Arf6 Activation Research Articles

P3.02-046 EGFR-Grb2-GEP100 Complex Promoted Its Invasive and Metastatic Potential via Arf6 Pathway in Lung Adenocarcinoma

Invasive and metastatic activities are the most challenging hallmark of cancer. We previously showed that GEP100-Arf6 pathway signals from stimulated ErbB family receptors was pivotal for epithelio-mesenchymal transition (EMT) and induced cancer invasive activity leading to nodal and distant metastasis. Here we have examined the enhancing effect of Grb2 on the binding of GEP100 with EGFR leading to Arf6 activation and EMT, and the significance of EGFR-Grb2-GEP100 binding complex on the invasive and metastatic potentials in lung cancer cells as well as in clinical settings.

Small GTPase ARF6 controls VEGFR2 trafficking and signaling in diabetic retinopathy.

The devastating sequelae of diabetes mellitus include microvascular permeability, which results in retinopathy. Despite clinical and scientific advances, there remains a need for new approaches to treat retinopathy. Here, we have presented a possible treatment strategy, whereby targeting the small GTPase ARF6 alters VEGFR2 trafficking and reverses signs of pathology in 4 animal models that represent features of diabetic retinopathy and in a fifth model of ocular pathological angiogenesis. Specifically, we determined that the same signaling pathway utilizes distinct GEFs to sequentially activate ARF6, and these GEFs exert distinct but complementary effects on VEGFR2 trafficking and signal transduction. ARF6 activation was independently regulated by 2 different ARF GEFs - ARNO and GEP100. Interaction between VEGFR2 and ARNO activated ARF6 and stimulated VEGFR2 internalization, whereas a VEGFR2 interaction with GEP100 activated ARF6 to promote VEGFR2 recycling via coreceptor binding. Intervening in either pathway inhibited VEGFR2 signal output. Finally, using a combination of in vitro, cellular, genetic, and pharmacologic techniques, we demonstrated that ARF6 is pivotal in VEGFR2 trafficking and that targeting ARF6-mediated VEGFR2 trafficking has potential as a therapeutic approach for retinal vascular diseases such as diabetic retinopathy.

Regulation of Arf activation occurs via distinct mechanisms at early and late Golgi compartments.

At the Golgi complex, the biosynthetic sorting center of the cell, the Arf GTPases are responsible for coordinating vesicle formation. The Arf-GEFs activate Arf GTPases and are therefore the key molecular decision-makers for trafficking from the Golgi. In Saccharomyces cerevisiae, three conserved Arf-GEFs function at the Golgi: Sec7, Gea1, and Gea2. Our group has described the regulation of Sec7, the trans-Golgi Arf-GEF, through autoinhibition, positive feedback, dimerization, and interactions with a suite of small GTPases. However, we lack a clear understanding of the regulation of the early Golgi Arf-GEFs Gea1 and Gea2. Here we demonstrate that Gea1 and Gea2 prefer neutral over anionic membrane surfaces in vitro, consistent with their localization to the early Golgi. We illustrate a requirement for a critical mass of either Gea1 or Gea2 for cell growth under stress conditions. We show that the C-terminal domains of Gea1 and Gea2 toggle roles in the cytosol and at the membrane surface, preventing membrane binding in the absence of a recruiting interaction but promoting maximum catalytic activity once recruited. We also identify the small GTPase Ypt1 as a recruiter for Gea1 and Gea2. Our findings illuminate core regulatory mechanisms unique to the early Golgi Arf-GEFs.

