The ubiquitin ligase gp78 promotes sarcoma metastasis by targeting KAI1 for degradation
ABSTRACT Metastasis is the primary cause of mortality from cancer, but the mechanisms leading to metastasis are poorly understood. In particular, relatively little is known about metastasis in cancers of mesenchymal origins, which are known as sarcomas. Approximately ten proteins have been characterized as 'metastasis suppressors', but how these proteins function and are regulated is, in general, not well understood. Gp78 (also known as AMFR or RNF45) is a RING finger E3 ubiquitin ligase that is integral to the endoplasmic reticulum (ER) and involved in ER-associated degradation (ERAD) of diverse substrates. Here we report that expression of gp78 has a causal role in the metastasis of an aggressive human sarcoma and that this prometastatic activity requires the E3 activity of gp78. Further, gp78 associates with and targets the transmembrane metastasis suppressor, KAI1 (also known as CD82), for degradation. Suppression of gp78 increases KAI1 abundance and reduces the metastatic potential of tumor cells, an effect that is largely blocked by concomitant suppression of KAI1. An inverse relationship between these proteins was confirmed in a human sarcoma tissue microarray. Whereas most previous efforts have focused on genetic mechanisms for the loss of metastasis suppressor genes, our results provide new evidence for post-translational downregulation of a metastasis suppressor by its ubiquitin ligase, resulting in abrogation of its metastasis-suppressing effects.
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- "As shown in Figure 3F, Amfr À/À MEFs showed an increased number of HSV-1-GFP-positive cells. AMFR Catalyzes K27-Linked Polyubiquitination of STING As an E3 ubiquitin ligase in ERAD (ER-associated protein degradation ), AMFR catalyzes the polyubiquitination of HMG-CoA reductase (HMGCR) and the metastasis suppressor KAI1, and promotes their proteasome-dependent degradation (Song et al., 2005; Tsai et al., 2007). Our in vitro ubiquitination assays confirmed that AMFR could catalyze the formation of polyubiquitin chains, whereas AMFR C2S could not (Figure 4B). "
ABSTRACT: Stimulator of interferon genes (STING, also known as MITA, ERIS, or MPYS) is essential for host immune responses triggered by microbial DNAs. However, the regulatory mechanisms underlying STING-mediated signaling are not fully understood. We report here that, upon cytoplasmic DNA stimulation, the endoplasmic reticulum (ER) protein AMFR was recruited to and interacted with STING in an insulin-induced gene 1 (INSIG1)-dependent manner. AMFR and INSIG1, an E3 ubiquitin ligase complex, then catalyzed the K27-linked polyubiquitination of STING. This modification served as an anchoring platform for recruiting TANK-binding kinase 1 (TBK1) and facilitating its translocation to the perinuclear microsomes. Depletion of AMFR or INSIG1 impaired STING-mediated antiviral gene induction. Consistently, myeloid-cell-specific Insig1(-/-) mice were more susceptible to herpes simplex virus 1 (HSV-1) infection than wild-type mice. This study uncovers an essential role of the ER proteins AMFR and INSIG1 in innate immunity, revealing an important missing link in the STING signaling pathway. Copyright © 2014 Elsevier Inc. All rights reserved.Immunity 12/2014; 41(6):919-933. DOI:10.1016/j.immuni.2014.11.011 · 19.75 Impact Factor
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- "Affinity-purified rabbit polyclonal rat gp78 antibody (aa 624–642; used for all experiments with Rat-1 cells; Pearce et al., 2007), affinitypurified rabbit polyclonal mouse/human gp78 antibody (gp78 Ab2; used for all experiments with mouse or human gp78 except Figure 1D; Tsai et al., 2007), rabbit polyclonal HMGCR antibody (Roitelman et al., 1992), and rabbit polyclonal calnexin antibody (Leichner et al., 2009) have been previously described. Mouse monoclonal HMGCR antibody (A9; Liscum et al., 1983b) was obtained from the American Type Culture Collection (Manassas, VA) and was prepared in mice as ascites fluid or harvested from conditioned medium. "
ABSTRACT: The endoplasmic reticulum (ER) resident enzyme HMG-CoA reductase (3-hydroxy-3-methylglutaryl CoA reductase) catalyzes the rate-limiting step in sterol production and is the therapeutic target of statins. Understanding HMG-CoA reductase regulation has tremendous implications for atherosclerosis. HMG-CoA reductase levels are regulated in response to sterols both transcriptionally, through a complex regulatory loop involving the ER Insig proteins, and posttranslationally by Insig-dependent protein degradation by the ubiquitin-proteasome system. The ubiquitin ligase (E3) gp78 has been implicated in the sterol-regulated degradation of HMG-CoA reductase and Insig-1 through ER-associated degradation (ERAD). More recently, a second ERAD E3, TRC8, has also been reported to play a role in the sterol-accelerated degradation of HMG-CoA reductase. We have interrogated this network in gp78(-/-) mouse embryonic fibroblasts and also assessed two fibroblast cell lines using RNAi. While we consistently observe involvement of gp78 in Insig-1 degradation, no substantive evidence to support roles for either gp78 or TRC8 in the robust sterol-accelerated degradation of HMG-CoA reductase was found. We discuss factors that might lead to such discrepant findings. Our results suggest a need for additional studies before definitive mechanistic conclusions are drawn that might set the stage for development of drugs to manipulate gp78 function in metabolic disorders.Molecular biology of the cell 10/2012; 23(23). DOI:10.1091/mbc.E12-08-0631 · 5.98 Impact Factor
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- "Error bars: standard deviations from the mean of 3 experimental replicates. metastasis (Joshi et al., 2010; Tsai et al., 2007), control of hepatitis C virus production (Saeed et al., 2011) and immunoevasion (Tortorella et al., 2000) to name few examples. "
ABSTRACT: Several regulators of endoplasmic reticulum (ER)-associated degradation (ERAD) have a shorter half-life compared to conventional ER chaperones. At steady state, they are selectively removed from the ER by poorly defined events collectively referred to as ERAD tuning. Here we identify the complex comprising the type-I transmembrane protein SEL1L and the cytosolic protein LC3-I as an ERAD tuning receptor regulating the COPII-independent, vesicle-mediated removal of the lumenal ERAD regulators EDEM1 and OS-9 from the ER. Expression of folding-defective polypeptides enhances the lumenal content of EDEM1 and OS-9 by inhibiting their SEL1L:LC3-I-mediated segregation. This raises ERAD activity in the absence of UPR-induction. The mouse hepatitis virus (MHV) subverts ERAD tuning for replication. Consistently, SEL1L or LC3 silencing impair the MHV life cycle. Collectively, our data provide new molecular information about the ERAD tuning mechanisms that regulate ERAD in mammalian cells at the post translational level and how these mechanisms are hijacked by a pathogen.Molecular cell 05/2012; 46(6):809-19. DOI:10.1016/j.molcel.2012.04.017 · 14.46 Impact Factor