ADP-ribosylation Factor Activation Research Articles

Hepatocyte growth factor attenuates high glucose-disturbed mitochondrial dynamics in podocytes by decreasing ARF6-dependent DRP1 translocation

Diabetic nephropathy (DN), one of the most common complications of Diabetes Mellitus, is the leading cause of end-stage renal diseases worldwide. Our previous study proved that hepatocyte growth factor (HGF) alleviated renal damages in mice with type 1 Diabetes Mellitus by suppressing overproduction of reactive oxygen species (ROS) in podocytes, while the further mechanism of how HGF lessens ROS production had not been clarified yet. ADP-ribosylation factor 6 (ARF6), the member of the small GTPases superfamilies, is widely spread among epithelial cells and can be activated by the HGF/c-Met signaling. Thus, this study was aimed to explore whether HGF could function on mitochondrial homeostasis, the main resource of ROS, in podocytes exposed to diabetic conditions via ARF6 activation. Our in vivo data showed that HGF markedly ameliorated the pathological damages in kidneys of db/db mice, especially the sharp decline of podocyte number, which was mostly blocked by the ARF6 inhibitor SecinH3. Correspondingly, our in vitro data revealed that HGF protected against high glucose-induced podocyte injuries by increasing ARF6 activity. Besides, this ARF6-dependent beneficial effect of HGF on podocytes was accompanied by improved mitochondrial dynamics and declined DRP1 translocation from cytosol to mitochondria. Collectively, our findings confirm the ability of HGF maintaining mitochondrial homeostasis in diabetic podocytes via decreasing ARF6-dependent DRP1 translocation and shed light on the novel mechanism of HGF treatment for DN.

Neuroinflammatory disease disrupts the blood-CNS barrier via crosstalk between proinflammatory and endothelial-to-mesenchymal-transition signaling

Breakdown of the blood-central nervous system barrier (BCNSB) is a hallmark of many neuroinflammatory disorders, such as multiple sclerosis (MS). Using a mouse model of MS, experimental autoimmune encephalomyelitis (EAE), we show that endothelial-to-mesenchymal transition (EndoMT) occurs in the CNS before the onset of clinical symptoms and plays a major role in the breakdown of BCNSB function. EndoMT can be induced by an IL-1β-stimulated signaling pathway in which activation of the small GTPase ADP ribosylation factor 6 (ARF6) leads to crosstalk with the activin receptor-like kinase (ALK)-SMAD1/5 pathway. Inhibiting the activation of ARF6 both prevents and reverses EndoMT, stabilizes BCNSB function, reduces demyelination, and attenuates symptoms even after the establishment of severe EAE, without immunocompromising the host. Pan-inhibition of ALKs also reduces disease severity in the EAE model. Therefore, multiple components of the IL-1β-ARF6-ALK-SMAD1/5 pathway could be targeted for the treatment of a variety of neuroinflammatory disorders.

Cytohesin-2 mediates group I metabotropic glutamate receptor-dependent mechanical allodynia through the activation of ADP ribosylation factor 6 in the spinal cord

Group I metabotropic glutamate receptors (mGluRs), mGluR1 and mGluR5, in the spinal cord are implicated in nociceptive transmission and plasticity through G protein-mediated second messenger cascades leading to the activation of various protein kinases such as extracellular signal-regulated kinase (ERK). In this study, we demonstrated that cytohesin-2, a guanine nucleotide exchange factor for ADP ribosylation factors (Arfs), is abundantly expressed in subsets of excitatory interneurons and projection neurons in the superficial dorsal horn. Cytohesin-2 is enriched in the perisynapse on the postsynaptic membrane of dorsal horn neurons and forms a protein complex with mGluR5 in the spinal cord. Central nervous system-specific cytohesin-2 conditional knockout mice exhibited reduced mechanical allodynia in inflammatory and neuropathic pain models. Pharmacological blockade of cytohesin catalytic activity with SecinH3 similarly reduced mechanical allodynia and inhibited the spinal activation of Arf6, but not Arf1, in both pain models. Furthermore, cytohesin-2 conditional knockout mice exhibited reduced mechanical allodynia and ERK1/2 activation following the pharmacological activation of spinal mGluR1/5 with 3,5-dihydroxylphenylglycine (DHPG). The present study suggests that cytothesin-2 is functionally associated with mGluR5 during the development of mechanical allodynia through the activation of Arf6 in spinal dorsal horn neurons.