ARF1 recruits RAC1 to leading edge in neutrophil chemotaxis

BackgroundThe small GTPase ARF1 mediates membrane trafficking mostly from the Golgi, and is essential for the G protein-coupled receptor (GPCR)-mediated chemotaxis of neutrophils. In this process, ARF1 is activated by the guanine nucleotide exchanger GBF1, and is inactivated by the GTPase-activating protein GIT2. Neutrophils generate the Gβγ-PAK1-αPIX-GIT2 linear complex during GPCR-induced chemotaxis, in which αPIX activates RAC1/CDC42, which then employs PAK1. However, it has remained unclear as to why GIT2 is included in this complex.ResultsWe investigated the association between ARF1 and RAC1/CDC42 during the fMLP-stimulated chemotaxis of HL60 cells. We found that the silencing of GBF1 significantly impaired the recruitment of RAC1 to the leading edges, but not PAK1, αPIX, RAC2, or CDC42. A significant population of RAC1 colocalized with ARF1 at the leading edges in stimulated cells, whereas fMLP activated both ARF1 and ARF5. Consistently, the silencing of ARF1, but not ARF5, impaired the recruitment of RAC1, whereas the silencing of RAC1 did not affect the recruitment of ARF1 to the leading edges.ConclusionsOur results indicated that the activation of ARF1 triggers the plasma membrane recruitment of RAC1 in GPCR-mediated chemotaxis, which is essential for cortical actin remodeling. Thus, membrane remodeling at the leading edges appears to precede actin remodeling in chemotaxis. Together with the fact that GIT2, which inactivates ARF1, is an integral component of the machinery activating RAC1, we proposed a model in which the ARF1-RAC1 linkage enables the regulation of ARF1 by repetitive on/off cycles during GPCR-mediated neutrophil chemotaxis.

Multiple interactions between an Arf/GEF complex and charged lipids determine activation kinetics on the membrane

Lipidated small GTPases and their regulators need to bind to membranes to propagate actions in the cell, but an integrated understanding of how the lipid bilayer exerts its effect has remained elusive. Here we focused on ADP ribosylation factor (Arf) GTPases, which orchestrate a variety of regulatory functions in lipid and membrane trafficking, and their activation by the guanine-nucleotide exchange factor (GEF) Brag2, which controls integrin endocytosis and cell adhesion and is impaired in cancer and developmental diseases. Biochemical and structural data are available that showed the exceptional efficiency of Arf activation by Brag2 on membranes. We determined the high-resolution crystal structure of unbound Brag2 containing the GEF (Sec7) and membrane-binding (pleckstrin homology) domains, revealing that it has a constitutively active conformation. We used this structure to analyze the interaction of uncomplexed Brag2 and of the myristoylated Arf1/Brag2 complex with a phosphatidylinositol bisphosphate (PIP2) -containing lipid bilayer, using coarse-grained molecular dynamics. These simulations revealed that the system forms a close-packed, oriented interaction with the membrane, in which multiple PIP2 lipids bind the canonical lipid-binding site and unique peripheral sites of the PH domain, the Arf GTPase and, unexpectedly, the Sec7 domain. We cross-validated these predictions by reconstituting the binding and kinetics of Arf and Brag2 in artificial membranes. Our coarse-grained structural model thus suggests that the high efficiency of Brag2 requires interaction with multiple lipids and a well-defined orientation on the membrane, resulting in a local PIP2 enrichment, which has the potential to signal toward the Arf pathway.

Inhibition of smooth muscle contraction and ARF6 activity by the inhibitor for cytohesin GEFs, secinH3, in the human prostate.

Prostate smooth muscle contraction is critical for etiology and treatment of male lower urinary tract symptoms (LUTS) and is promoted by small monomeric GTPases (RhoA and Rac). GTPases may be activated by guanosine nucleotide exchange factors (GEFs). GEFs of the cytohesin family may indirectly activate Rac, or ADP ribosylation factor (ARF) GTPases directly. Here we investigated the expression of cytohesin family GEFs and effects of the cytohesin inhibitor Sec7 inhibitor H3 (secinH3) on smooth muscle contraction and GTPase activities in human prostate tissues. Of all four cytohesin isoforms, cytohesin-1 and -2 showed the highest expression in real-time PCR. Western blot and fluorescence staining suggested that cytohesin-2 may be the predominant isoform in prostate smooth muscle cells. Contractions induced by norepinephrine, the α1-adrenoceptor agonist phenylephrine, the thromboxane A2 analog U-46619 , and endothelin-1 and -3, as well as neurogenic contractions induced by electric field stimulation (EFS), were reduced by secinH3 (30 µM). Inhibition of EFS-induced contractions appeared to have efficacy similar to that of inhibition by the α1-adrenoceptor antagonist tamsulosin (300 nM). Combined application of secinH3 plus tamsulosin caused larger inhibition of EFS-induced contractions than tamsulosin alone. Pull-down assays demonstrated inhibition of the small monomeric GTPase ARF6 by secinH3, but no inhibition of RhoA or Rac1. In conclusion, we suggest that a cytohesin-ARF6 pathway takes part in smooth muscle contraction. This may open attractive new possibilities in medical treatment of male LUTS.

