Basic Leucine Zipper Protein Cnc-C Is a Substrate and Transcriptional Regulator of the Drosophila 26S Proteasome

Abstract

While the 26S proteasome is a key proteolytic complex, little is known about how proteasome levels are maintained in higher eukaryotic cells. Here we describe an RNA interference (RNAi) screen of Drosophila melanogaster that was used to identify transcription factors that may play a role in maintaining levels of the 26S proteasome. We used an RNAi library against 993 Drosophila transcription factor genes to identify genes whose suppression in Schneider 2 cells stabilized a ubiquitin-green fluorescent protein reporter protein. This screen identified Cnc (cap 'n' collar [CNC]; basic region leucine zipper) as a candidate transcriptional regulator of proteasome component expression. In fact, 20S proteasome activity was reduced in cells depleted of cnc. Immunoblot assays against proteasome components revealed a general decline in both 19S regulatory complex and 20S proteasome subunits after RNAi depletion of this transcription factor. Transcript-specific silencing revealed that the longest of the seven transcripts for the cnc gene, cnc-C, was needed for proteasome and p97 ATPase production. Quantitative reverse transcription-PCR confirmed the role of Cnc-C in activation of transcription of genes encoding proteasome components. Expression of a V5-His-tagged form of Cnc-C revealed that the transcription factor is itself a proteasome substrate that is stabilized when the proteasome is inhibited. We propose that this single cnc gene in Drosophila resembles the ancestral gene family of mammalian nuclear factor erythroid-derived 2-related transcription factors, which are essential in regulating oxidative stress and proteolysis.

p62/Sequestosome-1, Autophagy-related Gene 8, and Autophagy in Drosophila Are Regulated by Nuclear Factor Erythroid 2-related Factor 2 (NRF2), Independent of Transcription Factor TFEB

“…6C). Consistent with a previous study (65), we observed accumulation of CncC upon proteasomal inhibition. This did not occur after BafA1 treatment (Fig.…”

“…This is most probably due to the lack of the N-terminal transactivation domain found in CncC. Overexpressed CncC is a proteasome substrate, not detectable under normal conditions, but is stabilized by inhibition or depletion of proteasome subunit S5a (65). Consistently, we could not easily detect overexpressed CncC by Western blot in cultured cells unless proteasomal degradation was inhibited.…”

Dual Regulation of the Transcriptional Activity of Nrf1 by β-TrCP- and Hrd1-Dependent Degradation Mechanisms

“…The degradation process of Skn-1 is mediated by the p38 mitogen-activated protein (MAP) kinase, GSK-3, and the insulin-like receptor pathways, suggesting the presence of phosphorylation-dependent regulation. CncC is controlled by both Keap1-dependent and -independent proteasomal pathways (6). Notably, CncC also regulates the gene expression of proteasome subunits.…”

“…The transcription factors Caenorhabditis elegans Skn-1 and Drosophila CncC are considered to be ancestral genes of NF-E2-related factors and activate the expression of oxidative stress response genes (32). Intriguingly, both factors are also regulated by proteasomal degradation (4,6,25). For instance, Skn-1 is degraded through polyubiquitination by the WDR-23/Cul4/DDB1 ubiquitin ligase under physiological conditions (4).…”

Understanding the Mechanisms Involved in the Regulation of Cytochrome P450 Gene Expression in Drosophila melanogaster (Diptera: Drosophilidae)

“…CnCC regulates the transcriptional responses to xenobiotic compounds whilst the CnCC-Keap1 (dKeap 1) protein complexes regulate the native cellular and developmental processes [18,57].…”