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Tsai, Y. C. et al. The ubiquitin ligase gp78 promotes sarcoma metastasis by targeting KAI1 for degradation. Nature Med. 13, 1504-1509

Laboratory of Protein Dynamics and Signaling, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, Maryland 21702, USA.
Nature medicine (Impact Factor: 27.36). 01/2008; 13(12):1504-9. DOI: 10.1038/nm1686
Source: PubMed

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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    • "We presume that the ERAD phenotypes observed in gp78 knockdown cells are not caused by an unknown off-target effect, because they were observed using siRNA/shRNAs with different targeting sequences. In addition, siRNA-mediated knockdown of each component of the gp78 complex (including gp78, UbxD8, and UBAC2) inhibited ERAD in all instances (Fang et al., 2001; Song et al., 2005; Shen et al., 2006; Tsai et al., 2007; Morito et al., 2008; Christianson et al., 2011; Jo et al., 2011a,b; Liu et al., 2014). By contrast, CRISPR cells lacking these proteins are functionally competent in ERAD, at least FIGURE 7: The functional relationship between gp78 and Hrd1. "
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    ABSTRACT: Eukaryotic cells eliminate misfolded proteins from the endoplasmic reticulum (ER) via a conserved process termed ER-associated degradation (ERAD). Central regulators of the ERAD system are membrane-bound ubiquitin ligases, which are thought to channel misfolded proteins through the ER membrane during retrotranslocation. Hrd1 and gp78 are mammalian ubiquitin ligases homologous to Hrd1p, an ubiquitin ligase essential for ERAD in S. cerevisiae. However, the functional relevance of these proteins to Hrd1p is unclear. Here we characterize the gp78-containing ubiquitin ligase complex and define its functional interplay with Hrd1 using biochemical and recently developed CRISPR-based genetic tools. Our data show that transient inactivation of the gp78 complex by short hairpin RNA-mediated gene silencing causes significant stabilization of both luminal and membrane ERAD substrates, but unlike Hrd1, which plays an essential role in retrotranslocation and ubiquitination of these ERAD substrates, knockdown of gp78 does not affect either of these processes. Instead, gp78 appears to act downstream of Hrd1 to promote ERAD via cooperation with the BAG6 chaperone complex. We conclude that the Hrd1 complex forms an essential retrotranslocation module that is evolutionarily conserved, but the mammalian ERAD system uses additional ubiquitin ligases to assist Hrd1 during retrotranslocation.
    Full-text · Article · Oct 2015 · Molecular biology of the cell
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    • "Gp78 substrates include T cell receptor subunit (CD3-delta, TCR alpha), ApoB lipoprotein, Insig-1, HMG CoA reductase, the Z variant of α1-antitrypsin, mutant cystic fibrosis transmembrane conductance regulator (CFTR∆508), SOD1, Ataxin 3 and the metastasis suppressor KAI1/CD82 linking Gp78 ERAD activity to lipid metabolism disorders, cystic fibrosis, neurodegenerative diseases and cancer (Fang et al., 2001; Kostova et al., 2007; Tsai et al., 2007; Ying et al., 2009). Increased expression of Gp78 and degradation of KAI1 is associated with preneoplastic hyperplasia in a transgenic breast cancer model (Joshi et al., 2010) and sarcoma metastasis (Tsai et al., 2007). More recently, we showed that Gp78 promoted degradation of the mitofusin mitochondrial fusion proteins, mitochondrial fission and, upon mitochondrial depolarization, mitophagy (Fu et al., 2013). "
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    ABSTRACT: Gp78 is an ERAD-associated E3 ubiquitin ligase that induces degradation of the mitofusin mitochondrial fusion proteins and mitochondrial fission. Gp78 is localized throughout the ER, however the anti-Gp78 3F3A monoclonal antibody (mAb) recognizes Gp78 selectively in mitochondria-associated ER domains. Epitope mapping localized the epitope of 3F3A and a commercial anti-Gp78 mAb to an eight amino acid (533-541 in mouse Gp78 isoform 2) motif, that forms part of a highly conserved 41 amino acid region containing 14-3-3 and WW binding domains and a p38 MAP kinase (p38 MAPK) consensus site on serine 538 (S538). 3F3A binds selectively to non-phosphorylated S538 Gp78. Using 3F3A as a reporter, Gp78 S538 phosphorylation was induced by serum starvation and shown to be mediated by p38 MAPK. Mass spectroscopy analysis of Gp78 phosphopeptides confirmed S538 as a major p38 MAPK phosphorylation site on Gp78. Gp78 S538 phosphorylation limited its ability to induce mitochondrial fusion and degrade MFN1 and MFN2 but did not impact in vitro Gp78 ubiquitin E3 ligase activity. Phosphomimetic Gp78 S538D mutation prevented Gp78 promotion of ER-mitochondria interaction and SB203580 inhibition of p38 MAPK increased ER-mitochondria association. p38 MAPK phosphorylation of Gp78 S538 therefore regulates Gp78-dependent ER-mitochondria association and mitochondria motility. © 2015 by The American Society for Cell Biology.
    Preview · Article · Sep 2015 · Molecular biology of the cell
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    • "Interestingly, substantially more ubiquitin conjugated MRP2 was detected in cells cotransfected with GFP-Ub K0 than with GFP-Ub WT , supporting that polyubiquitination of MRP2 leads to its degradation [25]. As ubiquitin conjugation is the rate limiting step in target protein degradation [26] [27], we examined which ubiquitin ligase E3s was responsible for MRP2 ubiquitination in human cholestatic livers, i.e., GP78, TEB4 and HRD1. As shown in Figure 6C and D, only GP78 expression was significantly increased at both mRNA and protein levels (2.1-flod, p<0.05 and 4.2-fold, p<0.01 of control livers, respectively), whereas hepatic expression of TEB4 and HRD1 did not change. "
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    ABSTRACT: Multidrug resistance-associated protein 2 (MRP2) excretes conjugated organic anions including bilirubin and bile acids. Malfunction of MRP2 leads to jaundice in patients. Studies in rodents indicate that Radixin plays a critical role in determining Mrp2 canalicular membrane expression. However, it is not known how human hepatic MRP2 expression is regulated in cholestasis. We assessed liver MRP2 expression in patients with obstructive cholestasis caused by gallstone blockage of bile ducts, and investigated the regulatory mechanism in HepG2 cells. Western blot detected that liver MRP2 protein expression in obstructive cholestatic patients (n=30) was significantly reduced to 25% of the non-cholestatic controls (n=23). Immunoprecipitation identified Ezrin but not Radixin associating with MRP2 in human livers, and the increased amount of phospho-Ezrin Thr567 was positively correlated with the amount of co-precipitated MRP2 in cholestatic livers, whereas Ezrin and Radixin total protein levels were unchanged in cholestasis. Further detailed studies indicate that Ezrin Thr567 phosphorylation plays an important role in MRP2 internalization in HepG2 cells. Since increased expression of PKCα, δ and ε were detected in these cholestatic livers, we further confirmed that these PKCs stimulated Ezrin phosphorylation and reduced MRP2 membrane expression in HepG2 cells. Finally, we identified GP78 as the key ubiquitin ligase E3 involved in MRP2 proteasome degradation. Activation of liver PKCs during cholestasis leads to Ezrin Thr567 phosphorylation resulting in MRP2 internalization and degradation where ubiquitin ligase E3 GP78 is involved. This process provides a mechanistic explanation for jaundice seen in patients with obstructive cholestasis. Copyright © 2015. Published by Elsevier B.V.
    Full-text · Article · Jul 2015 · Journal of Hepatology
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