Combined targeting of Arf1 and Ras potentiates anticancer activity for prostate cancer therapeutics

BackgroundAlthough major improvements have been made in surgical management, chemotherapeutic, and radiotherapeutic of prostate cancer, many prostate cancers remain refractory to treatment with standard agents. Therefore, the identification of new molecular targets in cancer progression and development of novel therapeutic strategies to target them are very necessary for achieving better survival for patients with prostate cancer. Activation of small GTPases such as Ras and Arf1 is a critical component of the signaling pathways for most of the receptors shown to be upregulated in advanced prostate cancer.MethodsThe drug effects on cell proliferation were measured by CellTiter 96® AQueous One Solution Cell Proliferation Assay. The drug effects on cell migration and invasion were determined by Radius™ 24-well and Matrigel-coated Boyden chambers. The drug effects on apoptosis were assessed by FITC Annexin V Apoptosis Detection Kit with 7-AAD and Western blot with antibodies against cleaved PARP and Caspase 3. A NOD/SCID mouse model generated by subcutaneous injection was used to assess the in vivo drug efficacy in tumor growth. ERK activation and tumor cell proliferation in xenografts were examined by immunohistochemistry.ResultsWe show that Exo2, a small-molecule inhibitor that reduces Arf1 activation, effectively suppresses prostate cancer cell proliferation by blocking ERK1/2 activation. Exo2 also has other effects, inhibiting migration and invasion of PCa cells and inducing apoptosis. The Ras inhibitor salirasib augments Exo2-induced cytotoxicity in prostate cancer cells partially by enhancing the suppression of ERK1/2 phosphorylation. In a xenograft mouse model of prostate cancer, Exo2 reduces prostate tumor burden and inhibits ERK1/2 activation at a dose of 20 mg/kg. Synergistic treatment of salirasib and Exo2 exhibits a superior inhibitory effect on prostate tumor growth compared with either drug alone, which may be attributed to the more efficient inhibition of ERK1/2 phosphorylation.ConclusionThis study suggests that simultaneous blockade of Arf1 and Ras activation in prostate cancer cells is a potential targeted therapeutic strategy for preventing prostate cancer development.

The ArfGAP protein MoGlo3 regulates the development and pathogenicity of Magnaporthe oryzae.

The ADP ribosylation factor (Arf) and the coat protein complex I (COPI) are involved in vesicle transport. Together with GTPase-activating proteins (ArfGAPs) and guanine exchange factors (ArfGEFs) that regulate the activity of Arf, they govern vesicle formation, COPI trafficking and the maintenance of the Golgi complex. In an ongoing effort to study the role of membrane trafficking in pathogenesis of the rice blast fungus Magnaporthe oryzae, we identified MoGlo3 as an ArfGAP protein that is homologous to Glo3p of the budding yeast Saccharomyces cerevisiae. As suspected, MoGlo3 partially complements the function of yeast Glo3p. Consistent with findings in S. cerevisiae, MoGlo3 is localized to the Golgi, and that the localization is dependent on the conserved BoCCS domain. We found that MoGlo3 is highly expressed during conidiation and early infection stages and is required for vegetative growth, conidial production and sexual development. We further found that the ΔMoglo3 mutant is defective in endocytosis, scavenging of the reactive oxygen species, and in the response to endoplasmic reticulum (ER) stress. The combined effects result in failed appressorium function and decreased pathogenicity. Moreover, we provided evidence showing that the domains including the GAP, BoCCS and GRM are all important for normal MoGlo3 functions. Our studies further illustrate the importance of normal membrane trafficking in the physiology and pathogenicity of the rice blast fungus.

Abstract 1357: The small GTPase Arf6 potentiates melanoma metastasis by activating Akt

