Parkin Suppresses Unfolded Protein Stress-induced Cell Death through Its E3 Ubiquitin-protein Ligase Activity

Abstract

Autosomal recessive juvenile parkinsonism (AR-JP) is caused by mutations in the parkingene. Parkin protein is characterized by a ubiquitin-like domain at its NH2-terminus and two RING finger motifs and an IBR (in between RING fingers) at its COOH terminus (RING-IBR-RING). Here, we show that Parkin is a RING-type E3 ubiquitin-protein ligase which binds to E2 ubiquitin-conjugating enzymes, including UbcH7 and UbcH8, through its RING-IBR-RING motif. Moreover, we found that unfolded protein stress induces up-regulation of both the mRNA and protein level of Parkin. Furthermore, overexpression of Parkin, but not a set of mutants without the E3 activity, specifically suppressed unfolded protein stress-induced cell death. These findings demonstrate that Parkin is an E3 enzyme and suggest that it is involved in the ubiquitination pathway for misfolded proteins derived from endoplasmic reticulum and contributes to protection from neurotoxicity induced by unfolded protein stresses. Autosomal recessive juvenile parkinsonism (AR-JP) is caused by mutations in the parkingene. Parkin protein is characterized by a ubiquitin-like domain at its NH2-terminus and two RING finger motifs and an IBR (in between RING fingers) at its COOH terminus (RING-IBR-RING). Here, we show that Parkin is a RING-type E3 ubiquitin-protein ligase which binds to E2 ubiquitin-conjugating enzymes, including UbcH7 and UbcH8, through its RING-IBR-RING motif. Moreover, we found that unfolded protein stress induces up-regulation of both the mRNA and protein level of Parkin. Furthermore, overexpression of Parkin, but not a set of mutants without the E3 activity, specifically suppressed unfolded protein stress-induced cell death. These findings demonstrate that Parkin is an E3 enzyme and suggest that it is involved in the ubiquitination pathway for misfolded proteins derived from endoplasmic reticulum and contributes to protection from neurotoxicity induced by unfolded protein stresses. autosomal recessive juvenile parkinsonism hemagglutinin antibody ubiquitin COOH-terminal hydrolase L1 glutathioneS-transferase the unfolded protein response endoplasmic reticulum-associated protein degradation glyceraldehyde-3-phosphate dehydrogenase green fluorescent protein ubiquitin-activating enzyme ubiquitin-conjugating enzyme ubiquitin-protein ligase endoplasmic reticulum reverse transcription polymerase chain reaction 2-mercaptoethanol AR-JP1 is one of the most common forms of the familial Parkinson's diseases and is characterized by juvenile onset, a recessive mode of inheritance and selective loss of the dopaminergic neurons in the substantia nigra without Lewy bodies (intraneuronal accumulations of aggregated proteins) (1Yamamura Y. Sobue I. Ando K. Iida M. Yanagi T. Kono C. Neurology. 1973; 23: 239-244Crossref PubMed Google Scholar). In 1998, the gene responsible for AR-JP was identified and designated parkin (2Kitada T. Asakawa S. Hattori N. Matsumine H. Yamamura Y. Minoshima S. Yokochi M. Mizuno Y. Shimizu N. Nature. 1998; 392: 605-608Crossref PubMed Scopus (4124) Google Scholar).Recently, several proteins with RING finger motifs have been identified as E3 ubiquitin ligases, which are responsible for substrate recognition and for promotion of substrate ubiquitination in conjunction with ubiquitin-conjugating enzymes (E2s) (3Dawson T.M. Cell. 2000; 101: 115-118Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 4Ciechanover A. Orian A. Schwartz A.L. Bioessays. 2000; 22: 442-451Crossref PubMed Scopus (692) Google Scholar, 5Yokouchi M. Kondo T. Houghton A. Bartkiewicz M. Horne W.C. Zhang H. Yoshimura A. Baron R. J. Biol. Chem. 1999; 274: 31707-31712Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar, 6Joazeiro C.A. Wing S.S. Huang H. Leverson J.D. Hunter T. Liu Y.C. Science. 1999; 286: 309-312Crossref PubMed Scopus (909) Google Scholar, 7Lorick K.L. Jensen J.P. Fang S. Ong A.M. Hatakeyama S. Weissman A.