Parkin Phosphorylation and Modulation of Its E3 Ubiquitin Ligase Activity

Abstract

Mutations in the PARKIN Parkinson's disease (PD)1 is the second most common neurodegenerative disorder. Parkinsonian symptoms are caused by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta (1). Although more than 90% of PD cases occur sporadically, the study of genetic mutations has offered great insight into the molecular mechanisms of PD (2). After the discovery that mutations in the PARKIN gene cause autosomal recessive juvenile parkinsonism (3), parkin mutations have been recognized as the most common cause of hereditary PD and possibly a risk factor for idiopathic PD (4, 5).The PARKIN gene comprises 12 exons and codes for a 465-amino acid protein that is widely expressed, most prominently in muscle and throughout the brain (3). The 52-kDa parkin protein comprises an N-terminal ubiquitin-like domain (aa 1-76), a unique parkin domain (aa 145-232), and two RING (really interesting new gene) fingers (aa 238 -293 and 418 -449, respectively) flanking an IBR (in-between RING) domain (aa 314 -377) at the C terminus. All of these domains appear to be functionally important, because PD mutations cluster in them (6).Parkin functions in the ubiquitin-proteasome system as an E3 ubiquitin-protein ligase together with the E2 ubiquitinconjugating co-enzymes UbcH7 or UbcH8 (7-9). Because this function appears to be defective in patients with parkin mutations, the identification of protein substrates is of great importance (6, 10). Parkin substrates include synaptic proteins (the septins CDCrel-1 and CDCrel-2, synaptotagmin XI, and the ␣-synuclein interactor synphilin-1), PaelR (parkin-associated endothelin-like receptor), cyclin E, ␣/␤ tubulin, and the p38 subunit of the aminoacyl-tRNA synthetase complex (9,(11)(12)(13)(14)(15)(16)(17). Parkin expression was found to be neuroprotective in Drosophila (18) and in cell culture models of dopamine neuron loss (19 -22). However, the exact molecular mechanisms of how parkin dysfunction causes PD remain to be elucidated (6, 10).Post-translational modifications often regulate enzymatic activity. Nitrosylation of parkin was recently found to occur in PD, leading to an inhibition of its ubiquitin ligase activity (23,24). Here we addressed the question whether phosphorylation of parkin occurred and, if so, by which kinases under what cellular conditions. Parkin was found to be phosphorylated on at least five serine residues. Casein kinase-1 (CK-1), protein kinase A (PKA) and protein kinase C (PKC) were identified as parkin kinases in vitro, and inhibition of CK-1 suppressed phosphorylation of parkin in cell lysates. Unfolded protein stress mediated by proteasomal inhibition or endoplasmatic reticulum (ER) stress, but not oxidative stress, reduced the overall phosphorylation of parkin. Unphosphorylated parkin isolated from eukaryotic cells or purified as recombinant fusion protein from bacteria showed a small but significant increase of autoubiquitin ligase activity, compared with parkin phosphorylated in vivo and in vitro. Thus, we suggest that modulat...

Structure of the Parkin in-between-ring domain provides insights for E3-ligase dysfunction in autosomal recessive Parkinson's disease

“…Based on the enhancement of UbcH7, UbcH8 and substrate binding (synphilin-1, Sept5, SIM2) in the presence of the IBR domain it is clear the T351P mutation would not only disrupt the binding site on the IBR domain surface but would spatially separate the RING1 and RING2 domains due to IBR unfolding. This idea, in combination with the Parkin IBR structure also agrees with the negative modulation of Parkin by either S-nitrosylation or phosphorylation (31)(32)(33). The only free thiol in the IBR domain (C323) a potential nitrosylation site, and the residue for phosphorylation (S378) are proximal to each other where the N and C termini meet near the edge of zinc site II.…”

Phosphorylation of Parkin by the Cyclin-dependent Kinase 5 at the Linker Region Modulates Its Ubiquitin-Ligase Activity and Aggregation

“…Parkin was shown to be phosphorylated in vivo at serines 101, 131, 136, and 296 by mass spectroscopy analysis (34). Ser-131 was found by the same authors to be phosphorylated in vitro by cAMP-dependent protein kinase.…”

“…For this, we first checked the levels of Parkin phosphorylation in the absence and presence of the phosphatase inhibitor okadaic acid, which was shown to increase Parkin phosphorylation levels (34). We found that the incubation with okadaic acid robustly increases the steady-state levels of Parkin phosphorylation in HEK 293 cells (Fig.…”

“…Yamamoto et al (34) have previously reported that Parkin is phosphorylated at the serine residues 101, 131, 136, 296, and 378, as determined by mass spectrometry analysis and in vitro phosphorylation assays. To determine the in vivo specificity of Ser-131 to Cdk5, we generated a Parkin construct where the serines 101, 136, 296, and 378 were mutated to alanine but not serine 131.…”

“…2A). We then carried out phosphorylation experiments in the absence of okadaic acid using untransfected HEK 293 cells, which were previously shown to express detectable levels of endogenous Parkin (34). We incubated the untransfected cells with the Ca 2ϩ ionophore A23187 in order to increase the pool of activated Cdk5 (36).…”