Diverse Effects of Pathogenic Mutations of Parkin That Catalyze Multiple Monoubiquitylation in Vitro

Mutational dysfunction of PARKIN gene, which encodes a double RING finger protein and has ubiquitin ligase E3 activity, is the major cause of autosomal recessive juvenile Parkinsonism. Although many studies explored the functions of Parkin, its biochemical character is poorly understood. To address this issue, we established an E3 assay system using maltose-binding protein-fused Parkin purified from Escherichia coli. Using this recombinant Parkin, we found that not the front but the rear RING finger motif is responsible for the E3 activity of Parkin, and it catalyzes multiple monoubiquitylation. Intriguingly, for autosomal recessive juvenile Parkinsonism-causing mutations of Parkin, whereas there was loss of E3 activity in the rear RING domain, other pathogenic mutants still exhibited E3 activity equivalent to that of the wild-type Parkin. The evidence presented allows us to reconsider the function of Parkin-catalyzed ubiquitylation and to conclude that autosomal recessive juvenile Parkinsonism is not solely attributable to catalytic impairment of the E3 activity of Parkin. Mutational dysfunction of PARKIN gene, which encodes a double RING finger protein and has ubiquitin ligase E3 activity, is the major cause of autosomal recessive juvenile Parkinsonism. Although many studies explored the functions of Parkin, its biochemical character is poorly understood. To address this issue, we established an E3 assay system using maltose-binding protein-fused Parkin purified from Escherichia coli. Using this recombinant Parkin, we found that not the front but the rear RING finger motif is responsible for the E3 activity of Parkin, and it catalyzes multiple monoubiquitylation. Intriguingly, for autosomal recessive juvenile Parkinsonism-causing mutations of Parkin, whereas there was loss of E3 activity in the rear RING domain, other pathogenic mutants still exhibited E3 activity equivalent to that of the wild-type Parkin. The evidence presented allows us to reconsider the function of Parkin-catalyzed ubiquitylation and to conclude that autosomal recessive juvenile Parkinsonism is not solely attributable to catalytic impairment of the E3 activity of Parkin. Recessive mutations in the human PARKIN gene are the most frequent cause of autosomal recessive juvenile parkinsonism, the common form of familial Parkinson disease (PD). 2The abbreviations used are: PD, Parkinson disease; E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin ligase; GST, glutathione S-transferase; MBP, maltose-binding protein. 2The abbreviations used are: PD, Parkinson disease; E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin ligase; GST, glutathione S-transferase; MBP, maltose-binding protein. It has been shown that almost 50% of patients with familial autosomal recessive juvenile parkinsonism carry a series of exon rearrangements or point mutations in PARKIN. Moreover, recent findings of the haploinsufficiency of parkin and S-nitrosylation also imply its association in sporadic PD (1Moore D.J. West A.B. Dawson V.L. Dawson T.M. Annu. Rev. Neurosci. 2005; 28: 57-87Crossref PubMed Scopus (1012) Google Scholar). The causal gene PARKIN encodes a double RING finger protein with ubiquitin ligase (E3) activity (2Giasson B.I. Lee V.M. Cell. 2003; 114: 1-8Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 3Ross C.A. Pickart C.M. Trends Cell Biol. 2004; 14: 703-711Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 4Tanaka K. Suzuki T. Hattori N. Mizuno 2004; PubMed Scopus Google Scholar, N. Mizuno 2004; Full Text Full Text PDF PubMed Scopus Google and mutations in the double RING finger motif in an of the disease mutations in other T. 2003; PubMed Scopus Google Scholar). To biochemical studies been to mutations in Parkin to its dysfunction and to pathogenic the biochemical of E3 activity of Parkin has been it is still the Parkin mutants E3 activity or of that Parkin its E3 activity Neurosci. 