ARF Protein Research Articles

Arf1-dependent LRBA recruitment to Rab4 endosomes is required for endolysosome homeostasis.

Deleterious mutations in the lipopolysaccharide responsive beige-like anchor protein (LRBA) gene cause severe childhood immune dysregulation. The complexity of the symptoms involving multiple organs and the broad range of unpredictable clinical manifestations of LRBA deficiency complicate the choice of therapeutic interventions. Although LRBA has been linked to Rab11-dependent trafficking of the immune checkpoint protein CTLA-4, its precise cellular role remains elusive. We show that LRBA, however, only slightly colocalizes with Rab11. Instead, LRBA is recruited by members of the small GTPase Arf protein family to the TGN and to Rab4+ endosomes, where it controls intracellular traffic. In patient-derived fibroblasts, loss of LRBA led to defects in the endosomal pathway promoting the accumulation of enlarged endolysosomes and lysosome secretion. Thus, LRBA appears to regulate flow through the endosomal system on Rab4+ endosomes. Our data strongly suggest functions of LRBA beyond CTLA-4 trafficking and provide a conceptual framework to develop new therapies for LRBA deficiency.

Quantitative imaging reveals the role of MpARF proteasomal degradation during gemma germination

The auxin signaling molecule controls a variety of growth and developmental processes in land plants. Auxin regulates gene expression through a nuclear auxin signaling pathway (NAP) consisting of the ubiquitin ligase auxin receptor TIR1/AFB, its Aux/IAA degradation substrate, and DNA-binding ARF transcription factors. Although extensive qualitative understanding of the pathway and its interactions has been obtained, mostly by studying the flowering plant Arabidopsis thaliana, it remains unknown how these translate to quantitative system behavior in vivo, a problem that is confounded by the large NAP gene families in most species. Here, we used the minimal NAP of the liverwort Marchantia polymorpha to quantitatively map NAP protein accumulation and dynamics in vivo through the use of knockin fluorescent fusion proteins. Beyond revealing the dynamic native accumulation profile of the entire NAP protein network, we discovered that the two central ARFs, MpARF1 and MpARF2, are proteasomally degraded. This auxin-independent degradation tunes ARF protein stoichiometry to favor gene activation, thereby reprogramming auxin response during the developmental progression. Thus, quantitative analysis of the entire NAP has enabled us to identify ARF degradation and the stoichiometries of activator and repressor ARFs as a potential mechanism for controlling gemma germination.

Toxoplasma and Plasmodium associate with host Arfs during infection.

The parasites Toxoplasma gondii and Plasmodium live complex intracellular lifestyles where they must acquire essential host nutrients while avoiding recognition. Although previous work has sought to identify the specific nutrients scavenged by apicomplexans, the mechanisms by which host materials are transported to and across the parasite vacuole membrane are largely unknown. Here, we examined members of the host vesicular trafficking network to identify specific pathways subverted by T. gondii and Plasmodium berghei. Our results indicate that T. gondii selectively internalizes host Arfs, a class of proteins involved in intracellular trafficking. For P. berghei, host Arfs were restricted by the parasite's vacuole membrane, but proteins involved in vesicular trafficking were identified as essential for liver stage development. A greater exploration into how and why apicomplexans subvert host vesicular trafficking could help identify targets for host-directed therapeutics.

Identification of ARF genes in Cucurbita pepo L and analysis of expression patterns, and functional analysis of CpARF22 under drought, salt stress

