Parkin is an E3 ubiquitin ligase that mediates the ubiquitination of protein substrates. The mutations in the parkin gene can lead to a loss of function of parkin and cause autosomal recessive juvenile onset parkinsonism. Recently, parkin was reported to be involved in the regulation of mitophagy. Here, we identify the Bcl-2, an anti-apoptotic and autophagy inhibitory protein, as a substrate for parkin. Parkin directly binds to Bcl-2 via its C terminus and mediates the mono-ubiquitination of Bcl-2, which increases the steady-state levels of Bcl-2. Overexpression of parkin, but not its ligase-deficient forms, decreases autophagy marker LC3 conversion, whereas knockdown of parkin increases LC3 II levels. In HeLa cells, a parkin-deficient cell line, knockdown of parkin does not change LC3 conversion. Moreover, overexpression of parkin enhances the interactions between Bcl-2 and Beclin 1. Our results provide evidence that parkin mono-ubiquitinates Bcl-2 and regulates autophagy via Bcl-2.
Parkinson disease (PD)2 is the second most common neurodegenerative disorder after Alzheimer disease (1) and is characterized by a distinct set of motor symptoms including tremor, muscle rigidity, postural instability, and bradykinesia (2). Although the cause of PD is poorly understood, there is evidence that both environmental factors and genetic factors contribute to its development. Recently, several genes have been reported to be associated with the pathogenesis of familial forms of PD. Mutations in the parkin gene (PARK2; OMIM600116) cause autosomal recessive juvenile onset parkinsonism (3). It has been shown that mutations in parkin account for nearly 50% of patients with the early onset familial PD cases (3-6) and more than 15% of sporadic PD cases with early onset (7).Parkin is a 465-amino acid protein that contains an ubiquitin-like domain at its N terminus and two RING finger domains separated by an in-between-ring domain at its C terminus. Similar to other RING finger-containing proteins, parkin is an E3 ubiquitin ligase. Parkin ubiquitinates several target proteins and enhances their degradation via the ubiquitin-proteasome system (8, 9). Ubiquitination of a substrate is usually processed by a multi-step involving the sequential activity of an E1 ubiquitin-activating enzyme, an E2 ubiquitin-conjugating enzyme, and an E3 ubiquitin-protein ligase (10). It was reported that parkin can selectively interact with the E2 enzymes, UbcH7 and UbcH8 (9,11,12). A number of protein substrates for parkin have been identified, including synphilin-1 (13, 14), CDCrel-1 and 2a (12, 15), Pael-R (16), synaptotagmin XI (17), ␣-and -tubulin (18), RanBP2 (19), cyclin E (20), the aminoacyl-tRNA synthetase cofactor p38/AIMP2 (21, 22), Eps15 (23), and far upstream sequence element-binding protein 1 (24). Within these substrates, p38/AIMP2 and far upstream sequence element-binding protein 1 were reported to be accumulated in brains of parkin null mice, MPTP (1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine) treated mice, and sporadic PD cases (22,24)...
