontaine S. Hill J. Firth S.D. Lockhart P.J. Mercer J.F. J. Biol. Chem. 1999; 274: 11170-11177Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar), and thus is substantially reducing copper efflux from these cells. The small reduction in 64Cu accumulation in mutant 115 (6 h, 189 μm copper; Fig. 5) may be because of copper efflux occurring as a consequence of a constitutive (copper-independent) recycling pool of catalytically active MNK (16Petris M.J. Mercer J.F. Culvenor J.G. Lockhart P. Gleeson P.A. Camakaris J. EMBO J. 1996; 15: 6084-6095Crossref PubMed Scopus (528) Google Scholar). Given the low expression of MNK in 115 cells (compared with 116), such a pool would be proportionately higher relative to the pool of catalytically active MNK that does not traffick in response to copper.The role of putative MBSs in the N terminus of MNK and other copper-transporting ATPases and in particular “the reason” for the six MBSs in the mammalian copper ATPases is not understood. Previous studies utilizing yeast complementation assays have suggested, in contrast to our results, that the MBSs are needed for the copper-transporting activity of MNK, WND, and Ccc2p (Refs. 12Payne A.S. Gitlin J.D. J. Biol. Chem. 1998; 273: 3765-3770Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 13Iida M. Terada K. Sambongi Y. Wakabayashi T. Miura N. Koyama K. Futai M. Sugiyama T. FEBS Lett. 1998; 428: 281-285Crossref PubMed Scopus (96) Google Scholar, 14Vulpe C. Yuan D. Ibom V. Gitschier J. Copper and Zinc Receptors in Signalling, Trafficking and Disease. ASBMB, Granlibakken, Lake Tahoe, CA1997: 35Google Scholar; see the Introduction). The yeast system involves an indirect measurement of the activity of MNK, WND, and Ccc2p through ability to form the [Fet 3-Cu] complex (12Payne A.S. Gitlin J.D. J. Biol. Chem. 1998; 273: 3765-3770Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 13Iida M. Terada K. Sambongi Y. Wakabayashi T. Miura N. Koyama K. Futai M. Sugiyama T. FEBS Lett. 1998; 428: 281-285Crossref PubMed Scopus (96) Google Scholar, 14Vulpe C. Yuan D. Ibom V. Gitschier J. Copper and Zinc Receptors in Signalling, Trafficking and Disease. ASBMB, Granlibakken, Lake Tahoe, CA1997: 35Google Scholar) in copper-deficient medium. Mutations of MNK or WND could reduce, but not abolish, their catalytic activities (as reported in this paper), but the reduced activity may be insufficient to complement the ΔCCC2 phenotype. In addition, MBSs may be required to scavenge and concentrate the low amounts of copper present used in the yeast ΔCCC2 complementation assay. It is noteworthy that, using a direct assay on the Cd2+-transporting P-type ATPase CadA in S. aureus, Nucifora et al. (26Nucifora G. Chu L. Misra T.K. Silver S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 3544-3548Crossref PubMed Scopus (288) Google Scholar) demonstrated that this protein contains only one MBS, and the mutations of cysteines in the sequence GFTCANC reduced but did not abolish the rates of Cd2+ transport (15Konings W.N. Kaback H.R. Lolkema J.S. Transport Processes in Eukaryotic and Prokaryotic Organisms. Elsevier Science Publishers B.V., Amsterdam1996: 3-4Google Scholar). Therefore it is clear that the metal-binding sites are required for some of the functions carried out by these enzymes, but our in vitro results show that copper translocation is possible without the N-terminal CXXC motifs. It is not possible to reconcile the results from the different groups at this stage. However, one should also consider the possibility that there may be a number of modes of delivery of copper to the ion channel, one involving the MBSs and another MBS independent, and the various assays are measuring these distinct modes of presentation.Related to this point is the involvement of copper chaperones,e.g. Atx1 in yeast and Atox1 in humans, in the delivery of copper to the enzymes. The inability of MBS mutants 115 and 116 to traffick, while being catalytically active suggests that one of the roles of MBSs is copper binding, possibly via the [Atox 1-Cu+] complex, followed by the recognition of copper-loaded MNK by the vesicle-assembling system. In the in vitro system described here, it is possible DTT functioned not only as a reducing agent for copper, but also as a copper chaperone as it can bind copper (27Kachur A.V. Held K.D. Koch C.J. Biaglow J.E. Radiat. Res. 1997; 147: 409-415Crossref PubMed Scopus (57) Google Scholar) in a complex similar to the CXXC motif (the structure of DTT is HS-CH2(CHOH)2CH2SH). One can argue that there