BackgroundAuxin transcription factor (ARF) is an important transcription factor that transmits auxin signals and is involved in plant growth and development as well as stress response. However, genome-wide identification and responses to abiotic and pathogen stresses of the ARF gene family in Cucurbita pepo L, especially pathogen stresses, have not been reported.ResultsFinally, 33 ARF genes (CpARF01 to CpARF33) were identified in C.pepo from the Cucurbitaceae genome database using bioinformatics methods. The putative protein contains 438 to 1071 amino acids, the isoelectric point is 4.99 to 8.54, and the molecular weight is 47759.36 to 117813.27 Da, the instability index ranged from 40.74 to 68.94, and the liposoluble index ranged from 62.56 to 76.18. The 33 genes were mainly localized in the nucleus and cytoplasm, and distributed on 16 chromosomes unevenly. Phylogenetic analysis showed that 33 CpARF proteins were divided into 6 groups. According to the amino acid sequence of CpARF proteins, 10 motifs were identified, and 1,3,6,8,10 motifs were highly conserved in most of the CpARF proteins. At the same time, it was found that genes in the same subfamily have similar gene structures. Cis-elements and protein interaction networks predicted that CpARF may be involved in abiotic factors related to the stress response. QRT-PCR analysis showed that most of the CpARF genes were upregulated under NaCl, PEG, and pathogen treatment compared to the control. Subcellular localization showed that CpARF22 was localized in the nucleus. The transgenic Arabidopsis thaliana lines with the CpARF22 gene enhanced their tolerance to salt and drought stress.ConclusionIn this study, we systematically analyzed the CpARF gene family and its expression patterns under drought, salt, and pathogen stress, which improved our understanding of the ARF protein of zucchini, and laid a solid foundation for functional analysis of the CpARF gene.

High-pressure studies reveal the conformational landscape of the small GTPase Arf6.

Arf6, a small GTPase, functions as a protein “switch” by coupling the binding and subsequent hydrolysis of GTP to a variety of signaling cascades within the cell. Despite having significant degrees of sequence and structural homology in both the GDP and GTP-bound forms to the more well-characterized protein Arf1, the two proteins exhibit distinct spatial and functional segregation: Arf1 is responsible for membrane trafficking between the Endoplasmic Reticulum and Golgi Apparatus, while Arf6 is responsible for trafficking between the plasma membrane and endosome. Furthermore, the accessory proteins required for in vivo functionality of the switch are not interchangeable. Arf proteins are known to undergo massive structural rearrangements during the nucleotide switch transition, especially in the N-terminal and switch regions. Thus, it is unlikely that Arf protein binding partners’ specificity stems purely from the end state structures. We hypothesize that the excited conformations sampled during this transition are responsible for the observed specificity. To characterize these excited conformational states, our lab utilizes pressure-induced perturbation techniques which locally unfold the protein. We gain insights into the state of the protein using a two-pronged approach consisting of both high-pressure NMR and high-pressure SAXS; this has allowed us to develop residue-specific unfolding curves for Arf6 as well as pair-distance distribution curves to understand the global shape of the protein at various pressures. Using these results, we have begun to structurally map the conformational landscape of Arf6.

Molecular Process of Stem Cells in Modern Biology

Stem cells have a wide range of traits, including the ability to self-renewal and differential interactions between various cell types that vary with their location within tissue and their immediate surroundings. Inhibitors of both are influenced by the cell cycle, genes involved in chromosomal rearrangements, critical developmental proteins, and signaling pathways. Wnt, Notch, Hedgehog Pathways, BMI-1, OCT3/4, ARF, NANOG, p16Ink4a, and this includes HOXB4, SOX2, and their homologous paralog, govern self-renewal. Stem cells and their molecular pathways that regulate self-renewal and differentiation hold great promise for theoretical and practical studies. Keyword: gene regulation of stem cell self-renewal and differentiation genes for BMI-1, NANOG, OCT3, OCT4, p16Ink4a, SOX2, microenvironment, ARF protein proteins 1 (hedgehog), proteins 2 (Wnt.), and receptors 3 (notch).

Helicobacter pylori pathogen inhibits cellular responses to oncogenic stress and apoptosis.

Helicobacter pylori (H. pylori) is a common gastric pathogen that infects approximately half of the world’s population. Infection with H. pylori can lead to diverse pathological conditions, including chronic gastritis, peptic ulcer disease, and cancer. The latter is the most severe consequence of H. pylori infection. According to epidemiological studies, gastric infection with H. pylori is the strongest known risk factor for non-cardia gastric cancer (GC), which remains one of the leading causes of cancer-related deaths worldwide. However, it still remains to be poorly understood how host-microbe interactions result in cancer development in the human stomach. Here we focus on the H. pylori bacterial factors that affect the host ubiquitin proteasome system. We investigated E3 ubiquitin ligases SIVA1 and ULF that regulate p14ARF (p19ARF in mice) tumor suppressor. ARF plays a key role in regulation of the oncogenic stress response and is frequently inhibited during GC progression. Expression of ARF, SIVA1 and ULF proteins were investigated in gastroids, H. pylori-infected mice and human gastric tissues. The role of the H. pylori type IV secretion system was assessed using various H. pylori isogenic mutants. Our studies demonstrated that H. pylori infection results in induction of ULF, decrease in SIVA1 protein levels, and subsequent ubiquitination and degradation of p14ARF tumor suppressor. Bacterial CagA protein was found to sequentially bind to SIVA1 and ULF proteins. This process is regulated by CagA protein phosphorylation at the EPIYA motifs. Downregulation of ARF protein leads to inhibition of cellular apoptosis and oncogenic stress response that may promote gastric carcinogenesis.

