Drosophila melanogaster Parkin ubiquitinates peanut and septin1 as an E3 ubiquitin-protein ligase.
Autosomal recessive juvenile parkinsonism (AR-JP), a common familial form of Parkinson's disease, is caused by mutations of human Parkin. To deepen the understanding of Parkin biology in an in vivo model of Drosophila, we attempted to characterize the function of Drosophila melanogaster Parkin and found that D. melanogaster Parkin exhibited UbcH8-dependent E3 ubiquitin-protein ligase activity. Using E2 binding and in vitro ubiquitination assays, UbcH8 preferentially was found to bind to Parkin mutants harboring functional RING1 domains, but failed to bind to mutants harboring point mutants with complete loss of function. This inability of UbcH8 binding to such mutants was accompanied by abrogation of an E3 ligase activity, indicating that D. melanogaster Parkin as an E3 ligase interacts with UbcH8 through its RING1 domain. An in vivo ubiquitination assay revealed that D. melanogaster Parkin existed in ubiquitinated form in vivo. Moreover, peanut and septin1, D. melanogaster septin proteins, were also ubiquitinated by D. melanogaster Parkin. Co-immunoprecipitation with membrane protein Syntaxin indicated direct binding of septin proteins to syntaxin, implicating their relevance in the exocytosis of dopamine in cells. Western blot analysis and DNA fragmentation indicated that the rate and efficiency of p53-dependent apoptosis were significantly higher in the presence of dopamine than without the septin proteins. Therefore, our findings in the present study demonstrate that Parkin possibly influences septin protein effects on p53-mediated apoptosis, helping to extend the utility of Drosophila as a model system for the study of neurodegeneration.
Bae
Young-Joo
YJ
Laboratory of Genetics, Department of Life Science, College of Natural Science, Seoul 120-750, Republic of Korea.
Kang
Soon-Ja
SJ
Park
Kwang Sook
KS
D020816:Amino Acid Motifs; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001665:Binding Sites; D015153:Blotting, Western; D053938:DNA Fragmentation; D004298:Dopamine / Q000494:pharmacology; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D004331:Drosophila melanogaster / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D008840:Microfilament Proteins / Q000378:metabolism*; D009154:Mutation; D017434:Protein Structure, Tertiary; D050765:Qa-SNARE Proteins / Q000378:metabolism; D058112:Septins; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism / Q000502:physiology*
D029721:Drosophila Proteins; D008840:Microfilament Proteins; D050765:Qa-SNARE Proteins; D016159:Tumor Suppressor Protein p53; D025801:Ubiquitin; C087195:pnut protein, Drosophila; C522168:UbcH8 protein, Drosophila; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D020558:GTP Phosphohydrolases; C522167:Sep1 protein, Drosophila; D058112:Septins; C487430:park protein, Drosophila; D004298:Dopamine