Deubiquitinating Enzyme USP20 Regulates Extracellular Signal-Regulated Kinase 3 Stability and Biological Activity

Abstract

Extracellular signal-regulated kinase 3 (ERK3) is an atypical mitogenactivated protein kinase (MAPK) whose regulatory mechanisms and biological functions remain superficially understood. Contrary to most protein kinases, ERK3 is a highly unstable protein that is subject to dynamic regulation by the ubiquitin-proteasome system. However, the effectors that control ERK3 ubiquitination and degradation are unknown. In this study, we carried out an unbiased functional loss-of-function screen of the human deubiquitinating enzyme (DUB) family and identified ubiquitin-specific protease 20 (USP20) as a novel ERK3 regulator. USP20 interacts with and deubiquitinates ERK3 both in vitro and in intact cells. The overexpression of USP20 results in the stabilization and accumulation of the ERK3 protein, whereas USP20 depletion reduces the levels of ERK3. We found that the expression levels of ERK3 correlate with those of USP20 in various cellular contexts. Importantly, we show that USP20 regulates actin cytoskeleton organization and cell migration in a manner dependent on ERK3 expression. Our results identify USP20 as a bona fide regulator of ERK3 stability and physiological functions.KEYWORDS deubiquitinating enzymes, ERK3, MAPKs E xtracellular signal-regulated kinase 3 (ERK3) along with its paralogous kinase ERK4 define a distinct subfamily of atypical mitogen-activated protein kinases (MAPKs) (1). ERK3 is expressed ubiquitously in adult mammalian tissues, with the highest levels being found in the central nervous system, skeletal muscle, lung, thymus, and testis (2-4). ERK4 shows a more restricted expression profile and is predominantly detected in brain tissue (4). Much remains to be learned about the substrates and physiological functions of these signaling enzymes. Unlike classical MAPKs, such as ERK1/ERK2, cJun NH 2 -terminal kinases, and p38s, that phosphorylate a large spectrum of substrates, ERK3 and ERK4 appear to have a restricted substrate specificity. Their best-characterized and validated substrate is the protein kinase MAPK-activated protein kinase 5 (MK5) (5-8). Genetic invalidation studies in mice have revealed important functions of ERK3 in fetal growth, pulmonary maturation, thymocyte development, and neuromuscular control (9-12). Biochemical and cellular studies also suggest that ERK3 plays key roles in transcriptional control, cell adhesion, cell migration, and the DNA damage response (13-16). The physiological functions of ERK4 are unknown.The activity of ERK3 and ERK4 is regulated in part by the phosphorylation of the Ser-Glu-Gly motif in the activation loop, which stimulates their intrinsic kinase activity and affinity for the substrate MK5 (17, 18). We and others have identified group I p21-activated kinases as ERK3/ERK4 activation loop kinases (19,20). Of note, the activating phosphorylation of ERK3/ERK4 is not modulated by classical MAPK stimuli or by any other extracellular stimuli tested, suggesting that other regulatory mechanisms control their biological activity (17). Citation Mathien S,...

The deubiquitinating enzyme USP20 stabilizes ULK1 and promotes autophagy initiation

“…Ever since USP20 was identified as a substrate of the cullin‐RING ligase family member CRL VHL , several signaling molecules, including hypoxia‐inducible factor 1α, Rad17, Claspin, TRAF6, and ERK3, have been reported as substrates of USP20 regarding hypoxia, DNA damage responses, and TLR4‐ and mitogen‐signaling pathways . In this study, we demonstrate that the deubiquitinating enzyme USP20 stabilizes ULK1 through deubiquitinating ULK1 at the basal level and is thus a critical factor in inducing autophagy initiation.…”

Network Analysis of UBE3A/E6AP-Associated Proteins Provides Connections to Several Distinct Cellular Processes

“…In addition, the link observed in the UBE3A network between UBE3A/the HUN complex and centrosomal and cytoskeleton-related functions becomes even more apparent in the HUN network, which highlights the association of NEURL4 with the centrosomal proteins CEP97, CCP110, CEP290, CEP170 and the pericentriolar protein PCM1. Furthermore, MAPK6, known for its regulatory role in actin cytoskeleton remodeling and cell migration [61–64], showed a strong functional enrichment for spindle and centriole-related GO terms due to the presence of the kinases AURKA and AURKB as well as the proteins ASPM, CEP170, and MAP7D1 amongst its HCIPs. ASPM, a centrosomal protein previously identified as interactor of UBE3A [65] was also coimmunoprecipitated by HERC2 [30] (Figure 5).…”

ERK3/MAPK6 controls IL-8 production and chemotaxis

“…It is tempting to hypothesize that LPS triggers alterations in the ubiquitin signaling machinery contributing to ERK3 ubiquitination and degradation, leading to the attenuation of IL-8 production and inflammation in primary epithelium. While Usp20 has been shown to function as a DUB for ERK3, the E3 ubiquitin ligase of ERK3 is not known (Mathien et al, 2017). Also, the kind of ubiquitin chains synthesized on ERK3 in response to LPS deserves further investigations.…”

“…The E3 ubiquitin ligases responsible for this process are currently unknown (Coulombe and Meloche, 2007;Coulombe et al, 2004;Coulombe et al, 2003). However, Usp20 was recently identified as the first deubiquitinating enzyme (DUB) for ERK3 (Mathien et al, 2017). The physiological stimuli that activate ERK3 remain unclear and the role of this atypical MAPK in the regulation of physiological processes such as innate immunity has not been studied.…”