Activation of glucagon-like peptide-1 receptor in microglia attenuates neuroinflammation-induced glial scarring via rescuing Arf and Rho GAP adapter protein 3 expressions after nerve injury.

Rationale: The neuroinflammation is necessary for glial group initiation and clearance of damaged cell debris after nerve injury. However, the proinflammatory polarization of excessive microglia amplifies secondary injury via enhancing cross-talk with astrocytes and exacerbating neurological destruction after spinal cord injury (SCI). The glucagon-like peptide-1 receptor (GLP-1R) agonist has been previously shown to have a neuroprotective effect in neurodegeneration, whereas its potency in microglial inflammation after SCI is still unknown.Methods: The effect and mechanism of GLP-1R activation by exendin-4 (Ex-4) were investigated in in vitro cultured glial groups and in vivo in SCI mice. Alterations in the gene expression after GLP-1R activation in inflammatory microglia were measured using mRNA sequencing. The microglial polarization, neuroinflammatory level, and astrocyte reaction were detected by using western blotting, flow cytometry, and immunofluorescence. The recoveries of neurological histology and function were also observed using imaging and ethological examinations.Results: GLP-1R activation attenuated microglia-induced neuroinflammation by reversing M1 subtypes to M2 subtypes in vitro and in vivo. In addition, activation of GLP-1R in microglia blocked production of reactive astrocytes. We also found less neuroinflammation, reactive astrocytes, corrected myelin integrity, ameliorated histology, and improved locomotor function in SCI mice treated with Ex-4. Mechanistically, we found that Ex-4 rescued the RNA expression of Arf and Rho GAP adapter protein 3 (ARAP3). Knockdown of ARAP3 in microglia reversed activation of RhoA and the pharmacological effect of Ex-4 on anti-inflammation in vitro.Conclusion: Ex-4 exhibited a previously unidentified role in reducing reactive astrocyte activation by mediation of the PI3K/ARAP3/RhoA signaling pathway, by neuroinflammation targeting microglia, and exerted a neuroprotective effect post-SCI, implying that activation of GLP-1R in microglia was a therapeutical option for treatment of neurological injury.

Arf GTPases Are Required for the Establishment of the Pre-Assembly Compartment in the Early Phase of Cytomegalovirus Infection.

Shortly after entering the cells, cytomegaloviruses (CMVs) initiate massive reorganization of cellular endocytic and secretory pathways, which results in the forming of the cytoplasmic virion assembly compartment (AC). We have previously shown that the formation of AC in murine CMV- (MCMV) infected cells begins in the early phase of infection (at 4–6 hpi) with the pre-AC establishment. Pre-AC comprises membranes derived from the endosomal recycling compartment, early endosomes, and the trans-Golgi network, which is surrounded by fragmented Golgi cisterns. To explore the importance of Arf GTPases in the biogenesis of the pre-AC, we infected Balb 3T3 cells with MCMV and analyzed the expression and intracellular localization of Arf proteins in the early phases (up to 16 hpi) of infection and the development of pre-AC in cells with a knockdown of Arf protein expression by small interfering RNAs (siRNAs). Herein, we show that even in the early phase, MCMVs cause massive reorganization of the Arf system of the host cells and induce the over-recruitment of Arf proteins onto the membranes of pre-AC. Knockdown of Arf1, Arf3, Arf4, or Arf6 impaired the establishment of pre-AC. However, the knockdown of Arf1 and Arf6 also abolished the establishment of infection. Our study demonstrates that Arf GTPases are required for different steps of early cytomegalovirus infection, including the establishment of the pre-AC.