2003; PubMed Google whereas the not E3 activity T. 2003; PubMed Google Scholar). the of other PD the is the of Parkin-catalyzed ubiquitylation poorly to is the using recombinant Parkin. of the biochemical been using in Parkin or Parkin. it is to of other that with Parkin. it has been that Parkin with other T. T. Cell. Full Text Full Text PDF PubMed Scopus Google and Biol. 2005; Full Text Full Text PDF PubMed Scopus Google and the of using or in Parkin To the E3 activity of Parkin, a biochemical using recombinant Parkin that is from other E3 is to a E3 assay system using Parkin purified from Escherichia of Parkin in it was to a Parkin many for that cause Parkin that to of for in Pickart C.M. PubMed Scopus Google Scholar). in which the was was to maltose-binding protein Parkin of and of and of the recombinant purified from the the using a and The was not Parkin to its E3 activity it was to the ubiquitylation assay to of Parkin also we E2, and the that in in the we not not purified the and a and of was a for of recombinant and in ubiquitylation assay was N. Suzuki T. K. Cell 114: PubMed Google Scholar, N. N. K. 2003; PubMed Scopus Google Scholar, N. K. T. T. K. K. 2005; PubMed Scopus Google Scholar). the purified of was in a and with of of recombinant and of purified or of for and to with Hattori N. T. N. Mizuno PubMed Scopus Google or or ubiquitin was used of The was for in the of and C.A. C.A. T. PubMed Scopus Google that glutathione Parkin purified from E3 we found that the E3 activity of this is and there is a for a E3 assay system for recombinant Parkin. studies of other RING finger we the of the to in RING finger that E3 N. Suzuki T. K. Cell 114: PubMed Google Scholar, N. N. K. 2003; PubMed Scopus Google Scholar, K. Suzuki T. N. K. T. T. T. K. 2003; PubMed Scopus Google Scholar, N. K. N. PubMed Scopus Google and to was purified using a and with E1, and of the in and we it to with from was with and the that the of or ubiquitin and are from To the is to the to with was used was double that the was was a was in the of and Pickart C.M. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). in this with the double in that the and to the of Moreover, the of ubiquitin with the of and and the of ubiquitin and we that catalyzes in with and the of the was to and that Parkin to its E3 activity in E3 activity shown in we used in the a to E3 of the or Parkin is To we purified in which a is and Parkin. in the is its ubiquitylation and but not its we to this recombinant protein was for not the of Parkin to and this was and was to an ubiquitylation assay and and the of was the in and that the is this that protein is the for The is protein was with this was not was with that the of Parkin but not MBP, and that Parkin a not of its but of its to Parkin. in the was using an the of the was also that and Although many of Parkin been the of a in biochemical that a to the E3 activity of a of Parkin. a of Parkin ubiquitylation of the is a in its the protein was to in and with The of was it is in the of Parkin. that in was not a of Parkin-catalyzed is also in the with was used in of mutations and of Parkin. E3 of Parkin mutations and that mutations the RING finger motif E3 activity of Parkin. that the is the catalytic of Parkin for pathogenic mutants other mutants E3 equivalent to that of the wild-type is an in the of Pickart C.M. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). that is and the findings that Parkin catalyzes K. Biol. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, C.A. Dawson V.L. Dawson T.M. Neurosci. 2005; PubMed Scopus Google and Parkin with in assay we the of Parkin-catalyzed Parkin multiple or has been most and it the to the the and a other Cell Biol. 2005; PubMed Scopus Google Scholar, Rev. Cell Biol. PubMed Scopus Google Scholar, C.M. Trends Full Text Full Text PDF PubMed Scopus Google Scholar). used ubiquitin to in which are and is of Parkin catalyzes the of the of ubiquitylation but the ubiquitylation the of and not the ubiquitylation that Parkin catalyzes multiple in The was was used and was and using in which to it form a the It is that of Parkin the this us to conclude that the of ubiquitylation Parkin in is multiple catalyzes multiple monoubiquitylation. and in ubiquitylation assay was in the or of ubiquitin or form was used in and in ubiquitylation in and that Parkin catalyzes multiple monoubiquitylation. and the was was was used in and in the was using was used in of E3 of Parkin with of mutations of Parkin been and the cause of autosomal recessive juvenile parkinsonism is to impairment of the E3 activity of Parkin it is still Parkin with its E3 activity or not of the of a assay system using recombinant Parkin. To this we the E3 activity of mutations and and mutations Parkin Parkin its and the other also mutants with E2, mutations the RING finger motif the E3 activity the rear RING finger motif E3 activity and the E3 activity that the RING finger motif is the catalytic for the E3 activity of Parkin. to was mutations other in still E3 equivalent to that of the wild-type Parkin The or was used not we used a recombinant Parkin, and