Preserving Vascular Integrity Protects Mice against Multidrug-Resistant Gram-Negative Bacterial Infection.

The rise in multidrug-resistant (MDR) organisms portends a serious global threat to the health care system with nearly untreatable infectious diseases, including pneumonia and its often fatal sequelae, acute respiratory distress syndrome (ARDS) and sepsis. Gram-negative bacteria (GNB), including Acinetobacter baumannii, Pseudomonas aeruginosa, and carbapenemase-producing Klebsiella pneumoniae (CPKP), are among the World Health Organization's and National Institutes of Health's high-priority MDR pathogens for targeted development of new therapies. Here, we show that stabilizing the host's vasculature by genetic deletion or pharmacological inhibition of the small GTPase ADP-ribosylation factor 6 (ARF6) increases survival rates of mice infected with A. baumannii, P. aeruginosa, and CPKP. We show that the pharmacological inhibition of ARF6-GTP phenocopies endothelium-specific Arf6 disruption in enhancing the survival of mice with A. baumannii pneumonia, suggesting that inhibition is on target. Finally, we show that the mechanism of protection elicited by these small-molecule inhibitors acts by the restoration of vascular integrity disrupted by GNB lipopolysaccharide (LPS) activation of the TLR4/MyD88/ARNO/ARF6 pathway. By targeting the host's vasculature with small-molecule inhibitors of ARF6 activation, we circumvent microbial drug resistance and provide a potential alternative/adjunctive treatment for emerging and reemerging pathogens.

Apigenin and Ethaverine Hydrochloride Enhance Retinal Vascular Barrier In Vitro and In Vivo.

PurposeThis study aims to develop an impedance-based drug screening platform that will help identify drugs that can enhance the vascular barrier function by stabilizing vascular endothelial cell junctions.MethodsChanges in permeability of cultured human retinal microvascular endothelial cells (HRMECs) monolayer were monitored in real-time with the xCELLigence RTCA system. Using this platform, we performed a primary screen of 2100 known drugs and confirmed hits using two additional secondary permeability assays: the transwell permeability assay and the XPerT assay. The cellular and molecular mechanisms of action and in vivo therapeutic efficacy were also assessed.ResultsEleven compounds blocked interleukin 1 beta (IL-1β) induced hyperpermeability in the primary screen. Two of 11 compounds, apigenin and ethaverine hydrochloride, reproducibly blocked multiple cytokines induced hyperpermeability. In addition to HRMEC monolayers, the two compounds stabilized three other types of primary vascular endothelial cell monolayers. Preliminary mechanistic studies suggest that the two compounds stabilize the endothelium by blocking ADP-ribosylation factor 6 (ARF6) activation, which results in enhanced VE-cadherin membrane localization. The two compounds showed in vivo efficacy in an animal model of retinal permeability.ConclusionsWe developed an impedance-based cellular phenotypic drug screening platform that can identify drugs that enhance vascular barrier function. We found apigenin and ethaverine hydrochloride stabilize endothelial cell junctions and enhance the vascular barrier by blocking ARF6 activation and increasing VE-cadherin membrane localization.Translational RelevanceThe drugs identified from the phenotypic screen would have potential therapeutic efficacy in retinal vascular diseases regardless of the underlying mechanisms that promote vascular leak.

Small GTPase ARF6 Is a Coincidence-Detection Code for RPH3A Polarization in Neutrophil Polarization.