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11364-11369Crossref PubMed Scopus (937) Google Scholar, 8Tyers M. Willems A.R. Science. 1999; 284 (, 603–604): 601Crossref PubMed Scopus (141) Google Scholar, 9Yang Y. Fang S. Jensen J.P. Weissman A.M. Ashwell J.D. Science. 2000; 288: 874-877Crossref PubMed Scopus (861) Google Scholar, 10Freemont P.S. Curr. Biol. 2000; 10: R84-R87Abstract Full Text Full Text PDF PubMed Google Scholar). In RING-type E3s, RING finger motifs serve as recruiting motifs for specific E2 ubiquitin-conjugating enzymes. These facts suggest that Parkin, which contains a RING-IBR-RING motif, is a new member of E3 ubiquitin ligases.On the other hand, the fact that the deletion of the parkingene causes the neuronal death of the substantia nigra in AR-JP patients suggests the cell-protective function of Parkin. Given that Parkin is involved in both the ubiquitin-proteasome pathway and cell death protection, an interesting possibility is that Parkin may inhibit a certain type of cell death through proteasome-mediated protein degradation. Accumulation of misfolded proteins in the endoplasmic reticulum (ER) would constitute an unfolded protein stress or ER stress, which may lead to cell death. Normal cells deal with unfolded protein stress by several mechanisms, including transcriptional induction of genes that facilitate protein folding or removal of misfolded proteins and degradation that is dependent on the cytosolic ubiquitin-proteasome pathway (11Mori K. Cell. 2000; 101: 451-454Abstract Full Text Full Text PDF PubMed Scopus (784) Google Scholar).Here, we provide evidence that Parkin is a RING-type E3 ubiquitin-protein ligase. Moreover, we show that Parkin is up-regulated in response to unfolded protein stress and suppresses unfolded protein stress-induced cell death via its E3 activity, suggesting that the physiological role of Parkin involves dealing with unfolded protein stress.RESULTS AND DISCUSSIONTo study the physiological function of Parkin in cells, we overexpressed Parkin with NH2-terminal FLAG tag (FLAG-Parkin) in several cell lines, including human kidney-derived 293(T) cells and dopaminergic neuroblastoma-derived SH-SY5Y cells. Overexpression of FLAG-Parkin in any cell line used led to the formation of slower migrating proteins that were recognized by a Western blot using anti-FLAG Ab (Fig.1 A). As this high molecular weight smear-like appearance of FLAG-Parkin seemed to be caused by polyubiquitin, we examined whether Parkin could be covalently modified by ubiquitin. An expression plasmid encoding ubiquitin with a hemagglutinin tag (HA-Ub) was transfected cells with or without plasmid for by with anti-FLAG Western blot of with Ab a high molecular weight and FLAG-Parkin were that FLAG-Parkin is Western of the using anti-FLAG Ab high molecular weight FLAG-Parkin was of the or of the of the was in the the of suggesting that FLAG-Parkin is modified with ubiquitin in the Overexpression of the which chain caused of ubiquitination of Parkin T. Cell. Biol. PubMed Scopus Google Scholar, S. Cell. Biol. PubMed Scopus Google (Fig.1 was with which was overexpressed in cells and ubiquitin hydrolase enzymes are to with unfolded including are to have to proteins M. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). molecular weight FLAG-Parkin as as were by overexpressed a human evidence that Parkin is overexpressed in cells several proteins the RING finger have been to be E3 ubiquitin-protein ubiquitination of of RING-type been K.L. Jensen J.P. Fang S. Ong A.M. Hatakeyama S. Weissman A.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11364-11369Crossref PubMed Scopus (937) Google Y. Fang S. Jensen J.P. Weissman A.M. Ashwell J.D. Science. 2000; 288: 874-877Crossref PubMed Scopus (861) Google Scholar). we examined whether Parkin is a RING-type a using FLAG-Parkin and a set of in SH-SY5Y cells. FLAG-Parkin with and but not with or was to that of a protein a RING-IBR-RING at its COOH which is to Parkin U. M. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google A). we to the of domain of Parkin. a or several mutants of FLAG-Parkin with in SH-SY5Y cells, with anti-FLAG antibody by Western by deletion the RING-IBR-RING domain to UbcH7 as as the other Parkin including deletion mutants and an IBR and the RING the and a with a in the RING to with the was with not These that the RING-IBR-RING domain of Parkin is responsible for with suggesting that Parkin is a RING-type E3 with UbcH7 and an E3 ubiquitin ligase SH-SY5Y cells were transfected with with or without were to with anti-FLAG and by Western with SH-SY5Y cells were transfected with or with is a deletion of the and was as in A. Parkin and its were from or SH-SY5Y cells transfected with FLAG-Parkin and a deletion of with FLAG An in was by and UbcH7 and to the and Parkin proteins in reaction at for were with 2-mercaptoethanol and by which Western with was Parkin and its were on the by Western with anti-FLAG Ab evidence that Parkin E3 activity, we an in ubiquitin ligase of FLAG-Parkin or an deletion from two cell and SH-SY5Y were with UbcH7 and Western using antibody a high molecular weight of with Parkin, but not with or the in E3 of Parkin. in this reaction are to be with Parkin, but not Parkin as the from that of Parkin. the other hand, ubiquitination of Parkin was not in this in suggesting that Parkin may other including to be with this and Parkin proteins are in cells, UbcH7 not to the E3 of Parkin, ubiquitination is in from SH-SY5Y cells in from cells, suggesting that expression of the for Parkin is neuronal cell the of this H. Hattori N. Mizuno Y. Asakawa S. Minoshima S. Shimizu N. K. T. K. T. 2000; PubMed Scopus Google that Parkin is an E3 protein with to that overexpressed Parkin was not in the for this are not it is that expression in cells to the ubiquitination of of Parkin is to lead to selective neuronal cell we evidence for a role for Parkin in neuronal cell death induced by including unfolded protein mRNA of Parkin in SH-SY5Y cells stress for were by A). or a was in the Parkin mRNA level a of stress including high the and In with the or the 2-mercaptoethanol of which is an of unfolded protein stress T. H. N. J. J. Nature. 2000; PubMed Scopus Google Scholar, M. C. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, A. Zhang Y. Science. 2000; PubMed Scopus Google in up-regulation of Parkin mRNA with we examined the expression of Parkin mRNA in cells with or for to up-regulation of Parkin mRNA was in or cells was in cells at with the of Parkin mRNA the Parkin protein expression level was the unfolded protein stress is up-regulated by unfolded protein SH-SY5Y cells were with a of stresses. was and to using a and level of was by glyceraldehyde-3-phosphate dehydrogenase mRNA of Parkin the with the from the from SH-SY5Y cells were with or for the Parkin mRNA was and as cell from SH-SY5Y cells were by Western with the protein of which was by Ab in it may a of Parkin. ER stress response was by the induction of protein level of was by the of on this we that the up-regulation of Parkin cells from unfolded protein whether Parkin suppresses unfolded protein stress-induced cell death in neuronal cells, we an unfolded protein stress-induced cell death using SH-SY5Y cells and cells were to unfolded protein stress-induced cell death with or for In Parkin proteins that E2 recruiting death at that the E3 ubiquitin ligase of Parkin is for the of unfolded protein stress-induced cell death the of cells in Parkin, Parkin and with for were the other hand, an of cell death 1999; PubMed Scopus Google suppressed cell death induced by any of stress These that Parkin specifically unfolded protein stress-induced cell death as a cell death with the that Parkin an death function through protein the of Parkin or cell death was by with a suppresses unfolded protein stress-induced cell death. SH-SY5Y cells were transfected with plasmid or with cells were with or without or for cells were cells with or were as cells or was the from of the cells in the of on cell death by Parkin SH-SY5Y cells transfected with FLAG-Parkin were with or as in A. to the cell death cells were with or without death was as response to stress in the the unfolded protein response which gene is study that ER and pathway including protein degradation genes in Weissman Cell. 2000; 101: Full Text Full Text PDF PubMed Scopus Google Scholar). misfolded ER proteins via degradation in the are the ER the are through the ubiquitin-proteasome pathway Sci. