Brefeldin A-inhibited Guanine Nucleotide-exchange Protein Research Articles

The Novel KLF4/BIG1 Regulates LPS-mediated Neuro-inflammation and Migration in BV2 Cells via PI3K/Akt/NF-kB Signaling Pathway

Excessive microglia activation occurred in many neurodegenerative diseases. Brefeldin A-inhibited guanine nucleotide-exchange protein 1 (BIG1, ARFGEF1) is involved in cell migration and neurite growth. In the present study, we aimed to explore the effects and potential mechanisms of BIG1 in LPS-mediated neuro-inflammation and migration in BV2 cells. Loss-of-function and gain-of-function experiments were performed. Inflammatory cytokines (TNF-α, IL-1β, IL-6, IL-10) mRNA levels and concentrations were analyzed by Quantitative Real-time PCR (RT-qPCR) and ELISA kits. The NO concentration was tested by ELISA kit. iNOS and COX-2 mRNA and protein levels were measured by RT-qPCR and western blot. Cell migration was determined by transwell assay. The results demonstrated that BIG1 silencing reduced TNF-α, IL-1β, and IL-6 expression, while increased IL-10 expression. The NO production, iNOS and COX-2 expression were clearly inhibited by BIG1 knockdown in the presence of LPS. Furthermore, ablation of BIG1 attenuated the migration capacity of BV2 cells. Overexpression of BIG1 displayed the opposite trends. Moreover, we found BIG1 suppression inhibited PI3K/Akt/NF-κB pathway activation. 740Y-P, an agonist of PI3K, abolished the roles of BIG1 silencing in neuro-inflammation and migration. Additionally, ChIP-qPCR and Dual-luciferase reporter assay determined that KLF4 binds to the promoter of BIG1, western blot analysis demonstrated that KLF4 could regulate BIG1 positively. In addition, we observed that BIG1 overexpression partly rescued the biological activities of KLF4 silencing in neuro-inflammation and migration in LPS-stimulated BV2 cells. Taken together, BIG1 was mediated by KLF4 regulated LPS-mediated neuro-inflammation and migration in BV2 cells via PI3K/Akt/NF-kB signaling pathway.

The miR-133b/brefeldin A-inhibited guanine nucleotide-exchange protein 1 (ARFGEF1) axis represses proliferation, invasion, and migration in cervical cancer cells

ABSTRACT Cervical cancer is a common gynecological malignancy, and miR-133b is an abnormally expressed cervical cancer gene, which suggests that miR-133b may be involved in the occurrence and development of cervical cancer. However, the underlying mechanism is still unclear. miR-133b was overexpressed or silenced in the cervical cancer cell line C33A. Brefeldin A-inhibited guanine nucleotide-exchange protein 1 (ARFGEF1) was combined with overexpression of miR-133b in C33A cells. Cell Counting Kit-8, clone formation, and Transwell assays were performed to determine the influence of miR-133b and ARFGEF1 on clone formation, proliferation, migration, and invasion of C33A cells. The interaction between miR-133b and ARFGEF1 was verified using a luciferase reporter assay. Finally, the mRNA and protein expression of miR-133b and ARFGEF1 in the tumor and adjacent normal tissues of cervical cancer patients was detected by real-time quantitative PCR, Western blotting, and immunohistochemistry. The results indicated that miR-133b up-regulation suppressed the proliferation, invasion, migration, and clone formation abilities of C33A cells (P < 0.05). However, silence of miR-133b promoted the proliferation, invasion, and migration of C33A cells (P < 0.05). Clone formation ability of C33A cells was also elevated by miR-133b deficiency (P < 0.05). Moreover, miR-133b interacted with ARFGEF1 and repressed ARFGEF1 expression in C33A cells (P < 0.05). ARFGEF1 overexpression weakened miR-133b overexpression-mediated inhibition of proliferation, invasion, and migration of C33A cells (P < 0.05). miR-133b expression was decreased, and ARFGEF1 was up-regulated in tumor tissues of cervical cancer patients (P < 0.05). All results revealed that miR-133b suppresses cervical cancer progression by inhibiting proliferation, invasion, and migration of cervical cancer cells via targeting ARFGEF1. Thus, our study determined the mechanism of miR-133b in cervical cancer, and confirmed miR-133b/ARFGEF1 may become a potential therapeutic target for cervical cancer.

