Ivan Robert Nabi

Professor

Relevant Degree Programs

 

Graduate Student Supervision

Doctoral Student Supervision (Jan 2008 - Mar 2019)
Endoplasmic reticulum: nanodomain organization and communication with mitochondria (2018)

No abstract available.

Caveolin-1 and membrane domain regulation of focal adhesions and tumor cell migration (2016)

Caveolin-1 (Cav1), a key protein component of cell surface invagination caveolae and a major substrate of Src kinase, has been shown to be associated with cancer malignancy. Galectin-3 (Gal3), a galactose-specific lectin, forms oligomers and crosslinks N-glycans on cell surface to form the galectin lattice. Gal3 and Cav1 function together to regulate focal adhesion dynamics and tumor cell migration. In this thesis we hypothesize that the galectin lattice, Cav1 membrane domain organization (caveolae, Cav1 scaffolds) and Cav1 molecular motifs (tyrosine 14 phosphorylation (pY14), the caveolin scaffolding domain (CSD)) are all involved in Cav1 promotion of focal adhesion dynamics and tumor cell motility.Firstly, we found a synergistic expression of Cav1 and Gal3 in malignant thyroid cancer cells, which was required for focal adhesion kinase (FAK) stabilization in focal adhesions (a measure of focal adhesion dynamics), RhoA activation and cell migration. Co-overexpression of Cav1 and Gal3, but not either alone, in an anaplastic thyroid cancer cell line stabilized FAK within focal adhesions. Therefore, co-function of Cav1 and Gal3 is required to promote focal adhesion dynamics and cell migration in thyroid cancer.Next we found that overexpression of PTRF/cavin-1 in PC3 prostate cancer cells, and consequent formation of caveolae, decreased cell motility by destabilizing FAK in focal adhesions. The impaired focal adhesion stabilization of FAK in PTRF/cavin-1-expressing PC3 cells was rescued by exogenous Gal3 in a Cav1-dependent manner. Hence the alteration of Cav1 microdomains by PTRF/cavin-1 overexpression decreases cell motility through affecting focal adhesion dynamics, which is overridden by reinforced Cav1-Gal3/galectin lattice co-function.Finally, using Cav1-positive but tyrosine 14-phosphorylated Cav1 (pY14Cav1)-negative DU145 prostate cancer cells, various Cav1 Y14 and CSD mutants and a CSD mimicking/competing peptide, we found a CSD-dependent pY14Cav1 regulation of focal adhesion dynamics and cell motility. Vinculin, a mechano-sensor at focal adhesions that was previously shown to recruit and stabilize other focal adhesion components, preferentially bound pY14Cav1 and was stabilized in focal adhesions by pY14Cav1 in a CSD-dependent manner. Vinculin tension was induced by pY14Cav1 in a CSD-dependent manner. Therefore, a novel interplay between pY14 and the CSD of Cav1 regulates focal adhesion dynamics and tension favouring cell migration.

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Unraveling the role of Gp78/AMFR : an E3 ubiquitin ligase and cell surface receptor, in cancer progression (2015)

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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Functional roles of ubiquitin ligase GP78 in endoplasmic reticulum domains (2013)

No abstract available.

Insight into ER quality control mechanisms : novel characterization of the E3 ubiquitin ligase gp78/autocrine motility factor receptor and the Gs subunit in the ER (2012)

This manuscript looks at ER Quality Control (ERQC) mechanisms and in particular, focuses on two cellular pathways: (1) the ER-associated degradation (ERAD) pathway and (2) the ER stress response. ERQC represents a complex assembly of pathways that are vital in maintaining proper cellular function and homeostasis, by helping the cell adapt to ER stress, prevent chronic imbalance in the ER and avoid many protein conformational diseases. Here, we investigate (1) a regulatory role for palmitoylation of the E3 ubiquitin ligase gp78/AMFR in the ERAD pathway; (2) the implication of G proteins in gp78/AMFR functions; and (3) the involvement of ER-localized Gαs in both substrate polyubiquitylation and ER stress.The dynamic posttranslational modification, palmitoylation, is important for receptor stability and intracellular trafficking. Using metabolic radiolabeling and Acyl-Biotinyl Exchange Chemistry, in chapter 2, we show that the E3 ubiquitin ligase gp78/AMFR is palmitoylated within the catalytic RING finger motif, a domain that is responsible for its ubiquitin ligase activity. We also discuss the modulatory implication of gp78/AMFR palmitoylation, showing that palmitoylation disrupts the RING finger motif, regulates its ER distribution and enhances its turnover. Whether palmitoylation of E3 ubiquitin ligases is gp78/AMFR-specific or a general mechanism to control the activity of RING finger ubiquitin ligases remains to be determined. Next, we look at the Gα subunit, a known component of the G protein-coupled receptor (GPCR) signal transduction pathway. In chapter 3, using immunoprecipitation and immunocytochemistry experiments, we report that the E3 ubiquitin ligase gp78/AMFR interacts with and recruits several G proteins to the ER, namely Gαi1 and Gαs. Thus, we reintroduce the possibility that gp78/AMFR is a novel ER-localized GPCR. The Gαs subunit is further discussed in chapter 4 where we characterize in detail its ER localization and its association with ERAD components, as well as we show a novel intracellular function, demonstrating the ability of Gαs to induce substrate polyubiquitylation and protect against ER stress. Together, these findings mark the beginning in understanding the physiological significance of (1) E3 ubiquitin ligase palmitoylation; (2) G protein binding to gp78/AMFR; (3) Gαs-mediated substrate polyubiquitylation and protection against ER stress, in ERQC mechanisms.