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). is an interesting that may be involved in it is in in to the and H. Hattori N. S. M. T. Matsumine H. Asakawa S. Minoshima S. Yamamura Y. Shimizu N. Mizuno Y. 1999; PubMed Scopus Google Scholar). deletion of the parkin gene may lead to of misfolded substrate in the in the cell death that causes AR-JP1 is one of the most common forms of the familial Parkinson's diseases and is characterized by juvenile onset, a recessive mode of inheritance and selective loss of the dopaminergic neurons in the substantia nigra without Lewy bodies (intraneuronal accumulations of aggregated proteins) (1Yamamura Y. Sobue I. Ando K. Iida M. Yanagi T. Kono C. Neurology. 1973; 23: 239-244Crossref PubMed Google Scholar). In 1998, the gene responsible for AR-JP was identified and designated parkin (2Kitada T. Asakawa S. Hattori N. Matsumine H. Yamamura Y. Minoshima S. Yokochi M. Mizuno Y. Shimizu N. Nature. 1998; 392: 605-608Crossref PubMed Scopus (4124) Google Scholar). several proteins with RING finger motifs have been identified as E3 ubiquitin ligases, which are responsible for substrate recognition and for promotion of substrate ubiquitination in conjunction with ubiquitin-conjugating enzymes (E2s) (3Dawson T.M. Cell. 2000; 101: 115-118Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 4Ciechanover A. Orian A. Schwartz A.L. Bioessays. 2000; 22: 442-451Crossref PubMed Scopus (692) Google Scholar, 5Yokouchi M. Kondo T. Houghton A. Bartkiewicz M. Horne W.C. Zhang H. Yoshimura A. Baron R. J. Biol. Chem. 1999; 274: 31707-31712Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar, 6Joazeiro C.A. Wing S.S. Huang H. Leverson J.D. Hunter T. Liu Y.C. Science. 1999; 286: 309-312Crossref PubMed Scopus (909) Google Scholar, 7Lorick K.L. Jensen J.P. Fang S. Ong A.M. Hatakeyama S. Weissman A.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11364-11369Crossref PubMed Scopus (937) Google Scholar, 8Tyers M. Willems A.R. Science. 1999; 284 (, 603–604): 601Crossref PubMed Scopus (141) Google Scholar, 9Yang Y. Fang S. Jensen J.P. Weissman A.M. Ashwell J.D. Science. 2000; 288: 874-877Crossref PubMed Scopus (861) Google Scholar, 10Freemont P.S. Curr. Biol. 2000; 10: R84-R87Abstract Full Text Full Text PDF PubMed Google Scholar). In RING-type E3s, RING finger motifs serve as recruiting motifs for specific E2 ubiquitin-conjugating enzymes. These facts suggest that Parkin, which contains a RING-IBR-RING motif, is a new member of E3 ubiquitin the other hand, the fact that the deletion of the parkingene causes the neuronal death of the substantia nigra in AR-JP patients suggests the cell-protective function of Parkin. Given that Parkin is involved in both the ubiquitin-proteasome pathway and cell death protection, an interesting possibility is that Parkin may inhibit a certain type of cell death through proteasome-mediated protein degradation. Accumulation of misfolded proteins in the endoplasmic reticulum (ER) would constitute an unfolded protein stress or ER stress, which may lead to cell death. Normal cells deal with unfolded protein stress by several mechanisms, including transcriptional induction of genes that facilitate protein folding or removal of misfolded proteins and degradation that is dependent on the cytosolic ubiquitin-proteasome pathway (11Mori K. Cell. 2000; 101: 451-454Abstract Full Text Full Text PDF PubMed Scopus (784) Google Scholar). Here, we provide evidence that Parkin is a RING-type E3 ubiquitin-protein ligase. Moreover, we show that Parkin is up-regulated in response to unfolded protein stress and suppresses unfolded protein stress-induced cell death via its E3 activity, suggesting that the physiological role of Parkin involves dealing with unfolded protein AND DISCUSSIONTo study the physiological