Biophysical characterization of the breast cancer-related BIG3-PHB2 interaction: Effect of non-conserved loop region of BIG3 on the structure and the interaction

Brefeldin A-inhibited guanine nucleotide-exchange protein 3 (BIG3) interacts with and inhibits the tumor suppressor function of prohibitin-2 (PHB2), and recent in vivo studies have demonstrated that the BIG3-PHB2 interaction is a promising target for breast cancer therapy. However, little biophysical characterization on BIG3 and its interaction with PHB2 has been reported. Here we compared the calculated 8-class secondary structure of the N-terminal domains of BIG family proteins and identified a loop region unique to BIG3. Our biophysical characterization demonstrated that this loop region significantly affects the colloidal and thermodynamic stability of BIG3 and the thermodynamic and kinetic profile of its interaction with PHB2. These results establish a model for the BIG3-PHB2 interaction and an entry for drug discovery for breast cancer.

Abstract 3813: PHB2 inactivation by AKAP-BIG3 is required for progression of HER2-overexpressing breast cancer

Abstract Overexpression of the human epidermal growth factor receptor 2 (HER2) in breast cancer is linked to aggressive tumor behavior and a poorer prognosis. Anti-HER2 monoclonal antibody, trastuzumab extends the overall survival of patients with HER2-overexpressing breast cancer, but resistance to trastuzumab and/or other HER2-targeted therapies remains a serious clinical problem. Our previous studies demonstrated that Brefeldin A-Inhibited Guanine nucleotide-exchange protein 3 (BIG3), which is exclusively overexpressed in the majority of breast cancers, functions as a cancer specific A-kinase anchoring protein that binds protein kinase A (PKA) and protein phosphatase 1 (PP1Cα), thereby inactivating prohibitin2 (PHB2) suppressive activity through its dephosphorylation on Ser39. We further developed dominant-negative stapling peptide (stERAP), which is designed to specifically disrupt binding of BIG3-PHB2 complex and improve duration of their anti-tumor effects. Here we newly report that stERAP has significant suppressive effects on the HER2 signaling network by activating the tumor suppressive ability of PHB2. stERAP resulted in intrinsic PHB2 release from BIG3, followed by rapid phosphorylation on Ser39, Thr42 and Thr169 via PKCα, TTK and MK5, respectively. Importantly, PHB2 released from BIG3 by stERAP treatment translocated into nucleus, followed by acting as a transcriptional co-repressor by recruiting HDAC1 and NcoR through its Ser39-phosphorylation. More importantly, we observed that serine/threonine phosphorylations of PHB2 are necessary for disruption of HER2-HER3 hetero-dimerization and NF-κB signalling activation which are associated with acquired resistance to trastuzumab. We demonstrated that weekly treatment of stERAP completely suppressed the proliferation of trastuzumab-resistant HER2-positive breast cancer cells in vitro and in vivo. We are currently examining the association of both BIG3 overexpression and PHB2 dephosphorylation with poor prognosis of patients with HER2-positive breast cancers by immunohistochemical staining, and will report these results at this meeting. We demonstrated for the first time that PHB2 inactivation by the AKAP-BIG3 (BIG3/PKA/PP1Cα tri-complex) is required for oncogenic HER2 signalling activation in breast cancer cells, and stERAP may be a promising anti-cancer drug for trastuzumab-resistant breast cancer. Citation Format: Tetsuro Yoshimaru, Yosuke Matsushita, Mitsunori Sasa, Yasuo Miyoshi, Toyomasa Katagiri. PHB2 inactivation by AKAP-BIG3 is required for progression of HER2-overexpressing breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3813.

Abstract 1837: Overcoming trastuzumab resistance in HER2-overexpressing breast cancer by utilizing PHB2, a tumor suppressor of multiple resistance pathways

