The Deubiquitinase USP47 Stabilizes MAPK by Counteracting the Function of the N-end Rule ligase POE/UBR4 in Drosophila

RAS-induced MAPK signaling is a central driver of the cell proliferation apparatus. Disruption of this pathway is widely observed in cancer and other pathologies. Consequently, considerable effort has been devoted to understanding the mechanistic aspects of RAS-MAPK signal transmission and regulation. While much information has been garnered on the steps leading up to the activation and inactivation of core pathway components, comparatively little is known on the mechanisms controlling their expression and turnover. We recently identified several factors that dictate Drosophila MAPK levels. Here, we describe the function of one of these, the deubiquitinase (DUB) USP47. We found that USP47 acts post-translationally to counteract a proteasome-mediated event that reduces MAPK half-life and thereby dampens signaling output. Using an RNAi-based genetic interaction screening strategy, we identified UBC6, POE/UBR4, and UFD4, respectively, as E2 and E3 enzymes that oppose USP47 activity. Further characterization of POE-associated factors uncovered KCMF1 as another key component modulating MAPK levels. Together, these results identify a novel protein degradation module that governs MAPK levels. Given the role of UBR4 as an N-recognin ubiquitin ligase, our findings suggest that RAS-MAPK signaling in Drosophila is controlled by the N-end rule pathway and that USP47 counteracts its activity.
In Drosophila, steady-state levels of the regulatory protein MAPK are found to be governed by the opposing actions of the deubiquitinase USP47 and components of the N-end rule pathway.
Author Summary The RAS-MAPK pathway plays a central role in the development of multicellular organisms, predominantly by regulating cell proliferation and differentiation. At its core, the pathway includes the RAS GTPase and three kinases (RAF, MEK, and MAPK) that transmit RAS signals through a phosphorylation cascade. Several factors have been discovered over the years that modulate signal transmission by altering the kinetics of phosphorylation/dephosphorylation of the core pathway components. In contrast, scant information is available on the mechanisms governing their expression and steady-state levels. Here, we report the characterization of a deubiquitinase (DUB) known as USP47, which stabilizes MAPK protein levels by opposing the activity of the proteasome, which is a major protein degradation machinery in eukaryotic cells. A search for enzymes that opposed USP47 activity identified several components of the N-end rule pathway centered on the poorly characterized POE/UBR4 E3 ligase. These components appear to work together to target MAPK for proteasome-mediated protein degradation and thereby reduce MAPK protein half-life. Together, this work identifies a new means by which RAS-MAPK signaling output is controlled in Drosophila. As the role of the N-end rule pathway remains largely uncharacterized in metazoans, this work opens new opportunities for studying its function in higher organisms.