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Master's Student Supervision (2010-2017)
Impact of y14 phosphorylation of caveolin-1 on its binding partners : a proteomic analysis (2016)

Caveolae, a special type of lipid-raft, are cave-like invaginations of plasma-membrane maintained and formed by Caveolins (Cav1, 2 and 3) and Cavin 1 and 2. Caveolae regulate various trafficking and signaling pathways. Cav1, a 178 amino acid protein has a Src-dependent tyrosine-14 phosphorylation site that regulates integrin signaling and focal adhesion dynamics, and a Caveolin Scaffolding Domain (CSD) that physically interacts with multiple proteins. Glutathione-S-transferase (GST) pull-downs and quantitative proteomics analysis (Maxquant) were performed using lysates from the DU145 prostate cancer cell line and GST-beads tagged with the N-terminal polypeptide of Cav1 (amino acids 1-101) incorporating phosphomimeitic (Y14D) and non-phosphorylatable (Y14F) mutations. Proteomic analysis showed 1.5 fold increased interaction of 196 and 78 proteins with Cav1(1-101)Y14D and Cav1(1-101)Y14F, respectively. Gene Ontology (GO) analysis revealed that Cav1(1-101)Y14D interacted more with proteins that regulate cell stress, proliferation, signal transduction, metabolic processes, apoptosis (Heat shock protein-90 (HSP90)) and focal adhesions, whereas Cav1(1-101)Y14F interacted more with proteins that regulate actin cytoskeleton and RNA processing. Pseudopod-enriched proteome list from DU145 cells revealed 84 proteins overlapping with the Cav1(1-101)Y14D interactome. Comparative proteomics analysis of the CSD mutants (F92A/V94A) suggested that one-third binding proteins of Cav1(1-101)Y14D and Cav1(1-101)Y14F were influenced by this mutation. Binding specificity of Y14 phosphorylation on Cav1 is partially affected by these CSD mutations. Pseudopod enriched HSP90 was one of the top hits in the Cav1(1-101)Y14D interactome. Inhibition of HSP90 with 17-N-allylamino-17-demethoxygeldanamycin (17AAG) increased expression of Protein Kinase B (also known as AKT) and Cav1 in pCav1 (Y14 phosphorylatedCav1) expressing DU145 and PC3 prostate cancer cell lines. However, there was no effect of HSP90 inhibition on pCav1 lacking DU145 and Cav1 knocked-down PC3 cells. Reduced cell migration and viability was observed after 17AAG treatment of DU145 (stably expressing various Cav1 mutants) and PC3 in pCav1 dependent manner. This suggests the HSP90 function in regulating cell migration rely on pCav1. This study reveals that Y14 phosphorylation impacts Cav1 interaction with different proteins and is partially affected by CSD mutants in our study. Also, pCav1 specific interaction with HSP90 decreases prostate cancer cell migration.

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Role and regulation of Gp78 E3 ubiquitin ligase and its ligand autocrine motility factor in mitochondrial dynamics and mitochondria-endoplasmic reticulum association (2015)

A ligand-receptor pair, autocrine motility factor (AMF) and Gp78, have been discovered to play multiple roles in mammalian cells. AMF functions as the essential glycolytic enzyme phosphoglucose isomerase in the cytoplasm, but when secreted acts a cytokine that stimulates cell motility, growth and survival. Gp78 serves as the cell surface receptor of AMF, and thus it is also known as AMFR. However, Gp78 can localize to the ER membrane and functions as an E3 ubiquitin ligase in the endoplasmic reticulum associated degradation (ERAD) pathway where it targets a wide variety of proteins for degradation. The concerted actions of AMF and Gp78 contribute to multiple aspects of cancer progression, and thus elevated levels of both proteins have been found in many types of cancers. Recently, it was discovered that AMF and Gp78 alter mitochondrial morphology and ER-mitochondria calcium coupling, processes that are essential in regulating mitochondrial metabolism and apoptosis. Furthermore, Gp78 has also been localized to ER-mitochondria contact sites where it targets the mitochondrial fusion proteins, mitofusin 1 and 2 (Mfn1/2), for degradation. In this dissertation, I show that during Gp78 induced mitophagy, autophagosome marker LC3 is recruited to mitochondria associated ER membrane. Moreover, I show that Gp78-dependent degradation of the mitofusins leads to diminished mitochondrial fusion and a perturbation of mitochondrial dynamics. I also report the ability of AMF to inhibit Gp78-induced mitochondrial fission. In my study of ER-mitochondrial association, I observed two types of ER-mitochondria contacts in HT-1080 fibrosarcoma cells: the rough and the smooth. Gp78 ubiquitin ligase activity selectively promotes rough ER-mitochondria association through the degradation of Mfn2. AMF treatment inhibits Gp78-dependent Mfn2 degradation and decreases rough ER-mitochondria contact sites. By dissecting the functions of AMF and Gp78 at the ER mitochondria contact sites, my thesis not only expands our understanding of the relationship between AMF and Gp78, it also provides novel insights into the intimate connection between the ER and mitochondria.

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