The Proteasome Ubiquitin Receptor hRpn13 and Its Interacting Deubiquitinating Enzyme Uch37 Are Required for Proper Cell Cycle Progression

Leah Randles,Ravi K Anchoori,Richard B.S Roden,Kylie J Walters

doi:10.1074/jbc.m115.694588

Abstract

Recently, we reported that bisbenzylidine piperidone RA190 adducts to Cys-88 of the proteasome ubiquitin receptor hRpn13, triggering accumulation of ubiquitinated proteins and endoplasmic reticulum stress-related apoptosis in various cancer cell lines. hRpn13 contains an N-terminal pleckstrin-like receptor for ubiquitin domain that binds ubiquitin and docks it into the proteasome as well as a C-terminal deubiquitinase adaptor (DEUBAD) domain that binds the deubiquitinating enzyme Uch37. Here we report that hRpn13 and Uch37 are required for proper cell cycle progression and that their protein knockdown leads to stalling at G0/G1 Moreover, serum-starved cells display reduced hRpn13 and Uch37 protein levels with hallmarks of G0/G1 stalling and recovery to their steady-state protein levels following release from nutrient deprivation. Interestingly, loss of hRpn13 correlates with a small but statistically significant reduction in Uch37 protein levels, suggesting that hRpn13 interaction may stabilize this deubiquitinating enzyme in human cells. We also find that RA190 treatment leads to a loss of S phase, suggesting a block of DNA replication, and G2 arrest by using fluorescence-activated cell sorting. Uch37 deprivation further indicated a reduction of DNA replication and G0/G1 stalling. Overall, this work implicates hRpn13 and Uch37 in cell cycle progression, providing a rationale for their function in cellular proliferation and for the apoptotic effect of the hRpn13-targeting molecule RA190.

Highlights

The ubiquitin-proteasome pathway is the major route for regulated proteolysis in eukaryotes, as reviewed in Refs. 1, 2
Prior to translocation into the proteasome core particle (CP), substrates are deubiquitinated by proteasome deubiquitinating enzymes, which include Rpn11 (24 –26), the aforementioned Ubp6/ Usp14 [27], and Uch37/UCHL5 [28], which is contributed to the proteasome by hRpn13 (29 –31)
We report here that hRpn13 and Uch37 are essential for proper cell cycle progression and provide insights into the cellular effects of the hRpn13-targeting bisbenzylidine piperidone RA190

Summary

Introduction

The ubiquitin-proteasome pathway is the major route for regulated proteolysis in eukaryotes, as reviewed in Refs. 1, 2. Cell lysates were resolved and immunoprobed for the cell cycle regulators p27Kip1, Cdc25c, Wee1, and p21Cip1 (Fig. 1C, left panel). This experiment was conducted independently four times to yield a p value of 0.039 for p27Kip1 stabilization following hRpn13 knockdown by two-tailed, two-sample equal variance Student’s t test analysis (Fig. 2A, right panel).

Results

Conclusion