Caspase-mediated parkin cleavage in apoptotic cell death.
The parkin protein is important for the survival of the neurons that degenerate in Parkinson's disease as demonstrated by disease-causing lesions in the parkin gene. The Chinese hamster ovary and the SH-SY5Y cell line stably expressing recombinant human parkin combined with epitope-specific parkin antibodies were used to investigate the proteolytic processing of human parkin during apoptosis by immunoblotting. Parkin is cleaved during apoptosis induced by okadaic acid, staurosporine, and camptothecin, thereby generating a 38-kDa C-terminal fragment and a 12-kDa N-terminal fragment. The cleavage was not significantly affected by the disease-causing mutations K161N, G328E, T415N, and G430D and the polymorphism R366W. Parkin and its 38-kDa proteolytic fragment is preferentially associated with vesicles, thereby indicating that cleavage is a membrane-associated event. The proteolysis is sensitive to inhibitors of caspases. The cleavage site was mapped by site-directed mutagenesis of potential aspartic residues and revealed that mutation of Asp-126 alone abrogated the parkin cleavage. The tetrapeptide aldehyde LHTD-CHO, representing the amino acid sequence N-terminal to the putative cleavage site was an efficient inhibitor of parkin cleavage. This suggests that parkin function is compromised in neuropathological states associated with an increased caspase activation, thereby further adding to the cellular stress.
Kahns
Soren
S
Department of Medical Biochemistry, Building 170, University of Aarhus, DK-8000 Aarhus-C, Denmark.
Lykkebo
Simon
S
Jakobsen
Lene Diness
LD
Nielsen
Morten S
MS
Jensen
Poul Henning
PH
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D017209:Apoptosis; D016466:CHO Cells; D020169:Caspases / Q000378:metabolism*; D003001:Cloning, Molecular; D006224:Cricetinae; D017931:DNA Primers; D006801:Humans; D008025:Ligases / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000737:chemistry; D010300:Parkinson Disease; D010446:Peptide Fragments / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases
D017931:DNA Primers; D009842:Oligopeptides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D044767:Ubiquitin-Protein Ligases; C111567:parkin protein; D020169:Caspases; D008025:Ligases