Abstract Overexpression of the human epidermal growth factor receptor 2 (HER2) is associated with aggressive tumor behavior and poor prognosis in breast cancer. Although anti-HER2 monoclonal antibody, trastuzumab shows considerable efficacy and extends the overall survival of patients with HER2 overexpressing breast cancer, about half of individuals with HER2-overexpressing breast cancer do not respond to trastuzumab-based therapies due to multiple resistance mechanisms. Therefore, acquired and de novo resistance to trastuzumab remains an important issue in the clinical treatment of HER2-overexpressing breast cancer. Our previous studies demonstrated that Brefeldin A-Inhibited Guanine nucleotide-exchange protein 3 (BIG3), which is exclusively overexpressed in the majority of breast cancers, functions as an A-kinase anchoring protein that binds protein kinase A (PKA) and protein phosphatase 1 (PP1Cα), thereby inactivating prohibitin2 (PHB2) suppressive activity through its dephosphorylation on Ser39. We further developed the stapled dominant-negative peptide (stERAP; stapled ERAP), which is designed to specifically inhibit the BIG3-PHB2 interaction and improve duration of their anti-tumor effects. Here we newly report that stERAP has significant suppressive effects on the HER2 signaling network by activating the tumor suppressive ability of PHB2. Intrinsic PHB2 released from BIG3 by stERAP was rapidly serine- and threonine-phosphorylated via PKCα and/or TTK, which are essential for its tumor suppressive activity, and effectively disrupted the HER2-HER3 interaction associated with acquired resistance to trastuzumab, resulting in significant reduction of proliferation of HER2-overexpressing breast cancer cells (IC50 = 54 nM in SK-BR-3 cells). We also confirm that stERAP inhibited HER2 phosphorylation on Thr488, HER2-HER3 interaction, and NF-κB pathway, which are associated with trastuzumab-resistance, respectively, resulting in significant suppression of growth of trastuzumab-resistant breast cancer cells. More importantly, stERAP treatment led to the enhancement of trastuzumab-sensitivity, resulting in a synergistic growth suppression of HER2-overexpressing and trastuzumab-resistant breast cancer cells, respectively. Our findings strongly suggest that stERAP is a novel promising anti-cancer drug to suppress the growth of trastuzumab-resistant breast cancer in clinical use. Citation Format: Tetsuro Yoshimaru, Yosuke Matsushita, Mitsunori Sasa, Yasuo Miyoshi, Toyomasa Katagiri. Overcoming trastuzumab resistance in HER2-overexpressing breast cancer by utilizing PHB2, a tumor suppressor of multiple resistance pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1837.

PKA-binding domain of AKAP8 is essential for direct interaction with DPY30 protein.

The main function of the A kinase-anchoring proteins (AKAPs) is to target the cyclic AMP-dependent protein kinase A (PKA) to its cellular substrates through the interaction with its regulatory subunits. Besides anchoring of PKA, AKAP8 participates in regulating the histone H3 lysine 4 (H3K4) histone methyltransferase (HMT) complexes. It is also involved in DNA replication, apoptosis, transcriptional silencing of rRNA genes, alternative splicing, and chromatin condensation during mitosis. In this study, we focused on the interaction between AKAP8 and the core subunit of all known H3K4 HMT complexes-DPY30 protein. Here, we demonstrate that the PKA-binding domain of AKAP8 and the C-terminal domain of DPY30, also called Dpy-30 motif, are crucial for the interaction between these proteins. We show that a single amino acid substitution in DPY30 L69D affects its dimerization andcompletely abolishes its interaction with AKAP8 and another DPY30-binding partner brefeldin A-inhibited guanine nucleotide-exchange protein 1 (BIG1), which is also AKAP domain-containing protein. We further demonstrate that AKAP8 interacts with DPY30 and the RII alpha regulatory subunit of PKA both in the interphase and in mitotic cells, and we show evidences that AKAP8L, a homologue of AKAP8, interacts with core subunits of the H3K4 HMT complexes, which suggests its role as a potential regulator of these complexes. The results presented here reinforce the analogy between AKAP8-RII alpha and AKAP8-DPY30 interactions, postulated before, and improve our understanding of the complexity of the cellular functions of the AKAP8 protein.

Abstract 3613: Development of chemically modified peptide inhibitor ERAP targeting BIG3-PHB2 complex on hormone-resistant breast tumor

Abstract Approximately 70% of breast cancer cells express estrogen receptor alpha (ERα), and depend on estrogen (E2) for proliferative and metastatic activity. The current endocrine therapies for breast cancer are mainly based on targeting ERα signaling using selective ERα modulators, ERα downregulators, and aromatase inhibitor. However, up to 50% of patients with ERα-positive tumors either initially do not respond or become resistant to these drugs. The precise molecular mechanisms for the endocrine resistance contributes to be an active area of research. Therefore, identifying the factors and pathways responsible for resistance and defining ways to overcome it lead to develop novel molecular-target therapies to endocrine resistance. Recent findings support that the Brefeldin A-inhibited guanine nucleotide-exchange protein 3-prohibitin 2 (BIG3-PHB2) complex plays a crucial role in E2/ERα signaling modulation in these cells. Moreover, specific inhibition of the BIG3-PHB2 interaction using ERα activity-regulator synthetic peptide (ERAP: 165-177 amino acids) derive from a helical BIG3 sequence, a dominant-negative peptide inhibitor leads to the significant anti-tumor effect. However, duration of its effect is very short for clinical use. Here, we report the development of the chemically modified ERAP using stapling methods (stapled ERAP) to improve duration of its antitumor effects. The stapled ERAP specifically inhibited the BIG3-PHB2 interaction, thereby exhibiting the long-lasting suppressive activity and their intracellular localization without membrane-permeable polyarginine sequence supposedly through the formation of stable α-helix structure induced by the stapling. Importantly, a combination of stapled ERAP and tamoxifen caused a synergistic inhibitory effect in breast cancer cell growth. Tumor bearing mice treated with every 7 days with stapled ERAP treatment effectively prevented the BIG3-PHB2 interaction as well as daily treatment, leading to the complete regression of E2-dependent tumor in vivo. Our findings suggest that the stapled ERAP may be a promising anti-tumor drug to suppress the growth of luminal-type breast cancer in clinical use. Citation Format: Toyomasa Katagiri, Tetsuro Yoshimaru. Development of chemically modified peptide inhibitor ERAP targeting BIG3-PHB2 complex on hormone-resistant breast tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3613. doi:10.1158/1538-7445.AM2017-3613