to this is the evidence that E3 activity of the Parkin pathogenic mutations is not biochemical studies been to the E3 activity of Parkin. it is to the of other the of a the to the E3 activity of Parkin. a E3 assay system using recombinant Parkin. assay system has we a of with E3 activity this protein was for ubiquitylation in the of a is purified from it is from of other The of this assay us to a biochemical of Parkin Parkin catalyzes multiple in Moreover, K. Biol. 2005; Full Text Full Text PDF PubMed Scopus Google that Parkin the in assay not the of and and and that the ubiquitylation Parkin functions not for but for in it is still to Although we that Parkin catalyzes multiple in in and this to the ubiquitin the ubiquitin of a a Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). an with Parkin in it is still that Parkin is used the for and functions for studies are in the and the function of Parkin-catalyzed ubiquitylation to was that most of the mutations not E3 activity of Parkin mutations in the rear RING finger motif the E3 activity, that not the but the RING finger motif is the catalytic of Parkin. studies that the of Parkin been 2003; PubMed Scopus Google Scholar, N. T. 2003; 14: PubMed Scopus Google Scholar, 2005; PubMed Scopus Google Scholar, N. T.M. Dawson T.M. 2005; PubMed Scopus Google Scholar, Dawson V.L. Dawson T.M. 2005; 14: PubMed Scopus Google Scholar). Although of Parkin are not with in the E3 assay for the in Parkin was used the of E3 in 2003; Full Text Full Text PDF PubMed Scopus Google and in other E3 activity of Parkin was or not of Parkin in the ubiquitylation of the Neurosci. 2003; PubMed Google Scholar, Dawson V.L. Dawson T.M. 2005; 14: PubMed Scopus Google Scholar). Although there is recent studies and the that the dysfunction of Parkin is not attributable to catalytic impairment of its E3 mutations cause Parkin to an and this in disease N. T. 2003; 14: PubMed Scopus Google Scholar, 2005; PubMed Scopus Google Scholar, N. T.M. Dawson T.M. 2005; PubMed Scopus Google Scholar, Dawson V.L. Dawson T.M. 2005; 14: PubMed Scopus Google Scholar). that mutations cause not of E3 activity but also a of and from its and a of to Parkin dysfunction and autosomal recessive juvenile is the most and of Parkin is in of a of Parkin been of not there for B.I. Dawson V.L. Dawson T.M. Lee V.M. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google the and a of the Parkin also for the E3 activity of Parkin, not the of we the activity of recombinant Parkin. Although is not we that biochemical using Parkin a for the Recessive mutations in the human PARKIN gene are the most frequent cause of autosomal recessive juvenile parkinsonism, the common form of familial Parkinson disease (PD). 2The abbreviations used are: PD, Parkinson disease; E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin ligase; GST, glutathione S-transferase; MBP, maltose-binding protein. 2The abbreviations used are: PD, Parkinson disease; E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin ligase; GST, glutathione S-transferase; MBP, maltose-binding protein. It has been shown that almost 50% of patients with familial autosomal recessive juvenile parkinsonism carry a series of exon rearrangements or point mutations in PARKIN. Moreover, recent findings of the haploinsufficiency of parkin and S-nitrosylation also imply its association in sporadic PD (1Moore D.J. West A.B. Dawson V.L. Dawson T.M. Annu. Rev. Neurosci. 2005; 28: 57-87Crossref PubMed Scopus (1012) Google Scholar). The causal gene PARKIN encodes a double RING finger protein with ubiquitin ligase (E3) activity (2Giasson B.I. Lee V.M. Cell. 2003; 114: 1-8Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 3Ross C.A. Pickart C.M. Trends Cell Biol. 2004; 14: 703-711Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 4Tanaka K. Suzuki T. Hattori N. Mizuno 2004; PubMed Scopus Google Scholar, N. Mizuno 2004; Full Text Full Text PDF PubMed Scopus Google and mutations in the double RING finger motif in an of the disease mutations in other T. 2003; PubMed Scopus Google Scholar). To biochemical studies been to mutations in Parkin to its dysfunction and to pathogenic the biochemical of E3 activity of Parkin has been it is still the Parkin mutants E3 activity or of that Parkin its E3 activity Neurosci. 