Differential Interaction of the E3 Ligase Parkin with the Proteasomal Subunit S5a and the Endocytic Protein Eps15

Abstract

Parkin is a multidomain E3 ligase associated with autosomal recessive Parkinson disease. The N-terminal ubiquitin-like domain (Ubld) of parkin functions with the S5a proteasomal subunit, positioning substrate proteins for degradation. In addition the parkin Ubld recruits the endocytotic protein Eps15, allowing the E3 ligase to ubiquinate Eps15 distal from its parkininteracting site. The recognition sequences in the S5a subunit and Eps15 for the parkin Ubld are ubiquitin-interacting motifs (UIM). Each protein has two UIM sequences separated by a 50-residue spacer in S5a, but only ϳ5 residues in Eps15. In this work we used NMR spectroscopy to determine how the parkin Ubld recognizes the proteasomal subunit S5a compared with Eps15, a substrate for ubiquitination. We show that Eps15 contains two flexible ␣-helices each encompassing a UIM sequence. The ␣-helix surrounding UIM II is longer than that for UIM I, a situation that is reversed from S5a. Furthermore, we show the parkin Ubld preferentially binds to UIM I in the S5a subunit. This interaction is strongly diminished in a K48A substitution, found near the center of the S5a interacting surface on the parkin Ubld. In contrast to S5a, parkin recruits Eps15 using both its UIM sequences resulting in a larger interaction surface that includes residues from ␤1 and ␤2, not typically known to interact with UIM sequences. These results show that the parkin Ubld uses differential surfaces to recruit UIM regions from the S5a proteasomal subunit compared with Eps15 involved in cell signaling.Modification of proteins by ubiquitin is an essential biochemical process that signals proteins for degradation via the 26 S proteasome and also for non-proteolytic processes such as cell cycle and cell division, protein trafficking, endocytosis, and DNA repair (1-3). Three enzymes in the ubiquitination pathway (E1, E2, and E3) label a targeted protein with ubiquitin. The E3 enzymes are important for mediating the transfer of ubiquitin onto the target protein through their interaction with both the E2 enzyme and substrate and provide the specificity for target protein recognition. Autosomal recessive juvenile parkinsonism (ARJP) 2 is an early-onset familial form of the disease that is clinically indistinguishable from the more prevalent idiopathic form of Parkinson disease. Mutations in several genes have been identified in ARJP patients, although the most commonly mutated gene encodes the E3 ubiquitin-protein ligase parkin (4 -6). Mutations in parkin account for ϳ50% of all ARJP cases. Parkin is a 465-residue multidomain E3 ligase comprising an N-terminal ubiquitin-like domain (Ubld) followed by a unique parkin-specific domain, two RING domains (RING0, RING1), an in-between RING (IBR) domain, and a C-terminal RING domain (RING2) (7,8). Mutations associated with ARJP are found throughout the parkin protein and have profound affects on the folding and functionality of the protein.For example, missense mutations in the C terminus of parkin have been shown to disrupt its function with E2 ...

The E3 Ubiquitin Ligase Parkin Is Recruited to the 26 S Proteasome via the Proteasomal Ubiquitin Receptor Rpn13

“…However, they could not confirm the interaction by co-immunoprecipitation or by in vitro binding experiments, suggesting either a very weak interaction or technical limitations of their assays. Interestingly, our NMR results showed the same pattern of chemical shifts perturbations in the parkin Ubl domain with the Rpn13 Pru domain as previously described with the UIM domains of Rpn10 (49). These involve a contiguous surface containing residues Phe-13, Arg-42, Ile-44, Ala-46, Lys-48, Glu-49, Arg-51, Leu-61, Ile-66, Val-67, and Gln-71.…”

“…However, the magnitude of the chemical shifts we observed was much higher when using the Rpn13 Pru domain compared with those obtained with the Rpn10 UIMs. In line with these observations, the calculated K d value between the parkin Ubl and the Rpn13 Pru domain was more than 1 order of magnitude lower than the one reported for parkin Ubl and Rpn10 UIMs (217 Ϯ 51 M) (49), further arguing in favor of our model in which Rpn13 acts as the major receptor for parkin within the proteasome.…”

“…The low micromolar affinity constant measured between the Ubl and Rpn13 is in qualitative agreement with the NMR results. Moreover, the calculated K d between parkin Ubl and Rpn13 Pru domain is ϳ2 orders of magnitude lower than the one reported for parkin Ubl binding to the Rpn10 UIMs (217 Ϯ 51 M) (49), likely explaining the difficulty in confirming the parkin-Rpn10 interaction by pulldown assays (32). We could also show that the Ubl domain of parkin directly interacts with the active purified bovine proteasome (Fig.…”

Molecular Basis for Impaired DNA Damage Response Function Associated with the RAP80 ΔE81 Defect

“…Considering the broad, vital role that multivalent interactions play in human biology (39) and the variety of different UIM-Ub pairs (40,41), it is of interest that near complete abolishment of multivalent binding is implicated in the development of cancer; that is, binding of polyUb by ⌬E81-RAP80 is not abolished. The C-UIM binds Ub normally, whereas abrogated binding of Ub by the N-UIM is responsible for the near total loss of multivalent recognition.…”

The Parkin-Like Human Homolog of Drosophila Ariadne-1 (HHARI) Can Induce Aggresome Formation in Mammalian Cells and Is Immunologically Detectable in Lewy Bodies

“…Parkin functions as an E3 ligase to regulate the abundance of itself and a number of other substrate proteins (Um and Chung 2006). Some parkin substrates include CDCrel-1 (Zhang et al 2000), synphilin-1 (Chung et al 2001), α-synuclein (Choi et al 2001), CASK (Fallon et al 2002;Dawson and Dawson 2010), the aminoacyl-tRNA synthetase cofactor, p38 (Ko et al 2005), PDZ protein PICK1 (Joch et al 2007), programmed cell death-2 isoform 1 (PDCD2-1; Fukae et al 2009), PLCgamma1 (Dehvari et al 2009), and S5a proteasomal Eps15 endocytic protein (Safadi and Shaw 2010).…”