Cell polarization is a key step for leukocytes adhesion and transmigration during leukocytes' inflammatory infiltration. Polarized localization of plasma membrane (PM) phosphatidylinositol-4-phosphate (PtdIns4P) directs the polarization of RPH3A, which contains a PtdIns4P binding site. Consequently, RPH3A mediates the RAB21 and PIP5K1C90 polarization, which is important for neutrophil adhesion to endothelia during inflammation. However, the mechanism by which RPH3A is recruited only to PM PtdIns4P rather than Golgi PtdIns4P remains unclear. By using ADP-ribosylation factor 6 (ARF6) small interfering RNA, ARF6 dominant-negative mutant ARF6(T27N), and ARF6 activation inhibitor SecinH3, we demonstrate that ARF6 plays an important role in the polarization of RPH3A, RAB21, and PIP5K1C90 in murine neutrophils. PM ARF6 is polarized and colocalized with RPH3A, RAB21, PIP5K1C90, and PM PtdIns4P in mouse and human neutrophils upon integrin stimulation. Additionally, ARF6 binds to RPH3A and enhances the interaction between the PM PtdIns4P and RPH3A. Consistent with functional roles of polarization of RPH3A, Rab21, and PIP5K1C90, ARF6 is also required for neutrophil adhesion on the inflamed endothelial layer. Our study reveals a previously unknown role of ARF6 in neutrophil polarization as being the coincidence-detection code with PM PtdIns4P. Cooperation of ARF6 and PM PtdIns4P direct RPH3A polarization, which is important for neutrophil firm adhesion to endothelia.

Involvement of BIG1 and BIG2 in regulating VEGF expression and angiogenesis.

VEGF stimulates the formation of new blood vessels by inducing endothelial cell (EC) proliferation and migration. Brefeldin A (BFA)-inhibited guanine nucleotide-exchange protein (BIG)1 and 2 accelerate the replacement of bound GDP with GTP to activate ADP-ribosylation factor (Arf)1, which regulates vesicular transport between the Golgi and plasma membrane. Although it has been reported that treating cells with BFA interferes with Arf1 activation to inhibit VEGF secretion, the role of BIG1 and BIG2 in VEGF trafficking and expression, EC migration and proliferation, and vascular development remains unknown. Here, we found that inactivation of Arf1 reduced VEGF secretion but did not affect the levels of VEGF protein. Interestingly, however, BIG1 and BIG2 knockdown significantly decreased the levels of VEGF mRNA and protein in glioblastoma U251 cells and HUVECs. Furthermore, depletion of BIG1 and BIG2 inhibited HUVEC angiogenesis by diminishing cell migration. Angioblast migration and intersegmental vessel sprouting were also impaired when the BIG2 homolog, Arf guanine nucleotide exchange factor (arfgef)2, was knocked down in zebrafish with endothelial expression of green fluorescent protein (GFP). Depletion of arfgef2 by clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) also caused defects in vascular development of zebrafish embryos. Taken together, these data reveal that BIG1 and BIG2 participate in endothelial cell angiogenesis.-Lu, F.-I., Wang, Y.-T., Wang, Y.-S., Wu, C.-Y., Li, C.-C. Involvement of BIG1 and BIG2 in regulating VEGF expression and angiogenesis.

Deletion of Class II ADP-Ribosylation Factors in Mice Causes Tremor by the Nav1.6 Loss in Cerebellar Purkinje Cell Axon Initial Segments.