2003; PubMed Google whereas the not E3 activity T. 2003; PubMed Google Scholar). the of other PD the is the of Parkin-catalyzed ubiquitylation poorly to is the using recombinant Parkin. of the biochemical been using in Parkin or Parkin. it is to of other that with Parkin. it has been that Parkin with other T. T. Cell. Full Text Full Text PDF PubMed Scopus Google and Biol. 2005; Full Text Full Text PDF PubMed Scopus Google and the of using or in Parkin To the E3 activity of Parkin, a biochemical using recombinant Parkin that is from other E3 is to a E3 assay system using Parkin purified from Escherichia coli. of Parkin in it was to a Parkin many for that cause Parkin that to of for in Pickart C.M. PubMed Scopus Google Scholar). in which the was was to maltose-binding protein Parkin of and of and of the recombinant purified from the the using a and The was not Parkin to its E3 activity it was to the ubiquitylation assay to of Parkin also we E2, and the that in in the we not not purified the and a and of was a for of recombinant and in ubiquitylation assay was N. Suzuki T. K. Cell 114: PubMed Google Scholar, N. N. K. 2003; PubMed Scopus Google Scholar, N. K. T. T. K. K. 2005; PubMed Scopus Google Scholar). the purified of was in a and with of of recombinant and of purified or of for and to with Hattori N. T. N. Mizuno PubMed Scopus Google or or ubiquitin was used of The was for in the of and of Parkin in it was to a Parkin many for that cause Parkin that to of for in Pickart C.M. PubMed Scopus Google Scholar). in which the was was to maltose-binding protein Parkin of and of and of the recombinant purified from the the using a and The was not Parkin to its E3 activity it was to the ubiquitylation assay to of Parkin also we E2, and the that in in the we not not purified the and a and of was a for of recombinant and in ubiquitylation assay was N. Suzuki T. K. Cell 114: PubMed Google Scholar, N. N. K. 2003; PubMed Scopus Google Scholar, N. K. T. T. K. K. 2005; PubMed Scopus Google Scholar). the purified of was in a and with of of recombinant and of purified or of for and to with Hattori N. T. N. Mizuno PubMed Scopus Google or or ubiquitin was used of The was for in the of and C.A. C.A. T. PubMed Scopus Google that glutathione Parkin purified from E3 we found that the E3 activity of this is and there is a for a E3 assay system for recombinant Parkin. studies of other RING finger we the of the to in RING finger that E3 N. Suzuki T. K. Cell 114: PubMed Google Scholar, N. N. K. 2003; PubMed Scopus Google Scholar, K. Suzuki T. N. K. T. T. T. K. 2003; PubMed Scopus Google Scholar, N. K. N. PubMed Scopus Google and to was purified using a and with E1, and of the in and we it to with from was with and the that the of or ubiquitin and are from To the is to the to with was used was double that the was was a was in the of and Pickart C.M. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). in this with the double in that the and to the of Moreover, the of ubiquitin with the of and and the of ubiquitin and we that catalyzes in with and the of the was to and that Parkin to its E3 activity in E3 activity shown in we used in the a to E3 of the or Parkin is To we purified in which a is and Parkin. in the is its ubiquitylation and but not its we to this recombinant protein was for not the of Parkin to and this was and was to an ubiquitylation assay and and the of was the in and that the is this that protein is the for The is protein was with this was not was with that the of Parkin but not MBP, and that Parkin a not of its but of its to Parkin. in the was using an the of the was also that and Although many of Parkin been the of a in biochemical that a to the E3 activity of of mutations and of Parkin. E3 of Parkin mutations and that mutations the RING finger motif E3 activity of Parkin. that the is the catalytic of Parkin for pathogenic mutants other mutants E3 equivalent to that of the wild-type is an in the of Pickart C.M. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). that is and the findings that Parkin catalyzes K. Biol. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, C.A. Dawson V.L. Dawson T.M. Neurosci. 