A-kinase anchoring protein BIG3 coordinates oestrogen signalling in breast cancer cells

Approximately 70% of breast cancer cells express oestrogen receptor alpha (ERα). Previous studies have shown that the Brefeldin A-inhibited guanine nucleotide-exchange protein 3–prohibitin 2 (BIG3-PHB2) complex has a crucial role in these cells. However, it remains unclear how BIG3 regulates the suppressive activity of PHB2. Here we demonstrate that BIG3 functions as an A-kinase anchoring protein that binds protein kinase A (PKA) and the α isoform of the catalytic subunit of protein phosphatase 1 (PP1Cα), thereby dephosphorylating and inactivating PHB2. E2-induced PKA-mediated phosphorylation of BIG3-S305 and -S1208 serves to enhance PP1Cα activity, resulting in E2/ERα signalling activation via PHB2 inactivation due to PHB2-S39 dephosphorylation. Furthermore, an analysis of independent cohorts of ERα-positive breast cancers patients reveal that both BIG3 overexpression and PHB2-S39 dephosphorylation are strongly associated with poor prognosis. This is the first demonstration of the mechanism of E2/ERα signalling activation via the BIG3–PKA–PP1Cα tri-complex in breast cancer cells.

Stapled BIG3 helical peptide ERAP potentiates anti-tumour activity for breast cancer therapeutics

Estradiol (E2) and the oestrogen receptor-alpha (ERα) signalling pathway play pivotal roles in the proliferative activity of breast cancer cells. Recent findings show that the brefeldin A-inhibited guanine nucleotide-exchange protein 3-prohibitin 2 (BIG3-PHB2) complex plays a crucial role in E2/ERα signalling modulation in breast cancer cells. Moreover, specific inhibition of the BIG3-PHB2 interaction using the ERα activity-regulator synthetic peptide (ERAP: 165–177 amino acids), derived from α-helical BIG3 sequence, resulted in a significant anti-tumour effect. However, the duration of this effect was very short for viable clinical application. We developed the chemically modified ERAP using stapling methods (stapledERAP) to improve the duration of its antitumour effects. The stapledERAP specifically inhibited the BIG3-PHB2 interaction and exhibited long-lasting suppressive activity. Its intracellular localization without the membrane-permeable polyarginine sequence was possible via the formation of a stable α-helix structure by stapling. Tumour bearing-mice treated daily or weekly with stapledERAP effectively prevented the BIG3-PHB2 interaction, leading to complete regression of E2-dependent tumours in vivo. Most importantly, combination of stapledERAP with tamoxifen, fulvestrant, and everolimus caused synergistic inhibitory effects on growth of breast cancer cells. Our findings suggested that the stapled ERAP may be a promising anti-tumour drug to suppress luminal-type breast cancer growth.

Selection and evaluation of new reference genes for RT-qPCR analysis in Epinephelus akaara based on transcriptome data.

Groupers are an economically important fish species in world fishery markets. Because many studies using RT-qPCR have addressed gene expression in groupers, appropriate reference genes are required to obtain reliable and accurate results. In this study, the most suitable reference genes were identified from eleven candidate genes of one of the most valuable species, Epinephelus akaara, in a range of different experimental conditions. Using the software packages geNorm, NormFinder, BestKeeper and refFinder, three traditionally used reference genes, β-actin (β-ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta-2-microglobulin (B2M), were identified as not suitable for E. akaara gene expression studies, whereas two newly identified reference genes, conserved oligomeric Golgi complex subunit 5 (Cog5) and brefeldin a-inhibited guanine nucleotide-exchange protein 1 (ARFGEF1), could be universally applied under all the tested conditions. These data provide the foundation for more precise results in RT-qPCR studies of gene expression in E. akaara and other Epinephelus species.