ADP-ribosylation factors (ARFs) are a family of small monomeric GTPases comprising six members categorized into three classes: class I (ARF1, 2, and 3), class II (ARF4 and 5), and class III (ARF6). In contrast to class I and III ARFs, which are the key regulators in vesicular membrane trafficking, the cellular function of class II ARFs remains unclear. In the present study, we generated class II ARF-deficient mice and found that ARF4+/-/ARF5-/- mice exhibited essential tremor (ET)-like behaviors. In vivo electrophysiological recordings revealed that ARF4+/-/ARF5-/- mice of both sexes exhibited abnormal brain activity when moving, raising the possibility of abnormal cerebellar excitability. Slice patch-clamp experiments demonstrated the reduced excitability of the cerebellar Purkinje cells (PCs) in ARF4+/-/ARF5-/- mice. Immunohistochemical and electrophysiological analyses revealed a severe and selective decrease of pore-forming voltage-dependent Na+ channel subunit Nav1.6, important for maintaining repetitive action potential firing, in the axon initial segment (AIS) of PCs. Importantly, this decrease in Nav1.6 protein localized in the AIS and the consequent tremors in ARF4+/-/ARF5-/- mice could be alleviated by the PC-specific expression of ARF5 using adeno-associated virus vectors. Together, our data demonstrate that the decreased expression of the class II ARF proteins in ARF4+/-/ARF5-/- mice, leading to a haploinsufficiency of ARF4 in the absence of ARF5, impairs the localization of Nav1.6 to the AIS and hence reduces the membrane excitability in PCs, resulting in the ET-like movement disorder. We suggest that class II ARFs function in localizing specific proteins, such as Nav1.6, to the AIS.SIGNIFICANCE STATEMENT We found that decreasing the expression of class II ARF proteins, through the generation of ARF4+/-/ARF5-/- mice, impairs Nav1.6 distribution to the axon initial segment (AIS) of cerebellar Purkinje cells (PCs), thereby resulting in the impairment of action potential firing of PCs. The ARF4+/-/ARF5-/- mutant mice exhibited movement-associated essential tremor (ET)-like behavior with pharmacological profiles similar to those in ET patients. The exogenous expression of ARF5 reduced the tremor phenotype and restored the localization of Nav1.6 immunoreactivity to the AIS in ARF4+/-/ARF5-/- mice. Thus, our results suggest that class II ARFs are involved in the localization of Nav1.6 to the AISs in cerebellar PCs and that the reduction of class II ARF activity leads to ET-like movement disorder.

Aberrant endocytosis leads to the loss of normal mitotic spindle orientation during epithelial glandular morphogenesis

Epithelial cells form tissues with many functions, including secretion and environmental separation and protection. Glandular epithelial tissues comprise cysts and tubules that are formed from a polarized, single-epithelial cell layer surrounding a central, fluid-filled lumen. The pathways regulating key processes in epithelial tissue morphogenesis such as mitotic spindle formation are incompletely understood, but are important to investigate, as their dysregulation is a signature of epithelial tumors. Here, we describe a signaling axis that manifests in a defect in mitotic spindle orientation during epithelial growth and cystogenesis. We found that activation of the small GTPase ADP-ribosylation factor 6 (ARF6) results in the sustained internalization of cell-surface components such as the cMet receptor and the cell-adhesion molecule E-cadherin. The spindle orientation defect arising from elevated levels of ARF6-GTP required an increase in cMet endocytosis, but was independent of E-cadherin internalization or elevated extracellular signal-regulated kinase (ERK) activity resulting from internalized receptor signaling on endosomes. Misorientation of the mitotic spindle resulted in the development of epithelial cysts with structural abnormalities, the most conspicuous of which was the presence of multiple intercellular lumens. Abnormal mitotic spindle orientation was necessary but insufficient to disrupt glandular development, as blocking the strong prosurvival signal resulting from ERK hyperactivation yielded structurally normal cysts despite continued manifestation of spindle orientation defects. Our findings highlight a previously unknown link between ARF6 activation, cMet receptor internalization, and mitotic spindle orientation during epithelial glandular morphogenesis.

Nonselective Chemical Inhibition of Sec7 Domain-Containing ARF GTPase Exchange Factors.

Small GTP-binding proteins from the ADP-ribosylation factor (ARF) family are important regulators of vesicle formation and cellular trafficking in all eukaryotes. ARF activation is accomplished by a protein family of guanine nucleotide exchange factors (GEFs) that contain a conserved catalytic Sec7 domain. Here, we identified and characterized Secdin, a small-molecule inhibitor of Arabidopsis thaliana ARF-GEFs. Secdin application caused aberrant retention of plasma membrane (PM) proteins in late endosomal compartments, enhanced vacuolar degradation, impaired protein recycling, and delayed secretion and endocytosis. Combined treatments with Secdin and the known ARF-GEF inhibitor Brefeldin A (BFA) prevented the BFA-induced PM stabilization of the ARF-GEF GNOM, impaired its translocation from the Golgi to the trans-Golgi network/early endosomes, and led to the formation of hybrid endomembrane compartments reminiscent of those in ARF-GEF-deficient mutants. Drug affinity-responsive target stability assays revealed that Secdin, unlike BFA, targeted all examined Arabidopsis ARF-GEFs, but that the interaction was probably not mediated by the Sec7 domain because Secdin did not interfere with the Sec7 domain-mediated ARF activation. These results show that Secdin and BFA affect their protein targets through distinct mechanisms, in turn showing the usefulness of Secdin in studies in which ARF-GEF-dependent endomembrane transport cannot be manipulated with BFA.