2005; PubMed Scopus Google and Parkin with in assay we the of Parkin-catalyzed Parkin multiple or has been most and it the to the the and a other Cell Biol. 2005; PubMed Scopus Google Scholar, Rev. Cell Biol. PubMed Scopus Google Scholar, C.M. Trends Full Text Full Text PDF PubMed Scopus Google Scholar). used ubiquitin to in which are and is of Parkin catalyzes the of the of ubiquitylation but the ubiquitylation the of and not the ubiquitylation that Parkin catalyzes multiple in The was was used and was and using in which to it form a the It is that of Parkin the this us to conclude that the of ubiquitylation Parkin in is multiple catalyzes multiple monoubiquitylation. and in ubiquitylation assay was in the or of ubiquitin or form was used in and in ubiquitylation in and that Parkin catalyzes multiple monoubiquitylation. and the was was was used in and in the was using was used in of E3 of Parkin with of mutations of Parkin been and the cause of autosomal recessive juvenile parkinsonism is to impairment of the E3 activity of Parkin it is still Parkin with its E3 activity or not of the of a assay system using recombinant Parkin. To this we the E3 activity of mutations and and mutations Parkin Parkin its and the other also mutants with E2, mutations the RING finger motif the E3 activity the rear RING finger motif E3 activity and the E3 activity that the RING finger motif is the catalytic for the E3 activity of Parkin. to was mutations other in still E3 equivalent to that of the wild-type Parkin The or was used not we used a recombinant Parkin, and to this is the evidence that E3 activity of the Parkin pathogenic mutations is not C.A. C.A. T. PubMed Scopus Google that glutathione Parkin purified from E3 we found that the E3 activity of this is and there is a for a E3 assay system for recombinant Parkin. studies of other RING finger we the of the to in RING finger that E3 N. Suzuki T. K. Cell 114: PubMed Google Scholar, N. N. K. 2003; PubMed Scopus Google Scholar, K. Suzuki T. N. K. T. T. T. K. 2003; PubMed Scopus Google Scholar, N. K. N. PubMed Scopus Google and to was purified using a and with E1, and of the in and we it to with from was with and the that the of or ubiquitin and are from To the is to the to with was used was double that the was was a was in the of and Pickart C.M. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). in this with the double in that the and to the of Moreover, the of ubiquitin with the of and and the of ubiquitin and we that catalyzes in with and the of the was to and that Parkin to its E3 activity in E3 activity shown in we used in the a to E3 of the or Parkin is To we purified in which a is and Parkin. in the is its ubiquitylation and but not its we to this recombinant protein was for not the of Parkin to and this was and was to an ubiquitylation assay and and the of was the in and that the is this that protein is the for The is protein was with this was not was with that the of Parkin but not MBP, and that Parkin a not of its but of its to Parkin. in the was using an the of the was also that and Although many of Parkin been the of a in biochemical that a to the E3 activity of Parkin. Parkin is an in the of Pickart C.M. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). that is and the findings that Parkin catalyzes K. Biol. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, C.A. Dawson V.L. Dawson T.M. Neurosci. 2005; PubMed Scopus Google and Parkin with in assay we the of Parkin-catalyzed Parkin multiple or has been most and it the to the the and a other Cell Biol. 2005; PubMed Scopus Google Scholar, Rev. Cell Biol. PubMed Scopus Google Scholar, C.M. Trends Full Text Full Text PDF PubMed Scopus Google Scholar). used ubiquitin to in which are and is of Parkin catalyzes the of the of ubiquitylation but the ubiquitylation the of and not the ubiquitylation that Parkin catalyzes multiple in The was was used and was and using in which to it form a the It is that of Parkin the this us to conclude that the of ubiquitylation Parkin in is multiple of E3 of Parkin with of mutations of Parkin been and the cause of autosomal recessive juvenile parkinsonism is to impairment of the E3 activity of Parkin it is still Parkin with its E3 activity or not of the of a assay system using recombinant Parkin. To this we the E3 activity of mutations and and mutations Parkin Parkin its and the other also mutants with E2, mutations the RING finger motif the E3 activity the rear RING finger motif E3 activity and the E3 activity that the RING finger motif is the catalytic for the E3 activity of Parkin. to was mutations other in still E3 equivalent to that of the wild-type Parkin The or was used not we used a recombinant Parkin, and to this is the evidence that E3 activity of the Parkin pathogenic mutations is not biochemical studies been to the E3 activity of Parkin. it is to the of other the of a the to the E3 activity of Parkin. a E3 assay system using recombinant Parkin. assay system has we a of with E3 activity this protein was for ubiquitylation in the of a is purified from it is from of other The of this assay us to a biochemical of Parkin Parkin catalyzes multiple in Moreover, K. Biol. 2005; Full Text Full Text PDF PubMed Scopus Google that Parkin the