is a potential for copper to bypass MBS(s) and be delivered to the channel directly, especially in MBS mutants and this may be the mechanism in cells exposed to high copper. As copper translocation for all the MNK proteins tested was stimulated by ATP and inhibited by orthovanadate, this direct delivery still has the properties of a P-type ATPase transport system.In conclusion, our studies provide biochemical and physiological evidence that MBSs of human MNK are not important for its catalytic activity, but the presence of at least MBS 4, 5, and 6, which may function as sensors for copper, appears to be essential for copper-regulated trafficking (19Strausak D. La Fontaine S. Hill J. Firth S.D. Lockhart P.J. Mercer J.F. J. Biol. Chem. 1999; 274: 11170-11177Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Copper-translocating activity and copper-stimulated trafficking of MNK together represent a finely tuned regulated intracellular copper homeostasis system. The current findings suggest that MNK can perform its physiological role in detoxification of copper and in the absorption of copper from small intestine into the blood stream only when the both functions are intact. It can be predicted that mutations, which result in the loss of catalytic activity and/or copper-stimulated trafficking ability of MNK, would result in copper accumulation and, potentially, Menkes disease. The present study provides the first detailed functional analysis of N-terminal putative copper-binding motifs (MBS) of MNK in mammalian cells by studying mutants where these motifs have been altered by site-directed mutagenesis. The overexpression of the normal and mutant MNKs enabled us to analyze the kinetics of MNK-mediated copper translocation using an in vitro vesicle assay. The results have shown that putative MBSs in the N-terminal domain of MNK were not essential for copper-translocating activity of MNK, as all the mutated proteins investigated retained their ATP-dependent copper-translocating activity (Table I, Fig. 4). The in vivo results (Fig. 5 and 6) are particularly noteworthy and are consistent with the retention of copper transport activity of the mutant MNKs in vitro, but those mutants which have lost the ability to traffick in response to copper (115 and 116) actually lead to enhanced copper accumulation in whole cells relative to the EV control. We suggest that this is a result of these mutant MNK proteins transporting copper into an intracellular compartment, presumably the TGN, from where there is little or no copper-regulated trafficking of MNK to the PM (19Strausak D. La Fontaine S. Hill J. Firth S.D. Lockhart P.J. Mercer J.F. J. Biol. Chem. 1999; 274: 11170-11177Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar), and thus is substantially reducing copper efflux from these cells. The small reduction in 64Cu accumulation in mutant 115 (6 h, 189 μm copper; Fig. 5) may be because of copper efflux occurring as a consequence of a constitutive (copper-independent) recycling pool of catalytically active MNK (16Petris M.J. Mercer J.F. Culvenor J.G. Lockhart P. Gleeson P.A. Camakaris J. EMBO J. 1996; 15: 6084-6095Crossref PubMed Scopus (528) Google Scholar). Given the low expression of MNK in 115 cells (compared with 116), such a pool would be proportionately higher relative to the pool of catalytically active MNK that does not traffick in response to copper. The role of putative MBSs in the N terminus of MNK and other copper-transporting ATPases and in particular “the reason” for the six MBSs in the mammalian copper ATPases is not understood. Previous studies utilizing yeast complementation assays have suggested, in contrast to our results, that the MBSs are needed for the copper-transporting activity of MNK, WND, and Ccc2p (Refs. 12Payne A.S. Gitlin J.D. J. Biol. Chem. 1998; 273: 3765-3770Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 13Iida M. Terada K. Sambongi Y. Wakabayashi T. Miura N. Koyama K. Futai M. Sugiyama T. FEBS Lett. 1998; 428: 281-285Crossref PubMed Scopus (96) Google Scholar, 14Vulpe C. Yuan D. Ibom V. Gitschier J. Copper and Zinc Receptors in Signalling, Trafficking and Disease. ASBMB, Granlibakken, Lake Tahoe, CA1997: 35Google Scholar; see the Introduction). The yeast system involves an indirect measurement of the activity of MNK, WND, and Ccc2p through ability to form the [Fet 3-Cu] complex (12Payne A.S. Gitlin J.D. J. Biol. Chem. 1998; 273: 3765-3770Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 13Iida M. Terada K. Sambongi Y. Wakabayashi