Abstract 5157: ARF6 controls WNT5A receptor internalization to promote uveal melanoma invasion and metastasis

Abstract Introduction: Uveal melanoma is the most common primary ocular malignancy and there is currently no effective treatment for metastatic uveal melanoma largely because of the lack of understanding of the molecular mechanisms underlying this cancer. Although activating oncogenic mutations in GNAQ and GNA11 are present in 95% of uveal melanoma tumors, virtually nothing is known about the molecular mechanisms that drive oncogenesis in the remaining 5% of uveal melanomas that possess only wild type Gαq alleles and the molecular and cellular mechanisms that promote metastasis have not yet been discovered. This latter deficit in knowledge is extremely vexing, given that metastatic disease causes death in most uveal melanoma patients. We have recently shown that ADP-ribosylation factor 6 (ARF6), a small GTPase, is an immediate downstream effector of oncogenic Gαq and controls the entire major signaling pathways known to drive Gαq-mediated tumor formation and growth in uveal melanoma cells that have an activating mutation in Gαq. Moreover, in cutaneous melanoma, stimulation of the receptor by WNT5A activates ARF6 and controls metastasis via the release of β-catenin from N-cadherin and β-catenin's subsequent nuclear transport. Previous studies have also demonstrated that ARF6 plays a role in the internalization of various classes of membrane receptors including receptor tyrosine kinase (RTK) and G protein coupled receptor (GPCR) to control signal transduction, and WNT5A and its receptor promote tumor invasion and metastasis via receptor internalization in human cancers. Based on these intriguing findings, we postulate that ARF6 might be functioning to control tumor invasion and metastasis in uveal melanomas. Experimental Procedures: To test our hypothesis, we conducted cell invasion assays on uveal melanoma cell lines while targeting ARF6 expression by siRNA or ARF6 activity by pharmacological inhibition. We then proceeded with cell fractionation to look for any differences in the properties of subcellular components in treated cells compared to untreated cells. Unpublished findings: Here we show that WNT5A is expressed at high levels in Gαq wild type uveal melanoma cells and that WNT5A activates ARF6 through ROR2 receptor. When we block ARF6 expression by knockdown or ARF6 activity with a small molecule inhibitor, uveal melanoma invasion is significantly reduced. Notably, ARF6 is necessary for WNT5A-mediated ROR2 internalization. Conclusions: This work indicates that ARF6 functions in the internalization of WNT5A receptor to activate the signaling pathways that drive invasion and metastasis in uveal melanoma. These results would suggest that targeting ARF6 activation might be an effective therapy for the treatment of all uveal melanomas, regardless of their mutational status for Gαq. Citation Format: Donghan Shin, Coulson P. Rich, Lehi Acosta, Jackson R. Richards, Jae Hyuk Yoo, Allie H. Grossmann, Zongzhong Tong, Alan L. Mueller, Weiquan Zhu, Dean Y. Li, Shannon J. Odelberg. ARF6 controls WNT5A receptor internalization to promote uveal melanoma invasion and metastasis [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 5157.

BIG2-ARF1-RhoA-mDia1 Signaling Regulates Dendritic Golgi Polarization in Hippocampal Neurons.

