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Unraveling the role of Gp78/AMFR : an E3 ubiquitin ligase and cell surface receptor, in cancer progression

University of British Columbia

Background/Aim: This thesis focuses on the role of Gp78/AMFR in cancer progression by (1) investigating the relationship between the dual functions of Gp78/AMFR as a cell surface receptor and intracellular ubiquitin ligase in ERAD; (2) unraveling the post-translational modification (PTM) of Gp78/AMFR selectively targeted by the cancer marker 3F3A mAb and (3) the impact of the PTM on the ubiquitin ligase activity, localization and degradation pathway of Gp78/AMFR. Results: Using microRNA mediated gene silencing technology we showed that a significant reduction of total and cell surface expression of Gp78/AMFR in Gp78/AMFR knockdown HEK293 cells; associated with decreased degradation of the established substrates KAI1, a tumor metastasis suppressor and preventing extracellular AMF/PGI dampening of thapsigargin and ATP-evoked ER calcium release and tunicamycin and thapsigargin induced ER-stress and pre-apoptosis. Next, we demonstrated that 3F3A selectively recognizes dephosphorylated S538 of Gp78/AMFR. Furthermore, we showed that serum starvation induced S538 phosphorylation of Gp78/AMFR via a p38 MAPK signaling pathway. Intriguingly, Gp78/AMFR phosphomimetic mutant S538D prevented Gp78/AMFR-dependent degradation of mitofusin 1 and 2, large GTPases essential for mitochondrial fusion. Serum starvation reduced mitofusin degradation by wild-type Gp78/AMFR, but not dominant negative Gp78/AMFR S538A, an effect that was reversed by p38 MAPK inhibition. We also found that S538A Gp78/AMFR mutation promoted peripheral ER distribution, which is consistent with previous reports that Gp78/AMFR mediated ubiquitylation is initiated from the peripheral ER labelled by 3F3A mAb. In addition, we showed that S538D did not alter the turnover of Gp78/AMFR but did signal its proteasomal degradation. In contrast, Gp78/AMFR S538A sensitized Gp78/AMFR for lysosomal degradation, indicating that S538 phosphorylation is a critical determinant of the degradation pathway of this E3 ubiquitin ligase. Conclusion: By dissecting the epitope of the malignancy associated 3F3A mAb based on immunohistochemical analysis, this thesis represents the beginning of an understanding of the relationship between the surface cytokine receptor of AMF/PGI and ER-localized E3 ubiquitin ligase Gp78/AMFR. Moreover, it reveals the important role of serine phosphorylation in the regulation of the distribution, ubiquitin ligase activity and degradation pathway of Gp78/AMFR, thereby furthering our understanding of the mechanism of Gp78/AMFR promotion of cancer progression and metastasis.

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2015-09-30

Attribution-NonCommercial-NoDerivs 2.5 Canada

http://hdl.handle.net/2429/52321

Cell and Developmental Biology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/2.5/ca/