in assay not the of and and and that the ubiquitylation Parkin functions not for but for in it is still to Although we that Parkin catalyzes multiple in in and this to the ubiquitin the ubiquitin of a a Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). an with Parkin in it is still that Parkin is used the for and functions for studies are in the and the function of Parkin-catalyzed ubiquitylation to was that most of the mutations not E3 activity of Parkin mutations in the rear RING finger motif the E3 activity, that not the but the RING finger motif is the catalytic of Parkin. studies that the of Parkin been 2003; PubMed Scopus Google Scholar, N. T. 2003; 14: PubMed Scopus Google Scholar, 2005; PubMed Scopus Google Scholar, N. T.M. Dawson T.M. 2005; PubMed Scopus Google Scholar, Dawson V.L. Dawson T.M. 2005; 14: PubMed Scopus Google Scholar). Although of Parkin are not with in the E3 assay for the in Parkin was used the of E3 in 2003; Full Text Full Text PDF PubMed Scopus Google and in other E3 activity of Parkin was or not of Parkin in the ubiquitylation of the Neurosci. 2003; PubMed Google Scholar, Dawson V.L. Dawson T.M. 2005; 14: PubMed Scopus Google Scholar). Although there is recent studies and the that the dysfunction of Parkin is not attributable to catalytic impairment of its E3 mutations cause Parkin to an and this in disease N. T. 2003; 14: PubMed Scopus Google Scholar, 2005; PubMed Scopus Google Scholar, N. T.M. Dawson T.M. 2005; PubMed Scopus Google Scholar, Dawson V.L. Dawson T.M. 2005; 14: PubMed Scopus Google Scholar). that mutations cause not of E3 activity but also a of and from its and a of to Parkin dysfunction and autosomal recessive juvenile is the most and of Parkin is in of a of Parkin been of not there for B.I. Dawson V.L. Dawson T.M. Lee V.M. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google the and a of the Parkin also for the E3 activity of Parkin, not the of we the activity of recombinant Parkin. Although is not we that biochemical using Parkin a for the To biochemical studies been to the E3 activity of Parkin. it is to the of other the of a the to the E3 activity of Parkin. a E3 assay system using recombinant Parkin. assay system has we a of with E3 activity this protein was for ubiquitylation in the of a is purified from it is from of other The of this assay us to a biochemical of Parkin protein. Parkin catalyzes multiple in Moreover, K. Biol. 2005; Full Text Full Text PDF PubMed Scopus Google that Parkin the in assay not the of and and and that the ubiquitylation Parkin functions not for but for in it is still to Although we that Parkin catalyzes multiple in in and this to the ubiquitin the ubiquitin of a a Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). an with Parkin in it is still that Parkin is used the for and functions for studies are in the and the function of Parkin-catalyzed ubiquitylation to was that most of the mutations not E3 activity of Parkin mutations in the rear RING finger motif the E3 activity, that not the but the RING finger motif is the catalytic of Parkin. studies that the of Parkin been 2003; PubMed Scopus Google Scholar, N. T. 2003; 14: PubMed Scopus Google Scholar, 2005; PubMed Scopus Google Scholar, N. T.M. Dawson T.M. 2005; PubMed Scopus Google Scholar, Dawson V.L. Dawson T.M. 2005; 14: PubMed Scopus Google Scholar). Although of Parkin are not with in the E3 assay for the in Parkin was used the of E3 in 2003; Full Text Full Text PDF PubMed Scopus Google and in other E3 activity of Parkin was or not of Parkin in the ubiquitylation of the Neurosci. 2003; PubMed Google Scholar, Dawson V.L. Dawson T.M. 2005; 14: PubMed Scopus Google Scholar). Although there is recent studies and the that the dysfunction of Parkin is not attributable to catalytic impairment of its E3 mutations cause Parkin to an and this in disease N. T. 2003; 14: PubMed Scopus Google Scholar, 2005; PubMed Scopus Google Scholar, N. T.M. Dawson T.M. 2005; PubMed Scopus Google Scholar, Dawson V.L. Dawson T.M. 2005; 14: PubMed Scopus Google Scholar). that mutations cause not of E3 activity but also a of and from its and a of to Parkin dysfunction and autosomal recessive juvenile PD is the most and of Parkin is in of a of Parkin been of not there for B.I. Dawson V.L. Dawson T.M. Lee V.M. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google the and a of the Parkin also for the E3 activity of Parkin, not the of we the activity of recombinant Parkin. Although is not we that biochemical using Parkin a for the of for recombinant protein. also of K. for the with with