Proper dendrite development is essential for establishing neural circuitry, and Rho GTPases play key regulatory roles in this process. From mouse brain lysates, we identified Brefeldin A-inhibited guanine exchange factor 2 (BIG2) as a novel Rho GTPase regulatory protein involved in dendrite growth and maintenance. BIG2 was highly expressed during early development, and knockdown of the ARFGEF2 gene encoding BIG2 significantly reduced total dendrite length and the number of branches. Expression of the constitutively active ADP-ribosylation factor 1 ARF1Q71L rescued the defective dendrite morphogenesis of ARFGEF2-null neurons, indicating that BIG2 controls dendrite growth and maintenance by activating ARF1. Moreover, BIG2 co-localizes with the Golgi apparatus and is required for Golgi deployment into major dendrites in cultured hippocampal neurons. Simultaneous overexpression of BIG2 and ARF1 activated RhoA, and treatment with the RhoA activator lysophosphatidic acid in neurons lacking BIG2 or ARF1 increased the number of cells with dendritic Golgi, suggesting that BIG2 and ARF1 activate RhoA to promote dendritic Golgi polarization. mDia1 was identified as a downstream effector of BIG2-ARF1-RhoA axis, mediating Golgi polarization and dendritic morphogenesis. Furthermore, in utero electroporation of ARFGEF2 shRNA into the embryonic mouse brain confirmed an in vivo role of BIG2 for Golgi deployment into the apical dendrite. Taken together, our results suggest that BIG2-ARF1-RhoA-mDia1 signaling regulates dendritic Golgi polarization and dendrite growth and maintenance in hippocampal neurons.

Highly conserved motifs within the large Sec7 ARF guanine nucleotide exchange factor GBF1 target it to the Golgi and are critical for GBF1 activity.

Cellular life requires the activation of the ADP-ribosylation factors (ARFs) by Golgi brefeldin A-resistant factor 1 (GBF1), a guanine nucleotide exchange factor (GEF) with a highly conserved catalytic Sec7 domain (Sec7d). In addition to the Sec7d, GBF1 contains other conserved domains whose functions remain unclear. Here, we focus on HDS2 (homology downstream of Sec7d 2) domain because the L1246R substitution within the HDS2 α-helix 5 of the zebrafish GBF1 ortholog causes vascular hemorrhaging and embryonic lethality (13). To dissect the structure/function relationships within HDS2, we generated six variants, in which the most conserved residues within α-helices 1, 2, 4, and 6 were mutated to alanines. Each HDS2 mutant was assessed in a cell-based "replacement" assay for its ability to support cellular functions normally supported by GBF1, such as maintaining Golgi homeostasis, facilitating COPI recruitment, supporting secretion, and sustaining cellular viability. We show that cells treated with the pharmacological GBF1 inhibitor brefeldin A (BFA) and expressing a BFA-resistant GBF1 variant with alanine substitutions of RDR1168 or LF1266 are compromised in Golgi homeostasis, impaired in ARF activation, unable to sustain secretion, and defective in maintaining cellular viability. To gain insight into the molecular mechanism of this dysfunction, we assessed the ability of each GBF1 mutant to target to Golgi membranes and found that mutations in RDR1168 and LF1266 significantly decrease targeting efficiency. Thus, these residues within α-helix 2 and α-helix 6 of the HDS2 domain in GBF1 are novel regulatory determinants that support GBF1 cellular function by impacting the Golgi-specific membrane association of GBF1.

Progesterone and calcitriol reduce invasive potential of endometrial cancer cells by targeting ARF6, NEDD9 and MT1-MMP.

Previously, we have demonstrated that progesterone and calcitriol synergistically inhibit growth of endometrial and ovarian cancer by enhancing apoptosis and causing cell cycle arrest. Metastasis is the main reason of mortality in cancer patients. Activation of ADP-Ribosylation Factor 6 (ARF6), Neural Precursor cell expressed Developmentally Downregulated 9 (NEDD9), and Membrane-Type-1 Matrix Metalloproteinase (MT1-MMP) have been implicated in promoting tumor growth and metastasis. We examined the effects of progesterone, calcitriol and progesterone-calcitriol combination on metastasis promoting proteins in endometrial cancer. Expression of ARF6, NEDD9, and MT1-MMP was enhanced in advanced-stage endometrial tumors and in cancer cell lines compared to normal tissues and immortalized EM-E6/E7-TERT endometrial epithelial cells. Knockdown of these proteins significantly inhibited the invasiveness of the cancer cells. The expression levels of all three proteins was reduced with progesterone and progesterone-calcitriol combination treatment, whereas calcitriol alone showed no effect on their expression but moderately decreased MT1-MMP activity. Fluorescence microscopy showed membrane expression of MT1-MMP in vehicle and calcitriol-treated endometrial cancer cells. However, progesterone and calcitriol-progesterone combination treatment revealed MT1-MMP in the cytoplasm. Furthermore, progesterone and calcitriol reduced the activity of MT1-MMP, MMP-9, and MMP-2. In addition, invadopodia regulatory proteins were attenuated in both progesterone and progesterone-calcitriol combination treated cells as well as in MT1-MMP knockdown cells. Thus, targeting the aberrant MT1-MMP signaling with progesterone-calcitriol may be a novel approach to impede MT1-MMP mediated cancer dissemination and may have therapeutic benefits for endometrial cancer patients.