function of Parkin in cells, we overexpressed Parkin with NH2-terminal FLAG tag (FLAG-Parkin) in several cell lines, including human kidney-derived 293(T) cells and dopaminergic neuroblastoma-derived SH-SY5Y cells. Overexpression of FLAG-Parkin in any cell line used led to the formation of slower migrating proteins that were recognized by a Western blot using anti-FLAG Ab (Fig.1 A). As this high molecular weight smear-like appearance of FLAG-Parkin seemed to be caused by polyubiquitin, we examined whether Parkin could be covalently modified by ubiquitin. An expression plasmid encoding ubiquitin with a hemagglutinin tag (HA-Ub) was transfected cells with or without plasmid for by with anti-FLAG Western blot of with Ab a high molecular weight and FLAG-Parkin were that FLAG-Parkin is Western of the using anti-FLAG Ab high molecular weight FLAG-Parkin was of the or of the of the was in the the of suggesting that FLAG-Parkin is modified with ubiquitin in the Overexpression of the which chain caused of ubiquitination of Parkin T. Cell. Biol. PubMed Scopus Google Scholar, S. Cell. Biol. PubMed Scopus Google (Fig.1 was with which was overexpressed in cells and ubiquitin hydrolase enzymes are to with unfolded including are to have to proteins M. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). molecular weight FLAG-Parkin as as were by overexpressed a human evidence that Parkin is overexpressed in cells several proteins the RING finger have been to be E3 ubiquitin-protein ubiquitination of of RING-type been K.L. Jensen J.P. Fang S. Ong A.M. Hatakeyama S. Weissman A.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11364-11369Crossref PubMed Scopus (937) Google Y. Fang S. Jensen J.P. Weissman A.M. Ashwell J.D. Science. 2000; 288: 874-877Crossref PubMed Scopus (861) Google Scholar). we examined whether Parkin is a RING-type a using FLAG-Parkin and a set of in SH-SY5Y cells. FLAG-Parkin with and but not with or was to that of a protein a RING-IBR-RING at its COOH which is to Parkin U. M. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google A). we to the of domain of Parkin. a or several mutants of FLAG-Parkin with in SH-SY5Y cells, with anti-FLAG antibody by Western by deletion the RING-IBR-RING domain to UbcH7 as as the other Parkin including deletion mutants and an IBR and the RING the and a with a in the RING to with the was with not These that the RING-IBR-RING domain of Parkin is responsible for with suggesting that Parkin is a RING-type E3 with UbcH7 and evidence that Parkin E3 activity, we an in ubiquitin ligase of FLAG-Parkin or an deletion from two cell and SH-SY5Y were with UbcH7 and Western using antibody a high molecular weight of with Parkin, but not with or the in E3 of Parkin. in this reaction are to be with Parkin, but not Parkin as the from that of Parkin. the other hand, ubiquitination of Parkin was not in this in suggesting that Parkin may other including to be with this and Parkin proteins are in cells, UbcH7 not to the E3 of Parkin, ubiquitination is in from SH-SY5Y cells in from cells, suggesting that expression of the for Parkin is neuronal cell the of this H. Hattori N. Mizuno Y. Asakawa S. Minoshima S. Shimizu N. K. T. K. T. 2000; PubMed Scopus Google that Parkin is an E3 protein with to that overexpressed Parkin was not in the for this are not it is that expression in cells to the ubiquitination of of Parkin is to lead to selective neuronal cell we evidence for a role for Parkin in neuronal cell death induced by including unfolded protein mRNA of Parkin in SH-SY5Y cells stress for were by A). or a was in the Parkin mRNA level a of stress including high the and In with the or the 2-mercaptoethanol of which is an of unfolded protein stress T. H. N. J. J. Nature. 2000; PubMed Scopus Google Scholar, M. C. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, A. Zhang Y. Science. 