Abstract 1710: Xanthohumol suppresses estrogen-signaling in endocrine resistant breast cancer through the specific inhibition of BIG3-PHB2 interactions

Abstract Estrogen-receptor-α (ERα) plays a pivotal role in the development and progression of breast cancer. The current endocrine therapies for breast cancer are mainly based on targeting ERα signaling using selective ERα modulators, ERα downregulators, and aromatase inhibitor (AI)1-3. However, tamoxifen treatment often fails, and patients succumb to recurrent, endocrine-resistant tumors. Moreover, some patients who have undergone AI treatment still relapse. The precise molecular events that determine alterations in the effectiveness of these endocrine therapies remain unknown. We previously reported that the oncoprotein brefeldin A-inhibited guanine nucleotide-exchange protein 3 (BIG3) and tumor suppressor prohibitin 2 (PHB2) complex play a pivotal role in E2 signaling modulation in ERα-positive breast cancer. Namely, BIG3 binds PHB2, thereby inhibiting the E2-dependent suppressive ability of PHB2 and resulting in constitutive ERα activation. Considering these findings, strategies utilising the tumour suppressive activity of PHB2 upon its release from BIG3 by inhibitors of protein-protein interaction may represent novel therapies for breast cancer. Accordingly, we focus on whether XN potentially binds to the tumor suppressor protein PHB2, forming a novel natural anti-tumor compound targeting the BIG3-PHB2 complex and acting as a pivotal modulator of E2 /ERα signaling in breast cancer cells. XN treatment effectively prevented the BIG3-PHB2 interaction, thereby releasing PHB2 to directly bind to both nuclear- and cytoplasmic ERα. This event led to the complete suppression of the E2-signaling pathways and ERα-positive breast cancer cell growth both in vitro and in vivo, but did not suppress the growth of normal mammary epithelial cells. Our findings suggest that XN may be a promising natural compound to suppress the growth of luminal-type breast cancers, especially endocrine resistant breast cancers. Citation Format: Toyomasa Katagiri. Xanthohumol suppresses estrogen-signaling in endocrine resistant breast cancer through the specific inhibition of BIG3-PHB2 interactions. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1710. doi:10.1158/1538-7445.AM2015-1710

BIG3 Inhibits the Estrogen-Dependent Nuclear Translocation of PHB2 via Multiple Karyopherin-Alpha Proteins in Breast Cancer Cells.

We recently reported that brefeldin A-inhibited guanine nucleotide-exchange protein 3 (BIG3) binds Prohibitin 2 (PHB2) in cytoplasm, thereby causing a loss of function of the PHB2 tumor suppressor in the nuclei of breast cancer cells. However, little is known regarding the mechanism by which BIG3 inhibits the nuclear translocation of PHB2 into breast cancer cells. Here, we report that BIG3 blocks the estrogen (E2)-dependent nuclear import of PHB2 via the karyopherin alpha (KPNA) family in breast cancer cells. We found that overexpressed PHB2 interacted with KPNA1, KPNA5, and KPNA6, thereby leading to the E2-dependent translocation of PHB2 into the nuclei of breast cancer cells. More importantly, knockdown of each endogenous KPNA by siRNA caused a significant inhibition of E2-dependent translocation of PHB2 in BIG3-depleted breast cancer cells, thereby enhancing activation of estrogen receptor alpha (ERα). These data indicated that BIG3 may block the KPNAs (KPNA1, KPNA5, and KPNA6) binding region(s) of PHB2, thereby leading to inhibition of KPNAs-mediated PHB2 nuclear translocation in the presence of E2 in breast cancer cells. Understanding this regulation of PHB2 nuclear import may provide therapeutic strategies for controlling E2/ERα signals in breast cancer cells.