Abstract Arf6 is a member of the Ras-superfamily of small GTPases and controls membrane trafficking and cytoskeletal remodeling, functioning mainly in endocytosis pathways at the cell periphery. Arf6 is activated by various extracellular signals and oncogenic events and has been shown to promote cell migration and pro-invasive phenotype in human cancer cells. Small molecule inhibition of ARF6 reduces spontaneous metastasis in xenograft models of human cutaneous melanoma, suggesting that ARF6 is necessary for disease progression. Using a genetically-engineered mouse model of BRAF-mutant melanoma, we determined whether activation of Arf6 is sufficient to induce spontaneous metastasis in vivo. For melanocyte-specific primary tumor induction, Cre recombinase was delivered via local injection of RCAS virus into DCT-TVA;Cdkn2alox/lox;BRAFV600E mice. In the experimental mice, a constitutively active mutant form of Arf6 (Q67L) was virally delivered with Cre. In this study, we observed a significant increase in spontaneous metastatic disease burden in Arf6 Q67L mice. Likewise, tail vein injection of melanoma cell lines derived from Arf6 Q67L tumors consistently show a diffuse pattern of pulmonary metastasis compared to controls that show rare, microscopic metastasis. Recently, it has been demonstrated that activation of Akt, but not Pten loss, leads to an aggressive phenotype in melanoma that includes the acquisition of brain metastases. Immunohistochemical analysis of phospho-Akt revealed that Arf6 Q67L is sufficient to induce Akt activation in tumors. In addition, ARF6 is necessary for AKT activation in human melanoma lines. We did not observe brain metastasis in DCT-TVA;Cdkn2alox/lox;BRAFV600E + Arf6 Q67L mice. When we added Ptenlox/lox allele to this genetic background, however, we observed microscopic brain metastases at a low frequency, suggesting that the combination of Pten loss and Arf6 activation reaches a threshold level of Akt activation that is sufficient to cause brain metastasis. Taken together our data indicate that activation of Arf6 is sufficient to potentiate melanoma metastasis, consistent with the proinvasive cellular phenotype attributed to Arf6. In addition, our data suggests a novel signaling mechanism by which Arf6, a small GTPase involved in trafficking, is somehow involved in Akt activation and that this step may be important for the acquisition of metastatic capacity. Citation Format: Lehi Acosta, Aaron Rogers, Jae H. Yoo, Shannon J. Odelberg, Dean Y. Li, Sheri Holmen, Allie H. Grossmann. The small GTPase Arf6 potentiates melanoma metastasis by activating Akt [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 1357. doi:10.1158/1538-7445.AM2017-1357

Essential role for GABARAP autophagy proteins in interferon-inducible GTPase-mediated host defense.

Mammalian autophagy-related 8 (Atg8) homologs consist of LC3 proteins and GABARAPs, all of which are known to be involved in canonical autophagy. In contrast, the roles of Atg8 homologs in noncanonical autophagic processes are not fully understood. Here we show a unique role of GABARAPs, in particular gamma-aminobutyric acid (GABA)-A-receptor-associated protein-like 2 (Gabarapl2; also known as Gate-16), in interferon-γ (IFN-γ)-mediated antimicrobial responses. Cells that lacked GABARAPs but not LC3 proteins and mice that lacked Gate-16 alone were defective in the IFN-γ-induced clearance of vacuolar pathogens such as Toxoplasma. Gate-16 but not LC3b specifically associated with the small GTPase ADP-ribosylation factor 1 (Arf1) to mediate uniform distribution of interferon-inducible GTPases. The lack of GABARAPs reduced Arf1 activation, which led to formation of interferon-inducible GTPase-containing aggregates and hampered recruitment of interferon-inducible GTPases to vacuolar pathogens. Thus, GABARAPs are uniquely required for antimicrobial host defense through cytosolic distribution of interferon-inducible GTPases.

The Arf activator GBF1 localizes to plasma membrane sites involved in cell adhesion and motility.

GBF1 is ubiquitously expressed in eukaryotic cells and is essential for cellular and organismal life. Depletion of GBF1 from cultured cells induces apoptosis, while mouse GBF1 knockout and D. melan...

The small GTPase Arf6 regulates sea urchin morphogenesis

The small GTPase Arf6 is a conserved protein that is expressed in all metazoans. Arf6 remodels cytoskeletal actin and mediates membrane protein trafficking between the plasma membrane in its active form and endosomal compartments in its inactive form. While a rich knowledge exists for the cellular functions of Arf6, relatively little is known about its physiological role in development. This study examines the function of Arf6 in mediating cellular morphogenesis in early development. We dissect the function of Arf6 with a loss-of-function morpholino and constitutively active Arf6-Q67L construct. We focus on the two cell types that undergo active directed migration: the primary mesenchyme cells (PMCs) that give rise to the sea urchin skeleton and endodermal cells that form the gut. Our results indicate that Arf6 plays an important role in skeleton formation and PMC migration, in part due to its ability to remodel actin. We also found that embryos injected with Arf6 morpholino have gastrulation defects and embryos injected with constitutively active Arf6 have endodermal cells detached from the gut epithelium with decreased junctional cadherin staining, indicating that Arf6 may mediate the recycling of cadherin. Thus, Arf6 impacts cells that undergo coordinated movement to form embryonic structures in the developing embryo.