T. Miura N. Koyama K. Futai M. Sugiyama T. FEBS Lett. 1998; 428: 281-285Crossref PubMed Scopus (96) Google Scholar, 14Vulpe C. Yuan D. Ibom V. Gitschier J. Copper and Zinc Receptors in Signalling, Trafficking and Disease. ASBMB, Granlibakken, Lake Tahoe, CA1997: 35Google Scholar) in copper-deficient medium. Mutations of MNK or WND could reduce, but not abolish, their catalytic activities (as reported in this paper), but the reduced activity may be insufficient to complement the ΔCCC2 phenotype. In addition, MBSs may be required to scavenge and concentrate the low amounts of copper present used in the yeast ΔCCC2 complementation assay. It is noteworthy that, using a direct assay on the Cd2+-transporting P-type ATPase CadA in S. aureus, Nucifora et al. (26Nucifora G. Chu L. Misra T.K. Silver S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 3544-3548Crossref PubMed Scopus (288) Google Scholar) demonstrated that this protein contains only one MBS, and the mutations of cysteines in the sequence GFTCANC reduced but did not abolish the rates of Cd2+ transport (15Konings W.N. Kaback H.R. Lolkema J.S. Transport Processes in Eukaryotic and Prokaryotic Organisms. Elsevier Science Publishers B.V., Amsterdam1996: 3-4Google Scholar). Therefore it is clear that the metal-binding sites are required for some of the functions carried out by these enzymes, but our in vitro results show that copper translocation is possible without the N-terminal CXXC motifs. It is not possible to reconcile the results from the different groups at this stage. However, one should also consider the possibility that there may be a number of modes of delivery of copper to the ion channel, one involving the MBSs and another MBS independent, and the various assays are measuring these distinct modes of presentation. Related to this point is the involvement of copper chaperones,e.g. Atx1 in yeast and Atox1 in humans, in the delivery of copper to the enzymes. The inability of MBS mutants 115 and 116 to traffick, while being catalytically active suggests that one of the roles of MBSs is copper binding, possibly via the [Atox 1-Cu+] complex, followed by the recognition of copper-loaded MNK by the vesicle-assembling system. In the in vitro system described here, it is possible DTT functioned not only as a reducing agent for copper, but also as a copper chaperone as it can bind copper (27Kachur A.V. Held K.D. Koch C.J. Biaglow J.E. Radiat. Res. 1997; 147: 409-415Crossref PubMed Scopus (57) Google Scholar) in a complex similar to the CXXC motif (the structure of DTT is HS-CH2(CHOH)2CH2SH). One can argue that there is a potential for copper to bypass MBS(s) and be delivered to the channel directly, especially in MBS mutants and this may be the mechanism in cells exposed to high copper. As copper translocation for all the MNK proteins tested was stimulated by ATP and inhibited by orthovanadate, this direct delivery still has the properties of a P-type ATPase transport system. In conclusion, our studies provide biochemical and physiological evidence that MBSs of human MNK are not important for its catalytic activity, but the presence of at least MBS 4, 5, and 6, which may function as sensors for copper, appears to be essential for copper-regulated trafficking (19Strausak D. La Fontaine S. Hill J. Firth S.D. Lockhart P.J. Mercer J.F. J. Biol. Chem. 1999; 274: 11170-11177Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Copper-translocating activity and copper-stimulated trafficking of MNK together represent a finely tuned regulated intracellular copper homeostasis system. The current findings suggest that MNK can perform its physiological role in detoxification of copper and in the absorption of copper from small intestine into the blood stream only when the both functions are intact. It can be predicted that mutations, which result in the loss of catalytic activity and/or copper-stimulated trafficking ability of MNK, would result in copper accumulation and, potentially, Menkes disease. Functional analysis of the N-terminal CXXC metal-binding motifs in the human Menkes copper-transporting P-type ATPase expressed in cultured mammalian cells.Journal of Biological ChemistryVol. 274Issue 50PreviewPage 22009, Table I: An arithmetical error occurred when converting units. Columns 2 and 4: pmol/min/mg should be nmol/min/mg. Footnotes a and c to this table: pmol of Cu/min/mg should be nmol of Cu/min/mg. The correct table is shown below. Full-Text PDF Open Access" @default.
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