Abstract 623: BIG3-PHB2 interaction is a key therapeutic target in luminal-type of breast cancer

Abstract More than 70% of primary breast tumors are estrogen receptor alpha (ERα)-positive, and the interactions between estrogen (E2) and ERα dramatically enhance the proliferative and metastatic activity of breast tumor cells. The selective estrogen receptor modulator tamoxifen directly inhibits E2 and ERα interactions and is a standard treatment offered to patients with ERα-positive breast cancer. The acquisition of endocrine resistance is a common obstacle in endocrine therapy for ERα-positive breast tumors. Therefore, identifying the factors and pathways responsible for resistance and defining ways to overcome it represent important therapeutic challenges in breast cancer research. Prohibitin 2 (PHB2) is known to function as a co-repressor of ERα because it regulates ERα transcriptional activity via its direct binding to ERα. However, it remains unclear how endogenous PHB2 is inactivated in ERα-positive breast cancer cells, despite its abundant expression and the lack of genomic mutations or methylations in breast cancer clinical specimens or cell lines. Here we report that brefeldin A-inhibited guanine nucleotide-exchange protein 3 (BIG3) protein interacts PP1α, and dephosphorylates the E2-dependent phosphorylation of PHB2, which is indispensable for its tumor suppressive ability, thereby causing an apparent “loss-of-function” of PHB2 protein, resulting in the constitutive activation of ERα signaling pathways. These findings demonstrate for the first time the novel mechanism for the inactivation of tumor suppressor protein. From a therapeutic perspective, intrinsic PHB2 released from BIG3 by the cell-permeable peptide inhibitor directly binds to both nuclear- and membrane-associated ERα, which leads to the inhibition of multiple ERα-signaling pathways, and the growth of ERα-positive breast cancer cells both in vitro and in vivo. More importantly, this peptide inhibitor treatment suppressed tamoxifen resistance and enhanced tamoxifen responsiveness in ERα-positive breast cancer cells. These findings mark the first step toward uncovering a new E2/ ERα signaling network in breast cancer and shed light on novel therapeutic strategies utilizing PHB2 protein functions for E2/ ERα-positive breast cancer. Citation Format: Toyomasa Katagiri, Tesuro Yoshimaru, Masato Komatsu, Taisuke Matsuo. BIG3-PHB2 interaction is a key therapeutic target in luminal-type of breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 623. doi:10.1158/1538-7445.AM2014-623

Brefeldin A-inhibited guanine nucleotide-exchange protein 3 (BIG3) is predicted to interact with its partner through an ARM-type α-helical structure.

BackgroundBrefeldin A-inhibited guanine nucleotide-exchange protein 3 (BIG3) has been identified recently as a novel regulator of estrogen signalling in breast cancer cells. Despite being a potential target for new breast cancer treatment, its amino acid sequence suggests no association with any well-characterized protein family and provides little clues as to its molecular function. In this paper, we predicted the structure, function and interactions of BIG3 using a range of bioinformatic tools.ResultsHomology search results showed that BIG3 had distinct features from its paralogues, BIG1 and BIG2, with a unique region between the two shared domains, Sec7 and DUF1981. Although BIG3 contains Sec7 domain, the lack of the conserved motif and the critical glutamate residue suggested no potential guaninyl-exchange factor (GEF) activity. Fold recognition tools predicted BIG3 to adopt an α-helical repeat structure similar to that of the armadillo (ARM) family. Using state-of-the-art methods, we predicted interaction sites between BIG3 and its partner PHB2.ConclusionsThe combined results of the structure and interaction prediction led to a novel hypothesis that one of the predicted helices of BIG3 might play an important role in binding to PHB2 and thereby preventing its translocation to the nucleus. This hypothesis has been subsequently verified experimentally.

BIG1, a brefeldin A-inhibited guanine nucleotide-exchange protein regulates neurite development via PI3K–AKT and ERK signaling pathways

The elongation of neuron is highly dependent on membrane trafficking. Brefeldin A (BFA)-inhibited guanine nucleotide-exchange protein 1 (BIG1) functions in the membrane trafficking between the Golgi apparatus and the plasma membrane. BFA, an uncompetitive inhibitor of BIG1 can inhibit neurite outgrowth and polarity development. In this study, we aimed to define the possible role of BIG1 in neurite development and to further investigate the potential mechanism. By immunostaining, we found that BIG1 was extensively colocalized with synaptophysin, a marker for synaptic vesicles in soma and partly in neurites. The amount of both protein and mRNA of BIG1 were up-regulated during rat brain development. BIG1 depletion significantly decreased the neurite length and inhibited the phosphorylation of phosphatidylinositide 3-kinase (PI3K) and protein kinase B (AKT). Inhibition of BIG1 guanine nucleotide-exchange factor (GEF) activity by BFA or overexpression of the dominant-negative BIG1 reduced PI3K and AKT phosphorylation, indicating regulatory effects of BIG1 on PI3K–AKT signaling pathway is dependent on its GEF activity. BIG1 siRNA or BFA treatment also significantly reduced extracellular signal-regulated kinase (ERK) phosphorylation. Overexpression of wild-type BIG1 significantly increased ERK phosphorylation, but the dominant-negative BIG1 had no effect on ERK phosphorylation, indicating the involvement of BIG1 in ERK signaling regulation may not be dependent on its GEF activity. Our result identified a novel function of BIG1 in neurite development. The newly recognized function integrates the function of BIG1 in membrane trafficking with the activation of PI3K–AKT and ERK signaling pathways which are critical in neurite development.

