Parkin phosphorylation and modulation of its E3 ubiquitin ligase activity.
Mutations in the PARKIN gene are the most common cause of hereditary parkinsonism. The parkin protein comprises an N-terminal ubiquitin-like domain, a linker region containing caspase cleavage sites, a unique domain in the central portion, and a special zinc finger configuration termed RING-IBR-RING. Parkin has E3 ubiquitin-protein ligase activity and is believed to mediate proteasomal degradation of aggregation-prone proteins. Whereas the effects of mutations on the structure and function of parkin have been intensely studied, post-translational modifications of parkin and the regulation of its enzymatic activity are poorly understood. Here we report that parkin is phosphorylated both in human embryonic kidney HEK293 cells and human neuroblastoma SH-SY5Y cells. The turnover of parkin phosphorylation was rapid, because inhibition of phosphatases with okadaic acid was necessary to stabilize phosphoparkin. Phosphoamino acid analysis revealed that phosphorylation occurred mainly on serine residues under these conditions. At least five phosphorylation sites were identified, including Ser101, Ser131, and Ser136 (located in the linker region) as well as Ser296 and Ser378 (located in the RING-IBR-RING motif). Casein kinase-1, protein kinase A, and protein kinase C phosphorylated parkin in vitro, and inhibition of casein kinase-1 caused a dramatic reduction of parkin phosphorylation in cell lysates. Induction of protein folding stress in cells reduced parkin phosphorylation, and unphosphorylated parkin had slightly but significantly elevated autoubiquitination activity. Thus, complex regulation of the phosphorylation state of parkin may contribute to the unfolded protein response in stressed cells.
Yamamoto
Ayako
A
Laboratory of Alzheimer's and Parkinson's Disease Research, Department of Metabolic Biochemistry, Ludwig Maximilians University, 80336 Munich, Germany.
Friedlein
Arno
A
Imai
Yuzuru
Y
Takahashi
Ryosuke
R
Kahle
Philipp J
PJ
Haass
Christian
C
D047389:Casein Kinase I / Q000378:metabolism; D045744:Cell Line, Tumor; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007668:Kidney / Q000166:cytology; D009447:Neuroblastoma; D019319:Okadaic Acid / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D017510:Protein Folding; D011493:Protein Kinase C / Q000378:metabolism; D017434:Protein Structure, Tertiary; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D004791:Enzyme Inhibitors; D019319:Okadaic Acid; D044767:Ubiquitin-Protein Ligases; C111567:parkin protein; D047389:Casein Kinase I; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C