EFA6 regulates selective polarised transport and axon regeneration from the axon initial segment.

ABSTRACTCentral nervous system (CNS) axons lose their intrinsic ability to regenerate upon maturity, whereas peripheral nervous system (PNS) axons do not. A key difference between these neuronal types is their ability to transport integrins into axons. Integrins can mediate PNS regeneration, but are excluded from adult CNS axons along with their Rab11 carriers. We reasoned that exclusion of the contents of Rab11 vesicles including integrins might contribute to the intrinsic inability of CNS neurons to regenerate, and investigated this by performing laser axotomy. We identify a novel regulator of selective axon transport and regeneration, the ARF6 guanine-nucleotide-exchange factor (GEF) EFA6 (also known as PSD). EFA6 exerts its effects from a location within the axon initial segment (AIS). EFA6 does not localise at the AIS in dorsal root ganglion (DRG) axons, and in these neurons, ARF6 activation is counteracted by an ARF GTPase-activating protein (GAP), which is absent from the CNS, ACAP1. Depleting EFA6 from cortical neurons permits endosomal integrin transport and enhances regeneration, whereas overexpressing EFA6 prevents DRG regeneration. Our results demonstrate that ARF6 is an intrinsic regulator of regenerative capacity, implicating EFA6 as a focal molecule linking the AIS, signalling and transport.This article has an associated First Person interview with the first author of the paper.

Podosome assembly is controlled by the GTPase ARF1 and its nucleotide exchange factor ARNO.

Podosomes represent a class of integrin-mediated cell-matrix adhesions formed by migrating and matrix-degrading cells. We demonstrate that in macrophage-like THP1 cells and fibroblasts stimulated to produce podosomes, down-regulation of the G-protein ARF1 or the ARF1 guanine nucleotide exchange factor, ARNO, by small, interfering RNA or pharmacological inhibitors led to striking podosome elimination. Concomitantly, treatments inducing podosome formation increased the level of guanosine triphosphate (GTP)-bound ARF1. ARNO was found to colocalize with the adhesive rings of podosomes, whereas ARF1 was localized to vesicular structures transiently contacting podosome rings. Inhibition of ARF1 led to an increase in RhoA-GTP levels and triggered assembly of myosin-IIA filaments in THP1 cells, whereas the suppression of myosin-IIA rescued podosome formation regardless of ARF1 inhibition. Finally, expression of constitutively active ARF1 in fibroblasts induced formation of putative podosome precursors: actin-rich puncta coinciding with matrix degradation sites and containing proteins of the podosome core but not of the adhesive ring. Thus, ARNO-ARF1 regulates formation of podosomes by inhibition of RhoA/myosin-II and promotion of actin core assembly.

Proangiogenic TF-FVIIa-PAR2 Signaling Requires Matriptase-Independent Integrin Interaction