Abstract 4377: The Golgi protein GLG1 coordinates ARF3 activation in tumor cells.

Abstract Increasing evidence suggests that endocytosis and membrane trafficking are deregulated in cancer cells. Because of their dependence on selected signaling pathways, tumor cells may need to rely on accelerated receptor recycling and increased secretion of a variety of molecules such as matrix components, adhesion molecules and growth factors. Mass spectrometry analysis of proteins associated with separase, recently shown to be an important regulator of membrane trafficking, identified the Golgi apparatus protein 1 (GLG1). GLG1 is primarily known as an E-selectin ligand that mediates the initial step of tethering and rolling of leukocytes on vascular endothelium. We therefore addressed the possibility that GLG1 could play a similar role in circulating tumor cells, facilitating arrest and extravasation into adjacent tissues. Consistent with this possibility, tail vein injection of stably GLG1-depleted tumor cells into NOD/SCID mice resulted in reduced numbers of metastatic nodules. Moreover, metastatic tumor growth was almost exclusively intravascular, indicating impaired extravasation of tumor cells. Further investigation, however, showed that, in tumor cells, GLG1 is almost exclusively localized to the Golgi apparatus, challenging the notion that it plays a direct role in mediating tumor cell adhesion to endothelium as in leukocytes. To elucidate the molecular mechanisms by which GLG1 could affect tumor cell migration through interactions at the Golgi level, we conducted various experiments using GLG1-depleted human tumor cell lines. Transient silencing of GLG1 by siRNA was observed to modify cell shape (elongated morphology), to reduce migration of a range of tumor cell lines and to markedly alter Golgi architecture. Interestingly, subsequent experiments led to the identification of a new interactor of GLG1, the Brefeldin A-inhibited guanine nucleotide-exchange protein 1 (BIG1), an ARF-GEF required to initiate membrane vesicle formation at the trans-Golgi compartment by promoting guanine-nucleotide exchange on ARF1 and ARF3. GLG1 was shown to participate in BIG1 and ARF3 recruitment to the Golgi membrane and subsequent ARF3 activation. Surprisingly, the observed morphological disruption of the Golgi apparatus and reduced ARF3 activation did not affect the secretory pathway in general, but rather seemed to impair secretion of selected molecules including MMP-9. Taken together, our observations provide insight into a novel and specific role of GLG1 in tumor progression by affecting, thanks to its privileged position in the secretory pathway, trafficking of key molecules implicated in cell migration. Citation Format: Anne Planche. The Golgi protein GLG1 coordinates ARF3 activation in tumor cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4377. doi:10.1158/1538-7445.AM2013-4377

BIG1, a Brefeldin A–Inhibited Guanine Nucleotide-Exchange Protein Modulates ATP-Binding Cassette Transporter A-1 Trafficking and Function

Cell-surface localization and intracellular trafficking are essential for the function of ATP-binding cassette transporter A-1 (ABCA1). However, regulation of these activities is still largely unknown. Brefeldin A, an uncompetitive inhibitor of brefeldin A-inhibited guanine nucleotide-exchange proteins (BIGs), disturbs the intracellular distribution of ABCA1, and thus inhibits cholesterol efflux. This study aimed to define the possible roles of BIGs in regulating ABCA1 trafficking and cholesterol efflux, and further to explore the potential mechanism. By vesicle immunoprecipitation, we found that BIG1 was associated with ABCA1 in vesicles preparation from rat liver. BIG1 depletion reduced surface ABCA1 on HepG2 cells, and inhibited by 60% cholesterol release. In contrast, BIG1 overexpression increased surface ABCA1 and cholesterol secretion. With partial restoration of BIG1 through overexpression in BIG1-depleted cells, surface ABCA1 was also restored. Biotinylation and glutathione cleavage revealed that BIG1 small interfering RNA dramatically decreased the internalization and recycling of ABCA1. This novel function of BIG1 was dependent on the guanine nucleotide-exchange activity and achieved through activation of ADP-ribosylation factor 1. BIG1, through its ability to activate ADP-ribosylation factor 1, regulates cell-surface levels and function of ABCA1, indicating a transcription-independent mechanism for controlling ABCA1 action.

