Brefeldin A-inhibited Guanine Nucleotide Exchange Research Articles

Transcriptome analysis revealed regulatory mechanisms of light and culture density on free-living sporangial filaments of Neopyropia yezoensis (Rhodophyta)

Previous research indicated that free-living sporangial filament keep hollow morph under high-culture density and form bipartite cells under low-culture density, while the following conchospore release was inhibited by high light. Here, we further explored the molecular bases of these affects caused by light and culture density using a transcriptome analysis. Many differentially expressed genes (DEGs) related to carbon dioxide concentration and fixation, photosynthesis, chlorophyll synthesis and nitrogen absorption were upregulated under high-light conditions compared with low-light conditions, indicating the molecular basis of rapid vegetative growth under the former. The stress response- and ion transport-related DEGs, as well as the gene encoding the vacuole formation–brefeldin A-inhibited guanine nucleotide exchange protein (BIG, py05721), were highly expressed under high-density conditions, indicating the molecular basis of the hollow morph of free-living sporangial filaments under high-culture density conditions. Additionally, the brefeldin A treatment indicated that the hollow morph was directly influenced by vacuole formation-related vesicle traffic. Others DEGs related to cell wall components, zinc-finger proteins, ASPO1527, cell cycle and cytoskeleton were highly expressed in the low density with low-light group, which might be related to the formation and release of conchospores. These results provide a deeper understanding of sporangial filaments in Neopyropia yezoensis and related species.

Distinct ADP-ribosylation factor-GTP exchange factors govern the opposite polarity of 2 receptor kinases.

Polarity of plasma membrane proteins is essential for cell morphogenesis and control of cell division and, thus, influences organ and whole plant development. In Arabidopsis (Arabidopsis thaliana) root endodermal cells, 2 transmembrane kinases, INFLORESCENCE AND ROOT APICES RECEPTOR KINASE (IRK) and KINASE ON THE INSIDE (KOIN), accumulate at opposite lateral domains. Their polarization is tightly linked to their activities regulating cell division and ground tissue patterning. The polarization of IRK and KOIN relies solely on the secretion of newly synthesized protein. However, the secretion machinery by which their opposite, lateral polarity is achieved remains largely unknown. Here, we show that different sets of ADP-ribosylation factor (ARF)-guanine-nucleotide exchange factors (ARF-GEFs) mediate their secretion. ARF-GEF GNOM-like-1 (GNL1) regulates KOIN secretion to the inner polar domain, thereby directing KOIN sorting early in the secretion pathway. For IRK, combined chemical and genetic analyses showed that the ARG-GEF GNL1, GNOM, and the BREFELDIN A-INHIBITED-GUANINE NUCLEOTIDE-EXCHANGE FACTORs 1 to 4 (BIG1-BIG4) collectively regulate its polar secretion. The ARF-GEF-dependent mechanisms guiding IRK or KOIN lateral polarity were active across different root cell types and functioned regardless of the protein's inner/outer polarity in those cells. Therefore, we propose that specific polar trafficking of IRK and KOIN occurs via distinct mechanisms that are not constrained by cell identity or polar axis and likely rely on individual protein recognition.

The survival and proliferation of osteosarcoma cells are dependent on the mitochondrial BIG3-PHB2 complex formation.

Previous studies reported the critical role of the brefeldin A–inhibited guanine nucleotide exchange protein 3–prohibitin 2 (BIG3‐PHB2) complex in modulating estrogen signaling activation in breast cancer cells, yet its pathophysiological roles in osteosarcoma (OS) cells remain elusive. Here, we report a novel function of BIG3‐PHB2 in OS malignancy. BIG3‐PHB2 complexes were localized mainly in mitochondria in OS cells, unlike in estrogen‐dependent breast cancer cells. Depletion of endogenous BIG3 expression by small interfering RNA (siRNA) treatment led to significant inhibition of OS cell growth. Disruption of BIG3‐PHB2 complex formation by treatment with specific peptide inhibitor also resulted in significant dose‐dependent suppression of OS cell growth, migration, and invasion resulting from G2/M‐phase arrest and in PARP cleavage, ultimately leading to PARP‐1/apoptosis‐inducing factor (AIF) pathway activation–dependent apoptosis in OS cells. Subsequent proteomic and bioinformatic pathway analyses revealed that disruption of the BIG3‐PHB2 complex might lead to downregulation of inner mitochondrial membrane protein complex activity. Our findings indicate that the mitochondrial BIG3‐PHB2 complex might regulate PARP‐1/AIF pathway–dependent apoptosis during OS cell proliferation and progression and that disruption of this complex may be a promising therapeutic strategy for OS.