The close link between coagulation activation and cancer progression is supported by clinical and experimental studies. A central molecular pathways by which tumor cells interact with the hemostatic system is through the expression of the cell surface receptor tissue factor (TF) that in complex with coagulation factor VIIa (FVIIa) triggers the extrinsic pathway of blood coagulation, contributes to cancer associated thrombosis, and promotes direct tumor cell signaling through protease-activated receptors (PARs). Genetic and pharmacological evidence shows that epithelial and tumor cell TF-FVIIa signaling induces a diverse set of proangiogenic and immune modulatory cytokines, chemokines and growth factors. However, the mechanistic details of this signaling pathway remain incompletely understood. Furthermore, a recent study has suggested that the TF-FVIIa complex does not directly cleave PAR2, but rather induces PAR signaling through initiating a cell surface protease cascade involving the transmembrane serine protease matriptase. Here, we employ mutants of FVIIa to characterize the roles of cell surface proteolysis and endosomal signaling by TF in keratinocytes and cancer cells.We studied TF-expressing MDA-MB-231 breast cancer cells, A7 melanoma cells transduced with TF and PAR2, and non-tumorigenic HaCaT keratinocytes. Stimulation with 10 nM FVIIa or 5 nM recombinant matriptase promoted productive PAR2 signaling in all three cell types as assessed by quantification of cytokine IL-8 mRNA up-regulation. Addition of the serine protease inhibitor aprotinin abolished IL-8 upregulation by recombinant matriptase, but not by FVIIa. Importantly, matriptase was expressed by HaCaT and A7 melanoma cells, whereas MDA-MB-231 cells did not express matriptase, while responding to FVIIa with efficient proangiogenic signaling. HaCaT and A7 cells showed shedding of the matriptase protease domain following incubation with FVIIa, suggesting activation of the serine protease. However, the signaling-defective FVIIa Q40A mutant also promoted matriptase shedding without inducing IL-8. We had previously shown that FVIIa induces association of TF with integrin β1 hetorodimers. We characterized an integrin-binding deficient FVIIa mutant, E26A, rendered incapable to interact with active integrin β1, resulting in defective TF-FVIIa internalization, TF-FVIIa stimulated cell migration and induction of IL-8. FVIIa E26A promoted matriptase shedding as observed with wild-type FVIIa. We found that inhibition of the small rho GTPase arf6, which controls cellular trafficking of β1 integrins, by pharmacological inhibitors or expression of a dominant negative arf6 mutant prevented IL-8 induction by TF-FVIIa, but not by direct stimulation of PAR2 with agonist peptide. Pharmacological activation of arf6 or expression of arf6 constitutive active mutant on the other hand promoted translocation of TF-FVIIa, which unlike PAR2 agonist peptide induced delayed and prolonged MAPK activation downstream of PAR2. This data show that TF-FVIIa triggers PAR2 signaling dependent on integrin-arf6-regulated trafficking and prolonged activation of endosomal signaling.Our data support the conclusion that the FVIIa induced interaction of TF with integrins and endocytosis of the TF-FVIIa-integrin complex plays a pivotal role in inducing proangiogenic cytokines downstream of proteolytic cleavage of PAR2. Mutants of FVIIa that are defective in supporting this pathway fully promote cell surface matriptase activation, but fail to upregulate tumor cell proangiogenic responses or tumor cell migration. These results argue against a role for matriptase as an intermediary in TF-FVIIa induced PAR2 activation, but do not exclude that matriptase modulates homeostatic epithelial responses through PAR2 signaling in non-malignant cells. These data furthermore suggest that matriptase inhibitors cannot substitute for targeting TF as potential therapeutic strategy to attenuate cancer proangiogenic signaling of the hemostatic system DisclosuresOstergaard:Novo Nordisk A/S: Employment. Ruf:Iconic Therapeutics: Consultancy.

The phosphatidyl inositol 3 kinase pathway does not suppress activation of the ARF and BIM genes by deregulated E2F1 activity

The transcription factor E2F plays crucial roles in tumor suppression by activating pro-apoptotic genes such as the tumor suppressor ARF. The regulation of the ARF gene is distinct from that of growth-related E2F targets, in that it is specifically activated by deregulated E2F activity, induced by over-expression of E2F or forced inactivation of pRB, but not by physiological E2F activity induced by growth stimulation. The phosphatidyl inositol 3 kinase (PI3K) pathway was reported to suppress expression of some atypical pro-apoptotic genes by over-expressed E2F1. However, the effects of the PI3K pathway on the distinct regulation of typical pro-apoptotic E2F targets have not been elucidated. We examined whether the PI3K pathway suppressed activation of the typical pro-apoptotic E2F targets ARF and BIM. Activation of the PI3K pathway by growth stimulation or introduction of a constitutively active Akt/PKB did not reduce induction of ARF or BIM gene expression or activation of their promoters by over-expressed E2F1. These results suggest that the PI3K pathway does not suppress induction of typical pro-apoptotic genes that are selectively activated by deregulated E2F1.

GTPase cross talk regulates TRAPPII activation of Rab11 homologues during vesicle biogenesis.

Rab guanosine triphosphatases (GTPases) control cellular trafficking pathways by regulating vesicle formation, transport, and tethering. Rab11 and its paralogs regulate multiple secretory and endocytic recycling pathways, yet the guanine nucleotide exchange factor (GEF) that activates Rab11 in most eukaryotic cells is unresolved. The large multisubunit transport protein particle (TRAPP) II complex has been proposed to act as a GEF for Rab11 based on genetic evidence, but conflicting biochemical experiments have created uncertainty regarding Rab11 activation. Using physiological Rab-GEF reconstitution reactions, we now provide definitive evidence that TRAPPII is a bona fide GEF for the yeast Rab11 homologues Ypt31/32. We also uncover a direct role for Arf1, a distinct GTPase, in recruiting TRAPPII to anionic membranes. Given the known role of Ypt31/32 in stimulating activation of Arf1, a bidirectional cross talk mechanism appears to drive biogenesis of secretory and endocytic recycling vesicles. By coordinating simultaneous activation of two essential GTPase pathways, this mechanism ensures recruitment of the complete set of effectors needed for vesicle formation, transport, and tethering.

