USP15 stabilizes the transcription factor Nrf1 in the nucleus, promoting the proteasome gene expression.

The transcriptional factor Nrf1 (NF-E2-related factor 1) sustains protein homeostasis (proteostasis) by regulating the expression of proteasome genes. Under physiological conditions, the transcriptional activity of Nrf1 is repressed by its sequestration into the endoplasmic reticulum (ER) and furthermore by two independent ubiquitin-proteasome pathways, comprising Hrd1 and ß-TrCP in the cytoplasm and nucleus, respectively. However, the molecular mechanisms underlying Nrf1 activation remain unclear. Here, we report that USP15 (Ubiquitin-Specific Protease 15) activates Nrf1 in the nucleus by stabilizing it through deubiquitination. We first identified USP15 as an Nrf1-associated factor through proteome analysis. USP15 physically interacts with Nrf1, and it markedly stabilizes Nrf1 by removing its ubiquitin moieties. USP15 activates the Nrf1-mediated expression of a proteasome gene luciferase reporter and endogenous proteasome activity. The siRNA-mediated knockdown of USP15 diminishes the Nrf1-induced proteasome gene expression in response to proteasome inhibition. These results uncover a new regulatory mechanism that USP15 activates Nrf1 against the ß-TrCP inhibition to maintain proteostasis.

Inhibitory mechanism of FAT4 gene expression in response to actin dynamics during Src-induced carcinogenesis.

Oncogenic transformation is characterized by morphological changes resulting from alterations in actin dynamics and adhesive activities. Emerging evidence suggests that the protocadherin FAT4 acts as a tumor suppressor in humans, and reduced FAT4 gene expression has been reported in breast and lung cancers and melanoma. However, the mechanism controlling FAT4 gene expression is poorly understood. In this study, we show that transient activation of the Src oncoprotein represses FAT4 mRNA expression through actin depolymerization in the immortalized normal human mammary epithelial cell line MCF-10A. Src activation causes actin depolymerization via the MEK/Erk/Cofilin cascade. The MEK inhibitor U0126 blocks the inhibitory effect of Src on FAT4 mRNA expression and Src-induced actin depolymerization. To determine whether actin dynamics act on the regulation of FAT4 mRNA expression, we treated MCF-10A cells with the ROCK inhibitor Y-27632. Y-27632 treatment decreased FAT4 mRNA expression. This suppressive effect was blocked by siRNA-mediated knockdown of Cofilin1. Furthermore, simultaneous administration of Latrunculin A (an actin depolymerizing agent), Y-27632, and Cofilin1 siRNA to the cells resulted in a marked reduction of FAT4 mRNA expression. Intriguingly, we also found that FAT4 mRNA expression was reduced under both low cell density and low stiffness conditions, which suggests that mechanotransduction affects FAT4 mRNA expression. Additionally, we show that siRNA-mediated FAT4 knockdown induced the activity of the Hippo effector YAP/TAZ in MCF-10A cells. Taken together, our results reveal a novel inhibitory mechanism of FAT4 gene expression through actin depolymerization during Src-induced carcinogenesis in human breast cells.

The casein kinase 2-nrf1 axis controls the clearance of ubiquitinated proteins by regulating proteasome gene expression.

Impairment of the ubiquitin-proteasome system (UPS) has been implicated in the pathogenesis of human diseases, including neurodegenerative disorders. Thus, stimulating proteasome activity is a promising strategy to ameliorate these age-related diseases. Here we show that the protein kinase casein kinase 2 (CK2) regulates the transcriptional activity of Nrf1 to control the expression of the proteasome genes and thus the clearance of ubiquitinated proteins. We identify CK2 as an Nrf1-binding protein and find that the knockdown of CK2 enhances the Nrf1-dependent expression of the proteasome subunit genes. Real-time monitoring of proteasome activity reveals that CK2 knockdown alleviates the accumulation of ubiquitinated proteins upon proteasome inhibition. Furthermore, we identify Ser 497 of Nrf1 as the CK2 phosphorylation site and demonstrate that its alanine substitution (S497A) augments the transcriptional activity of Nrf1 and mitigates proteasome dysfunction and the formation of p62-positive juxtanuclear inclusion bodies upon proteasome inhibition. These results indicate that the CK2-mediated phosphorylation of Nrf1 suppresses the proteasome gene expression and activity and thus suggest that the CK2-Nrf1 axis is a potential therapeutic target for diseases associated with UPS impairment.