Regulating the regulators: role of phosphorylation in modulating the function of the GBF1/BIG family of Sec7 ARF-GEFs.

Membrane traffic between secretory and endosomal compartments is vesicle-mediated and must be tightly balanced to maintain a physiological compartment size. Vesicle formation is initiated by guanine nucleotide exchange factors (GEFs) that activate the ARF family of small GTPases. Regulatory mechanisms, including reversible phosphorylation, allow ARF-GEFs to support vesicle formation only at the right time and place in response to cellular needs. Here, we review current knowledge of how the Golgi-specific brefeldin A-resistance factor 1 (GBF1)/brefeldin A-inhibited guanine nucleotide exchange protein (BIG) family of ARF-GEFs is influenced by phosphorylation and use predictive paradigms to propose new regulatory paradigms. We describe a conserved cluster of phosphorylation sites within the N-terminal domains of the GBF1/BIG ARF-GEFs and suggest that these sites may respond to homeostatic signals related to cell growth and division. In the C-terminal region, GBF1 shows phosphorylation sites clustered differently as compared with the similar configuration found in both BIG1 and BIG2. Despite this similarity, BIG1 and BIG2 phosphorylation patterns are divergent in other domains. The different clustering of phosphorylation sites suggests that the nonconserved sites may represent distinct regulatory nodes and specify the function of GBF1, BIG1, and BIG2.

Brefeldin A-inhibited guanine nucleotide exchange protein 3 is localized in lysosomes and regulates GABA signaling in hippocampal neurons.

ADP-ribosylation factor (ARF) family of guanine-nucleotide-binding (G) proteins regulates organelle biogenesis, structure and trafficking. The functions of ARF proteins are tightly controlled by guanine nucleotide exchange factors (GEFs) containing a conserved SEC7 domain. Based on sequence similarity to brefeldin A-inhibited guanine nucleotide exchange protein (BIG)/GBF of the Arf-GEF family, we recently identified BIG3 as a novel ARF GEF protein with a non-functional catalytic motif in the SEC7 domain. BIG3 is mainly expressed in pancreatic islets and brain. In the islets, depletion of BIG3 increases insulin and glucagon secretion because of enhanced biogenesis of insulin and glucagon granules in the absence of BIG3. Here, we investigate BIG3 functions in the brain, in particular its regulation of neurotransmitter release in hippocampal neurons from wild-type and BIG3 knockout mice. In hippocampal neurons, BIG3 is mainly localized in lysosomes, and its depletion selectively impairs inhibitory synaptic transmission. Our finding provides novel insights for a cell-specific function of BIG3 in regulating neurotransmission.

Increased biogenesis of glucagon-containing secretory granules and glucagon secretion in BIG3-knockout mice.

ObjectiveAlthough both insulin and glucagon are intimately involved in the regulation of glucose homeostasis, the intrinsic control of glucagon secretion, including the biogenesis and exocytosis of glucagon-containing granules, is far less understood compared with that of insulin. As Brefeldin A-inhibited guanine nucleotide exchange protein 3 (BIG3) is a negative regulator of insulin-granule biogenesis and insulin secretion, we investigated whether BIG3 plays any role in alpha-cells and glucagon secretion.MethodsWe examined the expression of BIG3 in islet cells by immuno-fluorescence and confocal microscopy, and measured glucagon production and secretion in BIG3-depleted and wild-type mice, islets and cells.ResultsBIG3 is highly expressed in pancreatic alpha-cells in addition to beta-cells, but is absent in delta-cells. Depletion of BIG3 in alpha-cells leads to elevated glucagon production and secretion. Consistently, BIG3-knockout (BKO) mice display increased glucagon release under hypoglycemic conditions.ConclusionsTogether with our previous studies, the current data reveal a conserved role for BIG3 in regulating alpha- and beta-cell functions. We propose that BIG3 negatively regulates hormone production at the secretory granule biogenesis stage and that such regulatory mechanism may be used in secretory pathways of other endocrine cells.

BIG1, a Brefeldin A-Inhibited Guanine Nucleotide-Exchange Factor, Is Required for GABA-Gated Cl– Influx Through Regulation of GABAA Receptor Trafficking