2000; PubMed Scopus Google in up-regulation of Parkin mRNA with we examined the expression of Parkin mRNA in cells with or for to up-regulation of Parkin mRNA was in or cells was in cells at with the of Parkin mRNA the Parkin protein expression level was the unfolded protein stress is up-regulated by unfolded protein SH-SY5Y cells were with a of stresses. was and to using a and level of was by glyceraldehyde-3-phosphate dehydrogenase mRNA of Parkin the with the from the from SH-SY5Y cells were with or for the Parkin mRNA was and as cell from SH-SY5Y cells were by Western with the protein of which was by Ab in it may a of Parkin. ER stress response was by the induction of protein level of was by the of on this we that the up-regulation of Parkin cells from unfolded protein whether Parkin suppresses unfolded protein stress-induced cell death in neuronal cells, we an unfolded protein stress-induced cell death using SH-SY5Y cells and cells were to unfolded protein stress-induced cell death with or for In Parkin proteins that E2 recruiting death at that the E3 ubiquitin ligase of Parkin is for the of unfolded protein stress-induced cell death the of cells in Parkin, Parkin and with for were the other hand, an of cell death 1999; PubMed Scopus Google suppressed cell death induced by any of stress These that Parkin specifically unfolded protein stress-induced cell death as a cell death with the that Parkin an death function through protein the of Parkin or cell death was by with a suppresses unfolded protein stress-induced cell death. SH-SY5Y cells were transfected with plasmid or with cells were with or without or for cells were cells with or were as cells or was the from of the cells in the of on cell death by Parkin SH-SY5Y cells transfected with FLAG-Parkin were with or as in A. to the cell death cells were with or without death was as response to stress in the the unfolded protein response which gene is study that ER and pathway including protein degradation genes in Weissman Cell. 2000; 101: Full Text Full Text PDF PubMed Scopus Google Scholar). misfolded ER proteins via degradation in the are the ER the are through the ubiquitin-proteasome pathway Sci. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). is an interesting that may be involved in it is in in to the and H. Hattori N. S. M. T. Matsumine H. Asakawa S. Minoshima S. Yamamura Y. Shimizu N. Mizuno Y. 1999; PubMed Scopus Google Scholar). deletion of the parkin gene may lead to of misfolded substrate in the in the cell death that causes study the physiological function of Parkin in cells, we overexpressed Parkin with NH2-terminal FLAG tag (FLAG-Parkin) in several cell lines, including human kidney-derived 293(T) cells and dopaminergic neuroblastoma-derived SH-SY5Y cells. Overexpression of FLAG-Parkin in any cell line used led to the formation of slower migrating proteins that were recognized by a Western blot using anti-FLAG Ab (Fig.1 A). As this high molecular weight smear-like appearance of FLAG-Parkin seemed to be caused by polyubiquitin, we examined whether Parkin could be covalently modified by ubiquitin. An expression plasmid encoding ubiquitin with a hemagglutinin tag (HA-Ub) was transfected cells with or without plasmid for by with anti-FLAG Western blot of with Ab a high molecular weight and FLAG-Parkin were that FLAG-Parkin is Western of the using anti-FLAG Ab high molecular weight FLAG-Parkin was of the or of the of the was in the the of suggesting that FLAG-Parkin is modified with ubiquitin in the Overexpression of the which chain caused of ubiquitination of Parkin T. Cell. Biol. PubMed Scopus Google Scholar, S. Cell. Biol. PubMed Scopus Google (Fig.1 was with which was overexpressed in cells and ubiquitin hydrolase enzymes are to with unfolded including are to have to proteins M. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). molecular weight FLAG-Parkin as as were by overexpressed a human evidence that Parkin is overexpressed in cells several proteins the RING finger have been to be E3 ubiquitin-protein ubiquitination of of RING-type been K.L. Jensen J.P. Fang S. Ong A.M. Hatakeyama S. Weissman A.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11364-11369Crossref PubMed Scopus (937) Google Y. Fang S. Jensen J.P. Weissman A.M. Ashwell J.D. Science. 