AKT-dependent phosphorylation of the SAM domain induces oligomerization and activation of the scaffold protein CNK1

Scaffold proteins are hubs for the coordination of intracellular signaling networks. The scaffold protein CNK1 promotes several signal transduction pathway. Here we demonstrate that sterile motif alpha (SAM) domain-dependent oligomerization of CNK1 stimulates CNK1-mediated signaling in growth factor-stimulated cells. We identified Ser22 located within the SAM domain as AKT-dependent phosphorylation site triggering CNK1 oligomerization. Oligomeric CNK1 increased the affinity for active AKT indicating a positive AKT feedback mechanism. A CNK1 mutant lacking the SAM domain and the phosphorylation-defective mutant CNK1S22A antagonizes oligomerization and prevents CNK1-driven cell proliferation and matrix metalloproteinase 14 promoter activation. The phosphomimetic mutant CNK1S22D constitutively oligomerizes and stimulates CNK1 downstream signaling. Searching the COSMIC database revealed Ser22 as putative target for oncogenic activation of CNK1. Like the phosphomimetic mutant CNK1S22D, the oncogenic mutant CNK1S22F forms clusters in serum-starved cells comparable to clusters of CNK1 in growth factor-stimulated cells. CNK1 clusters induced by activating Ser22 mutants correlate with enhanced cell invasion and binding to and activation of ADP ribosylation factor 1 associated with tumor formation. Mutational analysis indicate that EGF-triggered phosphorylation of Thr8 within the SAM domain prevents AKT binding and antagonizes CNK1-mediated AKT signaling. Our findings reveal SAM domain-dependent oligomerization by AKT as switch for CNK1 activation.

One target to quench many

Inhibiting the GTPase ARF6 chokes the diverse output of mutant G αq proteins in uveal melanoma.

The GTPase ARF6 Controls ROS Production to Mediate Angiotensin II-Induced Vascular Smooth Muscle Cell Proliferation.

High reactive oxygen species (ROS) levels and enhanced vascular smooth muscle cells (VSMC) proliferation are observed in numerous cardiovascular diseases. The mechanisms by which hormones such as angiotensin II (Ang II) acts to promote these cellular responses remain poorly understood. We have previously shown that the ADP-ribosylation factor 6 (ARF6), a molecular switch that coordinates intracellular signaling events can be activated by the Ang II receptor (AT1R). Whether this small GTP-binding protein controls the signaling events leading to ROS production and therefore Ang II-dependent VSMC proliferation, remains however unknown. Here, we demonstrate that in rat aortic VSMC, Ang II stimulation led to the subsequent activation of ARF6 and Rac1, a key regulator of NADPH oxidase activity. Using RNA interference, we showed that ARF6 is essential for ROS generation since in conditions where this GTPase was knocked down, Ang II could no longer promote superoxide anion production. In addition to regulating Rac1 activity, ARF6 also controlled expression of the NADPH oxidase 1 (Nox 1) as well as the ability of the EGFR to become transactivated. Finally, ARF6 also controlled MAPK (Erk1/2, p38 and Jnk) activation, a key pathway of VSMC proliferation. Altogether, our findings demonstrate that Ang II promotes activation of ARF6 to controls ROS production by regulating Rac1 activation and Nox1 expression. In turn, increased ROS acts to activate the MAPK pathway. These signaling events represent a new molecular mechanism by which Ang II can promote proliferation of VSMC.