GABAA receptors (GABAARs) mediate the majority of fast synaptic inhibition. Trafficking regulation and protein-protein interactions that maintain the appropriate number of GABAARs at the cell surface are considered to be important mechanisms for controlling the strength of synaptic inhibition. Here, we report that BIG1, a brefeldin A (BFA)-inhibited guanine nucleotide-exchange factor (GEF) which has a known role in vesicle trafficking, is a new binding partner of GABAARs. Treatment of neurons with BFA, an uncompetitive inhibitor of BIG1 GEF activity, or depletion of BIG1 by small RNA interference (siRNA) significantly decreased GABAARs at the neuronal surface and suppressed GABA-gated influx of chloride ions. Over-expression of HA-tagged BIG1-E793K, a dominant-negative mutant, also significantly decreased GABAARs at the neuronal surface, but had no effect on the total amount of GABAARs. Inhibition of GABAAR endocytosis by muscimol increased both GABAARs and BIG1 at the neuronal surface in a time-dependent fashion, and this increase could be abolished by bicuculline. Finally, depletion of BIG1 by siRNA inhibited the muscimol-stimulated increase of GABAARs. Those data suggest an important function of BIG1 in trafficking of GABAARs to the cell surface through its GEF activity. Thus, we identify an important role of BIG1 in modulating GABA-gated Cl(-) influx through the regulation of cell surface expression of GABAARs.

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.

Arf3 Is Activated Uniquely at thetrans-Golgi Network by Brefeldin A-inhibited Guanine Nucleotide Exchange Factors

It is widely assumed that class I and II Arfs function interchangeably throughout the Golgi complex. However, we report here that in vivo, Arf3 displays several unexpected properties. Unlike other Golgi-localized Arfs, Arf3 associates selectively with membranes of the trans-Golgi network (TGN) in a manner that is both temperature-sensitive and uniquely dependent on guanine nucleotide exchange factors of the BIGs family. For example, BIGs knockdown redistributed Arf3 but not Arf1 from Golgi membranes. Furthermore, shifting temperature to 20 degrees C, a temperature known to block cargo in the TGN, selectively redistributed Arf3 from Golgi membranes. Arf3 redistribution occurred slowly, suggesting it resulted from a change in membrane composition. Arf3 knockdown and overexpression experiments suggest that redistribution is not responsible for the 20 degrees C block. To investigate in more detail the mechanism for Arf3 recruitment and temperature-dependent release, we characterized several mutant forms of Arf3. This analysis demonstrated that those properties are readily separated and depend on pairs of residues present at opposite ends of the protein. Furthermore, phylogenetic analysis established that all four critical residues were absolutely conserved and unique to Arf3. These results suggest that Arf3 plays a unique function at the TGN that likely involves recruitment by a specific receptor.

CAMP-dependent Protein Kinase A (PKA) Signaling Induces TNFR1 Exosome-like Vesicle Release via Anchoring of PKA Regulatory Subunit RIIβ to BIG2

The 55-kDa TNFR1 (type I tumor necrosis factor receptor) can be released to the extracellular space by two mechanisms, the proteolytic cleavage and shedding of soluble receptor ectodomains and the release of full-length receptors within exosome-like vesicles. We have shown that the brefeldin A-inhibited guanine nucleotide exchange protein BIG2 associates with TNFR1 and selectively modulates the release of TNFR1 exosome-like vesicles via an ARF1- and ARF3-dependent mechanism. Here, we assessed the role of BIG2 A kinase-anchoring protein (AKAP) domains in the regulation of TNFR1 exosome-like vesicle release from human vascular endothelial cells. We show that 8-bromo-cyclic AMP induced the release of full-length, 55-kDa TNFR1 within exosome-like vesicles via a protein kinase A (PKA)-dependent mechanism. Using RNA interference to decrease specifically the levels of individual PKA regulatory subunits, we demonstrate that RIIbeta modulates both the constitutive and cAMP-induced release of TNFR1 exosome-like vesicles. Consistent with its AKAP function, BIG2 was required for the cAMP-induced PKA-dependent release of TNFR1 exosome-like vesicles via a mechanism that involved the binding of RIIbeta to BIG2 AKAP domains B and C. We conclude that both the constitutive and cAMP-induced release of TNFR1 exosome-like vesicles occur via PKA-dependent pathways that are regulated by the anchoring of RIIbeta to BIG2 via AKAP domains B and C. Thus, BIG2 regulates TNFR1 exosome-like vesicle release by two distinct mechanisms, as a guanine nucleotide exchange protein that activates class I ADP-ribosylation factors and as an AKAP for RIIbeta that localizes PKA signaling within cellular TNFR1 trafficking pathways.

