β-TrCP1 Is a Vacillatory Regulator of Wnt Signaling

Abstract

Summary Simultaneous hyperactivation of Wnt and antioxidant response (AR) are often observed during oncogenesis. However, it remains unclear how the β-catenin-driven Wnt and the Nrf2-driven AR mutually regulate each other. The situation is compounded because many players in these two pathways are redox-sensors, rendering bolus-redox-signal-dosing methods uninformative. Herein we examine the ramifications of single-protein-target-specific AR-upregulation in various knockdown lines. Our data document that Nrf2/AR strongly inhibits β-catenin/Wnt. The magnitude and mechanism of this negative regulation are dependent on the direct interaction between β-catenin-N-terminus and β-TrCP1 (an antagonist of both Nrf2 and β-catenin), and independent of binding between Nrf2 and β-TrCP1. Intriguingly, β-catenin positively regulates AR. Because AR is a negative regulator of Wnt regardless of β-catenin-N-terminus, this switch of function is likely sufficient to establish a new Wnt/AR equilibrium during tumorigenesis.

Precision Electrophile Tagging in Caenorhabditis elegans

“…We describe several C. elegans lines amenable to this procedure and show that single-protein targeting with diffusible reactive signaling electrophiles can be achieved in live worms with no observable negative effects. We have shown similar results in cultured cells 1−5 and fish. 6 These results in C. elegans , one of the most powerful and genetically tractable systems for the study of stress and aging, 7,8 represent a key step in developing model systems for interrogating simultaneously the genetics and biochemistry of electrophile signaling.…”

Post-transcriptional regulation of Nrf2-mRNA by the mRNA-binding proteins HuR and AUF1

“…For both HuR and AUF1, which we also identified as a novel regulator of Nrf2 activity, we found 2-3-fold suppression in Nrf2 activity in both HuR-and AUF1-knockdown cells relative to control cells ( Figures 1F, 2A, 2B, and S5). These changes are indicative of significant regulatory events because we and others have previously documented the narrow dynamic range (2-4-fold) of Nrf2 activity modulation in various readouts, including luciferase reporter assays, flow cytometry-based analyses, as well as qRT-PCR and western blot analyses which assay endogenous Nrf2-driven genes (Huang et al, 2012;Levonen et al, 2004;Long et al, 2017;Parvez et al, 2015). Consistent with this logic, knockdown of Nrf2 gave a similar fold change in Nrf2 activity to HuR knockdown.…”

The mRNA-binding protein HuR is a kinetically-privileged electrophile sensor

“…Intriguingly, T-REX delivery did not achieve particularly high HNE occupancy on HuR. The 11 ± 1% modification efficiency of HuR achieved under T-REX is not as high as we have observed for several other RES-sensor proteins under T-REX including Keap1 (20-55%, with Halo-fusion at either N-or C-terminus), [24,[38][39][40] HSPB7 (30 ± 10%), [49] Akt3 (20 ± 5%), [41] and Ube2V2 (15± 6%) [34] (Figure 2I). Consistent with these observations, labeling of HuR (40 μM) by HNE (40 μM) in vitro took longer time to saturate than other proteins we have reported to sense HNE by T-REX [such as labeling of Ube2V2 by HNE [34] (approximately 1 min, concentrations of both Ube2V2 and HNE = 12 μM); and HSPB7 [44] (2-4 min, concentrations of both HSPB7 and HNE = 12 μM)].…”