Brefeldin A-inhibited ADP-ribosylation factor activator BIG2 regulates cell migration via integrin β1 cycling and actin remodeling

Brefeldin A-inhibited guanine nucleotide-exchange protein (BIG)2 activates ADP-ribosylation factors, ∼20-kDa GTPase proteins critical for continuity of intracellular vesicular trafficking by accelerating the replacement of ADP-ribosylation factor-bound GDP with GTP. Mechanisms of additional BIG2 function(s) are less clear. Here, the participation of BIG2 in integrin β1 cycling through actin dynamics during cell migration was identified using small interfering RNA (siRNA) and difference gel electrophoresis analyses. After a 72-h incubation with BIG2 siRNA, levels of cytosolic Arp2, Arp3, cofilin-1, phosphocofilin, vinculin, and Grb2, known to be involved in the effects of integrin β1-extracellular matrix interactions on actin function and cell translocation, were increased. Treatment of HeLa cells with BIG2 siRNA resulted in perinuclear accumulation of integrin β1 and its delayed return to the cell surface. Motility of BIG2-depleted cells was simultaneously decreased, as were actin-based membrane protrusions and accumulations of Arp2, Arp3, cofilin, and phosphocofilin at the leading edges of migrating cells, in wound-healing assays. Taken together, these data reveal a mechanism(s) through which BIG2 may coordinate actin cytoskeleton mechanics and membrane traffic in cell migration via integrin β1 action and actin functions.

BIG2, an ARF guanine nucleotide‐exchange protein, regulates cell migration via effects integrin β1 cycling and actin cytoskeleton remodeling

Brefeldin A‐inhibited guanine nucleotide‐exchange protein (BIG)2 activates ADP‐ribosylation factors (ARFs), a family of ~20‐kDa GTPase proteins involved in regulation of vesicular trafficking, by accelerating the replacement of ARF‐bound GDP with GTP. Mechanisms of additional BIG2 function(s) are less clear. We identified an involvement of BIG2 in integrin β1 action during cell migration using small interfering RNA (siRNA) and Difference Gel Electrophoresis (DIGE) analysis. Amounts of Arp2, Arp3, and cofilin, proteins known to act downstream of integrin β1 in cell movement, were increased in the cytosol fraction after BIG2 depletion. Treatment of HeLa cells with BIG2 siRNA, but not control siRNAs, resulted in perinuclear accumulation of integrin β1 and delayed return of internalized integrin β1 to the cell surface. At the same time, motility of BIG2‐depleted cells was decreased, and formation of the actin‐based membrane protrusions at leading edges of migrating cells was blocked, as was recruitment of Arp2, Arp3, and cofilin to the leading edge. Taken together, these data reveal a novel mechanism through which BIG2 may serve to coordinate cytoskeleton and membrane traffic, in cell migration, via integrin β1 action.

Effects of brefeldin A-inhibited guanine nucleotide-exchange (BIG) 1 and KANK1 proteins on cell polarity and directed migration during wound healing

Brefeldin A-inhibited guanine nucleotide-exchange protein (BIG) 1 activates class I ADP ribosylation factors (ARFs) by accelerating the replacement of bound GDP with GTP to initiate recruitment of coat proteins for membrane vesicle formation. Among proteins that interact with BIG1, kinesin family member 21A (KIF21A), a plus-end-directed motor protein, moves cargo away from the microtubule-organizing center (MTOC) on microtubules. Because KANK1, a protein containing N-terminal KN, C-terminal ankyrin-repeat, and intervening coiled-coil domains, has multiple actions in cells and also interacts with KIF21A, we explored a possible interaction between it and BIG1. We obtained evidence for a functional and physical association between these proteins, and found that the effects of BIG1 and KANK1 depletion on cell migration in wound-healing assays were remarkably similar. Treatment of cells with BIG1- or KANK1-specific siRNA interfered significantly with directed cell migration and initial orientation of Golgi/MTOC toward the leading edge, which was not mimicked by KIF21A depletion. Although colocalization of overexpressed KANK1 and endogenous BIG1 in HeLa cells was not clear microscopically, their reciprocal immunoprecipitation (IP) is compatible with the presence of small percentages of each protein in the same complexes. Depletion or overexpression of BIG1 protein appeared not to affect KANK1 distribution. Our data identify actions of both BIG1 and KANK1 in regulating cell polarity during directed migration; these actions are consistent with the presence of both BIG1 and KANK1 in dynamic multimolecular complexes that maintain Golgi/MTOC orientation, differ from those that might contain all three proteins (BIG1, KIF21A, and KANK1), and function in directed transport along microtubules.