Abstract
The c-Myc oncogene is a transcription factor that regulates the expression of a very large set of genes mainly involved in cell growth and proliferation. It is overexpressed in more than 70% of human cancers, illustrating the importance of keeping its levels and activity under control. The ubiquitin proteasome system is a major regulator of MYC levels in humans as well as in model organisms such as Drosophila melanogaster. Although the E3 ligases that promote MYC ubiquitination have been largely investigated, the identity and the role of the deubiquitinating enzymes, which counteract their action is only beginning to be unraveled. Using isoform-specific CRISPR-Cas9 mutagenesis, we show that the Drosophila homolog of the Ubiquitin Specific Protease USP36 has different isoforms with specific sub-cellular localizations and that the nucleolar dUSP36-D isoform is specifically required for cell and organismal growth. We also demonstrate that this isoform interacts with dMYC and the E3 ligase AGO and regulates their stability and ubiquitination levels. Furthermore, we show that dUSP36 is ubiquitinated by AGO and is able to self-deubiquitinate. Finally, we provide in vivo evidence supporting the functional relevance of these regulatory relationships. Together these results reveal that dMYC, AGO and dUSP36 form a tripartite, evolutionary conserved complex that acts as a regulatory node to control dMYC protein levels.
Highlights
The c-Myc oncogene encodes a pleiotropic transcription factor controlling the expression of a very large number of genes involved in differentiation, apoptosis, angiogenesis, metabolism, ribosomal biogenesis, cell growth and proliferation (van Riggelen et al, 2010; Sabo and Amati, 2014; Sabo et al, 2014)
When transfected into Drosophila S2 cells, V5-tagged isoforms display different subcellular localizations (Figures 1B–D): while the dUSP36-B isoform accumulates in the cytoplasm and at the nuclear membrane as shown by colocalization with Lamin (Figure 1B), the dUSP36-C and -D isoforms are localized in the nucleus (Figures 1C,D)
The 110–670 construct, which contains the Ubiquitin Specific Protease (USP) catalytic domain, is present in the nucleus and in the cytoplasm (Figure 2D). These data place the sequence(s) responsible for the nuclear localization of the dUSP36-C and -D isoforms in the 670–1038 C-terminal domain, which is consistent with the identification of two putative Nuclear Localization Sequences (NLS, represented by black bars in Figure 2A) by NLS prediction programs (PSORT, NLS Mapper and SeqNLS). As these sequences are present in the dUSP36-B isoform that is not localized in the nucleus, we hypothesized that the N-terminal forty amino-acid long domain specific of this isoform (Figure 2B) acts as a Nuclear Export Sequence (NES)
Summary
The c-Myc oncogene encodes a pleiotropic transcription factor controlling the expression of a very large number of genes involved in differentiation, apoptosis, angiogenesis, metabolism, ribosomal biogenesis, cell growth and proliferation (van Riggelen et al, 2010; Sabo and Amati, 2014; Sabo et al, 2014). The expression level, stability and activity of MYC are tightly controlled to ensure proper cell growth and proliferation. In Drosophila, partial loss-of-function mutations of Nucleolar dUSP36 Deubiquitinates and Stabilizes dMYC the Myc ortholog (dMyc synonymous diminutive) result in delayed development and smaller than normal adult flies while null mutations strongly affect cell and organismal growth resulting in developmental lethality (Johnston et al, 1999; Pierce et al, 2004; Gallant, 2013; Grifoni and Bellosta, 2015). The Drosophila Fbw ortholog Archipelago (Ago) is an important regulator of dMYC stability: loss-of-function mutations of Ago result in strongly elevated dMYC protein levels and increased tissue growth (Moberg et al, 2004)
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