2000; 288: 874-877Crossref PubMed Scopus (861) Google Scholar). we examined whether Parkin is a RING-type a using FLAG-Parkin and a set of in SH-SY5Y cells. FLAG-Parkin with and but not with or was to that of a protein a RING-IBR-RING at its COOH which is to Parkin U. M. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google A). we to the of domain of Parkin. a or several mutants of FLAG-Parkin with in SH-SY5Y cells, with anti-FLAG antibody by Western by deletion the RING-IBR-RING domain to UbcH7 as as the other Parkin including deletion mutants and an IBR and the RING the and a with a in the RING to with the was with not These that the RING-IBR-RING domain of Parkin is responsible for with suggesting that Parkin is a RING-type E3 with UbcH7 and evidence that Parkin E3 activity, we an in ubiquitin ligase of FLAG-Parkin or an deletion from two cell and SH-SY5Y were with UbcH7 and Western using antibody a high molecular weight of with Parkin, but not with or the in E3 of Parkin. in this reaction are to be with Parkin, but not Parkin as the from that of Parkin. the other hand, ubiquitination of Parkin was not in this in suggesting that Parkin may other including to be with this and Parkin proteins are in cells, UbcH7 not to the E3 of Parkin, ubiquitination is in from SH-SY5Y cells in from cells, suggesting that expression of the for Parkin is neuronal cell the of this H. Hattori N. Mizuno Y. Asakawa S. Minoshima S. Shimizu N. K. T. K. T. 2000; PubMed Scopus Google that Parkin is an E3 protein with to that overexpressed Parkin was not in the for this are not it is that expression in cells to the ubiquitination of Parkin. As of Parkin is to lead to selective neuronal cell we evidence for a role for Parkin in neuronal cell death induced by including unfolded protein mRNA of Parkin in SH-SY5Y cells stress for were by A). or a was in the Parkin mRNA level a of stress including high the and In with the or the 2-mercaptoethanol of which is an of unfolded protein stress T. H. N. J. J. Nature. 2000; PubMed Scopus Google Scholar, M. C. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, A. Zhang Y. Science. 2000; PubMed Scopus Google in up-regulation of Parkin mRNA with we examined the expression of Parkin mRNA in cells with or for to up-regulation of Parkin mRNA was in or cells was in cells at with the of Parkin mRNA the Parkin protein expression level was the unfolded protein stress on this we that the up-regulation of Parkin cells from unfolded protein whether Parkin suppresses unfolded protein stress-induced cell death in neuronal cells, we an unfolded protein stress-induced cell death using SH-SY5Y cells and cells were to unfolded protein stress-induced cell death with or for In Parkin proteins that E2 recruiting death at that the E3 ubiquitin ligase of Parkin is for the of unfolded protein stress-induced cell death the of cells in Parkin, Parkin and with for were the other hand, an of cell death 1999; PubMed Scopus Google suppressed cell death induced by any of stress These that Parkin specifically unfolded protein stress-induced cell death as a cell death with the that Parkin an death function through protein the of Parkin or cell death was by with a In response to stress in the the unfolded protein response which gene is study that ER and pathway including protein degradation genes in Weissman Cell. 2000; 101: Full Text Full Text PDF PubMed Scopus Google Scholar). misfolded ER proteins via degradation in the are the ER the are through the ubiquitin-proteasome pathway Sci. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). is an interesting that may be involved in it is in in to the and H. Hattori N. S. M. T. Matsumine H. Asakawa S. Minoshima S. Yamamura Y. Shimizu N. Mizuno Y. 1999; PubMed Scopus Google Scholar). deletion of the parkin gene may lead to of misfolded substrate in the in the cell death that causes Y. Mizuno for the human Parkin S. for the and cells, M. for the cells, T. for the SH-SY5Y cells, A. for the K. for the of for Parkin, and S. Hatakeyama for and the of ubiquitin. T. H. and N. Hattori for of this