Brefeldin A exerts differential effects on anaplastic lymphoma kinase positive anaplastic large cell lymphoma and classical Hodgkin lymphoma cell lines.

To obtain further insights into the biological differences of anaplastic lymphoma kinase positive anaplastic large cell lymphoma (ALK+ ALCL) and classical Hodgkin lymphoma (HL), we screened microbial culture filtrates to search for compounds that would exert a significantly greater effect on the viability of ALK+ ALCL cell lines compared to HL cell lines and identified Brefeldin A (BFA) as a suitable candidate. BFA inhibited phosphorylation of ALK and its downstream molecule, signal transducer and activator of transcription 3 (STAT3), one of the central pathways for the survival of ALK+ ALCL cells. In HL cell lines BFA did not affect CD30 expression orconstitutive nuclear factor (NF)-κB activity, both of which are critical forHL cell survival. BFA induced disruption of the Golgi apparatus in ALK+ ALCL cell lines, which was accompanied by a decrease in active ADP-ribosylation factor 1 (ARF1), whereas BFA had no significant effect on these parameters in HL cell lines. These results add extra insights into the biological distinction between ALK+ ALCL and HL cells and highlight the Golgi apparatus as a target for the treatment of ALK+ ALCL.

Inhibition of Cytohesins Protects against Genetic Models of Motor Neuron Disease

Mutant genes that underlie Mendelian forms of amyotrophic lateral sclerosis (ALS) and biochemical investigations of genetic disease models point to potential driver pathophysiological events involving endoplasmic reticulum (ER) stress and autophagy. Several steps in these cell biological processes are known to be controlled physiologically by small ADP-ribosylation factor (ARF) signaling. Here, we investigated the role of ARF guanine nucleotide exchange factors (GEFs), cytohesins, in models of ALS. Genetic or pharmacological inhibition of cytohesins protects motor neurons in vitro from proteotoxic insults and rescues locomotor defects in a Caenorhabditis elegans model of disease. Cytohesins form a complex with mutant superoxide dismutase 1 (SOD1), a known cause of familial ALS, but this is not associated with a change in GEF activity or ARF activation. ER stress evoked by mutant SOD1 expression is alleviated by antagonism of cytohesin activity. In the setting of mutant SOD1 toxicity, inhibition of cytohesin activity enhances autophagic flux and reduces the burden of misfolded SOD1. These observations suggest that targeting cytohesins may have potential benefits for the treatment of ALS.

ADP Ribosylation Factor 6 (ARF6) Promotes Acrosomal Exocytosis by Modulating Lipid Turnover and Rab3A Activation

Regulated secretion is a central issue for the specific function of many cells; for instance, mammalian sperm acrosomal exocytosis is essential for egg fertilization. ARF6 (ADP-ribosylation factor 6) is a small GTPase implicated in exocytosis, but its downstream effectors remain elusive in this process. We combined biochemical, functional, and microscopy-based methods to show that ARF6 is present in human sperm, localizes to the acrosomal region, and is required for calcium and diacylglycerol-induced exocytosis. Results from pulldown assays show that ARF6 exchanges GDP for GTP in sperm challenged with different exocytic stimuli. Myristoylated and guanosine 5'-3-O-(thio)triphosphate (GTPγS)-loaded ARF6 (active form) added to permeabilized sperm induces acrosome exocytosis even in the absence of extracellular calcium. We explore the ARF6 signaling cascade that promotes secretion. We demonstrate that ARF6 stimulates a sperm phospholipase D activity to produce phosphatidic acid and boosts the synthesis of phosphatidylinositol 4,5-bisphosphate. We present direct evidence showing that active ARF6 increases phospholipase C activity, causing phosphatidylinositol 4,5-bisphosphate hydrolysis and inositol 1,4,5-trisphosphate-dependent intra-acrosomal calcium release. We show that active ARF6 increases the exchange of GDP for GTP on Rab3A, a prerequisite for secretion. We propose that exocytic stimuli activate ARF6, which is required for acrosomal calcium efflux and the assembly of the membrane fusion machinery. This report highlights the physiological importance of ARF6 as a key factor for human sperm exocytosis and fertilization.