Machineries regulating the activity of the small GTPase Arf6 in cancer cells are potential targets for developing innovative anti-cancer drugs.

The Small GTPase ADP-ribosylation factor 6 (Arf6) functions as the molecular switch in cellular signaling pathways by cycling between GDP-bound inactive and GTP-bound active form, which is precisely regulated by two regulators, guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Numerous studies have shown that these machineries play critical roles in tumor angiogenesis/growth and cancer cell invasion/metastasis through regulating the cycling of Arf6. Here, we summarize accumulating knowledge for involvement of Arf6 GEFs/GAPs and small molecule inhibitors of Arf6 signaling/cycling in cancer progression, and discuss possible strategies for developing innovative anti-cancer drugs targeting Arf6 signaling/cycling.

Study of Ethanol-Induced Golgi Disorganization Reveals the Potential Mechanism of Alcohol-Impaired N-Glycosylation.

It is known that ethanol (EtOH) and its metabolites have a negative effect on protein glycosylation. The fragmentation of the Golgi apparatus induced by alteration of the structure of largest Golgi matrix protein, giantin, is the major consequence of damaging effects of EtOH-metabolism on the Golgi; however, the link between this and abnormal glycosylation remains unknown. Because previously we have shown that Golgi morphology dictates glycosylation, we examined the effect EtOH administration has on function of Golgi residential enzymes involved in N-glycosylation. HepG2 cells transfected with mouse ADH1 (VA-13 cells) were treated with 35mM EtOH for 72hours. Male Wistar rats were pair-fed Lieber-DeCarli diets for 5 to 8weeks. Characterization of Golgi-associated mannosyl (α-1,3-)-glycoprotein beta-1,2-N-acetylglucosaminyltransferase (MGAT1), α-1,2-mannosidase (Man-I), and α-mannosidase II (Man-II) were performed in VA-13 cells and rat hepatocytes followed by three-dimensional structured illumination microscopy (3D SIM). First, we detected that EtOH administration results in the loss of sialylated N-glycans on asialoglycoprotein receptor; however, the high-mannose-type N-glycans are increased. Further analysis by 3D SIM revealed that EtOH treatment despite Golgi disorganization does not change cis-Golgi localization for Man-I, but does induce medial-to-cis relocation of MGAT1 and Man-II. Using different approaches, including electron microscopy, we revealed that EtOH treatment results in dysfunction of ADP-ribosylation factor 1 (Arf1) GTPase followed by a deficiency in COPI vesicles at the Golgi. Silencing beta-COP or expression of GDP-bound mutant Arf1(T31N) mimics the EtOH effect on retaining MGAT1 and Man-II at the cis-Golgi, suggesting that (i) EtOH specifically blocks activation of Arf1, and (ii) EtOH alters the proper localization of Golgi enzymes through impairment of COPI. Importantly, the level of MGAT1 was reduced, because likely MGAT1, contrary to Man-I and Man-II, is giantin sensitive. Thus, we provide the mechanism by which EtOH-induced Golgi remodeling may significantly modify formation of N-glycans.

C9ORF72 interaction with cofilin modulates actin dynamics in motor neurons.

Intronic hexanucleotide expansions in C9ORF72 are common in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia, but it is unknown whether loss of function, toxicity by the expanded RNA or dipeptides from non-ATG-initiated translation are responsible for the pathophysiology. We determined the interactome of C9ORF72 in motor neurons and found that C9ORF72 was present in a complex with cofilin and other actin binding proteins. Phosphorylation of cofilin was enhanced in C9ORF72-depleted motor neurons, in patient-derived lymphoblastoid cells, induced pluripotent stem cell-derived motor neurons and post-mortem brain samples from ALS patients. C9ORF72 modulates the activity of the small GTPases Arf6 and Rac1, resulting in enhanced activity of LIM-kinases 1 and 2 (LIMK1/2). This results in reduced axonal actin dynamics in C9ORF72-depleted motor neurons. Dominant negative Arf6 rescues this defect, suggesting that C9ORF72 acts as a modulator of small GTPases in a pathway that regulates axonal actin dynamics.