Phosphorylation of Tyr-950 in the proteasome scaffolding protein RPN2 modulates its interaction with the ubiquitin receptor RPN13

Abstract

Protein substrates are targeted to the 26S proteasome through several ubiquitin receptors. One of these receptors, RPN13, is recruited to the proteasome by binding of its N-terminal pleckstrin-like receptor of ubiquitin (PRU) domain to C-terminal residues of the scaffolding protein RPN2. The RPN13 PRU domain is followed by a flexible linker and a C-terminal deubiquitylase adaptor (DEUBAD) domain, which recruits and activates the deubiquitylase UCH37. Both RPN13 and UCH37 have been implicated in human cancers, and inhibitors of the RPN2–RPN13 interaction are being developed as potential therapeutic anticancer agents. Our current study builds on the recognition that a residue central to the RPN2–RPN13 interaction, RPN2 Tyr-950, is phosphorylated in Jurkat cells. We found that the Tyr-950 phosphorylation enhances binding to RPN13. The crystal structure of the RPN2–RPN13 pTyr-950–ubiquitin complex was determined at 1.76-Å resolution and reveals specific interactions with positively charged side chains in RPN13 that explain how phosphorylation increases binding affinity without inducing conformational change. Mutagenesis and quantitative binding assays were then used to validate the crystallographic interface. Our findings support a model in which RPN13 recruitment to the proteasome is enhanced by phosphorylation of RPN2 Tyr-950, have important implications for efforts to develop specific inhibitors of the RPN2–RPN13 interaction, and suggest the existence of a previously unknown stress-response pathway.

26S Proteasome Non-ATPase Regulatory Subunits 1 (PSMD1) and 3 (PSMD3) as Putative Targets for Cancer Prognosis and Therapy

“…We previously speculated that the difference in sensitivity between CML and normal progenitors to knockdown of PSMD1 or PSMD3 may be due to differences in post-translational modifications, which could alter peptide targets in malignant versus normal tissues [ 31 ]. Indeed, Hemmis et al recently reported that PSMD1 can be phosphorylated at tyrosine 950, and that this modulates interactions between ubiquitin receptors [ 42 ].…”

Chirality and asymmetry increase the potency of candidate ADRM1/RPN13 inhibitors

“…RA413S and RA414 also both show stronger binding to their 42kDa protein target, previously identified as RPN13, in ovarian cancer cell lysates than RA183. Although these reactive compounds have potential for off-target effects [50], RA190 and related inhibitors bind to cysteine 88 within a groove of RPN13 that mediates binding to the proteasome via RPN2 [16,17]. This suggests that drug blockade of RPN13 association to the 19S RP inhibits both proteasomal degradation and deubiquitinase activity of its binding partner UCH37.…”

“…The small molecule RA190 [11][12][13] was identified as the prototypic inhibitor of RPN13, and more potent analogs RA183 [14] and RA375 [15] were generated. Each of these molecules is a Michael acceptor that adducts RPN13 Cysteine 88 that resides within a groove required for binding of RPN13 to the proteasome via RPN2 [16,17], as well as other cellular proteins at higher concentrations [18,19]. However, in cytotoxicity assays, these RPN13 inhibitors (iRPN13) are not as potent as bortezomib based upon their IC50 against cancer cell lines.…”

Structure-function analyses of candidate small molecule RPN13 inhibitors with antitumor properties

“…Since RPN13 is over expressed in cancer [17], Kisselev has hypothesized that the covalent RA inhibitors block both ends of the proteasome, whereas in normal cells only one end is block and the other can utilize alternative ubiquitin receptors to maintain some proteasome function [14]. However, recent structural data suggest that these RA inhibitors act to prevent RPN2 binding and thus association with the 19S RP [4,59].…”