The Brefeldin A-inhibited Guanine Nucleotide-exchange Protein, BIG2, Regulates the Constitutive Release of TNFR1 Exosome-like Vesicles

The type I, 55-kDa tumor necrosis factor receptor (TNFR1) is released from cells to the extracellular space where it can bind and modulate TNF bioactivity. Extracellular TNFR1 release occurs by two distinct pathways: the inducible proteolytic cleavage of TNFR1 ectodomains and the constitutive release of full-length TNFR1 in exosome-like vesicles. Regulation of both TNFR1 release pathways appears to involve the trafficking of cytoplasmic TNFR1 vesicles. Vesicular trafficking is controlled by ADP-ribosylation factors (ARFs), which are active in the GTP-bound state and inactive when bound to GDP. ARF activation is enhanced by guanine nucleotide-exchange factors that catalyze replacement of GDP by GTP. We investigated whether the brefeldin A (BFA)-inhibited guanine nucleotide-exchange proteins, BIG1 and/or BIG2, are required for TNFR1 release from human umbilical vein endothelial cells. Effects of specific RNA interference (RNAi) showed that BIG2, but not BIG1, regulated the release of TNFR1 exosome-like vesicles, whereas neither BIG2 nor BIG1 was required for the IL-1beta-induced proteolytic cleavage of TNFR1 ectodomains. BIG2 co-localized with TNFR1 in diffusely distributed cytoplasmic vesicles, and the association between BIG2 and TNFR1 was disrupted by BFA. Consistent with the preferential activation of class I ARFs by BIG2, ARF1 and ARF3 participated in the extracellular release of TNFR1 exosome-like vesicles in a nonredundant and additive fashion. We conclude that the association between BIG2 and TNFR1 selectively regulates the extracellular release of TNFR1 exosome-like vesicles from human vascular endothelial cells via an ARF1- and ARF3-dependent mechanism.

BIG2, a guanine nucleotide exchange factor for ADP-ribosylation factors: its localization to recycling endosomes and implication in the endosome integrity.

Small GTPases of the ADP-ribosylation factor (ARF) family play a key role in membrane trafficking by regulating coated vesicle formation, and guanine nucleotide exchange is essential for the ARF function. Brefeldin A blocks the ARF-triggered coat assembly by inhibiting the guanine nucleotide exchange on ARFs and causes disintegration of the Golgi complex and tubulation of endosomal membranes. BIG2 is one of brefeldin A-inhibited guanine nucleotide exchange factors for the ARF GTPases and is associated mainly with the trans-Golgi network. In the present study, we have revealed that another population of BIG2 is associated with the recycling endosome and found that expression of a catalytically inactive BIG2 mutant, E738K, selectively induces membrane tubules from this compartment. We also have shown that BIG2 has an exchange activity toward class I ARFs (ARF1 and ARF3) in vivo and inactivation of either ARF exaggerates the BIG2(E738K)-induced tubulation of endosomal membranes. These observations together indicate that BIG2 is implicated in the structural integrity of the recycling endosome through activating class I ARFs.

Purification and Cloning of a Brefeldin A-inhibited Guanine Nucleotide-exchange Protein for ADP-ribosylation Factors

Activation of ADP-ribosylation factors (ARFs), approximately 20-kDa guanine nucleotide-binding proteins that play an important role in intracellular vesicular trafficking, depends on guanine nucleotide-exchange proteins (GEPs), which accelerate replacement of bound GDP with GTP. Two major families of ARF GEPs are known: approximately 200-kDa molecules that are inhibited by brefeldin A (BFA), a fungal metabolite that blocks protein secretion and causes apparent disintegration of Golgi structure, and approximately 50-kDa GEPs that are insensitive to BFA. We describe here two human brain cDNAs that encode BFA-inhibited GEPs. One is a approximately 209-kDa protein 99.5% identical in deduced amino acid sequence (1, 849 residues) to a BFA-inhibited ARF GEP (p200) from bovine brain. The other smaller protein, which is approximately 74% identical (1, 785 amino acids), represents a previously unknown gene. We propose that the former, p200, be named BIG1 for (brefeldin A-inhibited GEP1) and the second, which encodes a approximately 202-kDa protein, BIG2. A protein containing sequences found in BIG2 had been purified earlier from bovine brain. Human tissues contained a 7.5-kilobase BIG1 mRNA and a 9.4-kilobase BIG2 transcript. The BIG1 and BIG2 genes were localized, respectively, to chromosomes 8 and 20. BIG2, synthesized as a His6 fusion protein in Sf9 cells, accelerated guanosine 5'-3-O-(thio)triphosphate binding by recombinant ARF1, ARF5, and ARF6. It activated native ARF (mixture of ARF1 and ARF3) more effectively than it did any of the nonmyristoylated recombinant ARFs. BIG2 activity was inhibited by BFA in a concentration-dependent manner but not by B17, a structural analog without effects on Golgi function. Although several clones for approximately 50-kDa BFA-insensitive ARF GEPs are known, these new clones for the approximately 200-kDa BIG1 and BIG2 should facilitate characterization of this rather different family of proteins as well as the elucidation of mechanisms of regulation of BFA-sensitive ARF function in Golgi transport.