Comprehensive analysis of the regulatory network of differentially expressed mRNAs, lncRNAs and circRNAs in gastric cancer

Gastric cancer (GC) is one of the most common types of human cancers. However, the mechanisms underlying GC remained largely unclear. To determine whether the differentially expressed mRNAs, lncRNAs and circRNAs in GC, we screened conducted SBC-ceRNA microarray analysis in 3 pairs of GC and normal tissues. Furthermore, differentially expressed mRNAs mediated protein protein interaction (PPI) networks, lncRNAs mediated cis-regulatory network, and circRNA mediated ceRNA network were for the first time constructed to reveal their potential functions and mechanisms in GC. Quantitative real-time polymerase chain reaction analysis (qRT-PCR) was conducted to validate the microarray analysis. A total of 922 mRNAs, 2112 lncRNAs and 2896 circRNAs were observed to be dysregulated in GC samples. Bioinformatics analysis showed these differentially expressed genes (DEGs) were significantly associated with regulating branched − chain amino acid catabolic process, Glycolysis/Gluconeogenesis and ARF protein signal transduction. Moreover, we found the dysregulation of key mRNAs and lncRNAs were associated with the overall survival time in GC patients. We believe this study provides useful information for understanding the mechanism underlying GC progression and exploring potential therapeutic and prognostic targets for GC.

Identification of De Novo Enhancers Activated by TGFβ to Drive Expression of CDKN2A and B in HeLa Cells.

Disruption of the CDKN2A (INK4A/ARF) and B (INK4B) genes, which encode three function-independent tumor suppressors, is one of the most common events in human cancer. Because their relative importance in tumor prevention appears to be species- and context-specific, studying their regulation can shed light on mechanisms by which they are bypassed in malignant transformation. We previously unveiled a new pathway in which TGFβ selectively induces Arf at mouse Cdkn2a in eye development and cultured fibroblasts. As TGFβ signaling is often derailed in cancer development or progression, we investigated its control of CDKN2A/B in human cancer. Computational analyses of sequencing and array data from nearly 11,000 patients with cancer in TCGA showed discordant expression of ARF and INK4A in most cancer subtypes, with gene copy-number loss and promoter methylation involved in only a subset. Using HeLa cells as a model, we found that exogenous TGFβ induced ARF mRNA and protein, and ARF knockdown limited TGFβ-mediated growth suppression. TGFβ-mediated ARF mRNA induction required SMAD2/3, p38MAPK, and SP1, and ARF mRNA was induced without added RNAPII recruitment. Chromatin immunoprecipitation unveiled a remote enhancer element engaged by TGFβ by a mechanism that partially depended on p38MAPK. CRISPR-based editing of this enhancer limited induction of ARF and INK4B by TGFβ, but not by oncogenic RAS. IMPLICATIONS: Our findings reveal new molecular mechanisms by which CDKN2A/B regulation is coupled to external cues, and those findings represent entry points to further explore pharmacologic strategies to restore their expression in cancer.

TZAP plays an inhibitory role in the self-renewal of porcine mesenchymal stromal cells and is implicated the regulation of premature senescence via the p53 pathway

BackgroundMesenchymal stromal cells (MSCs) were originally characterized by the ability to differentiate into different mesenchymal lineages in vitro, and their immunomodulatory and trophic functions have recently aroused significant interest in the application of MSCs in cell-based regenerative medicine. However, a major problem in clinical practice is the replicative senescence of MSCs, which limits the cell proliferation potential of MSCs after large-scale expansion. Telomeric zinc finger-associated protein (TZAP), a novel specific telomere-binding protein, was recently found to stimulate telomere trimming and prevent excessive telomere elongation. The aim of this study was to elucidate the role of TZAP in regulating MSCs senescence, differentiation and proliferation.MethodPrimary porcine mesenchymal stromal cells (pMSCs) were isolated from the bone marrow of Tibet minipigs by a noninvasive method in combination with frequent medium changes (FMCs). The deterioration of the pMSCs’ proliferation capacity and their resultant entry into senescence were analyzed by using CCK8 and EdU incorporation assays, SA-β-gal staining and comparisons of the expression levels of cellular senescence markers (p16INK14 and p21) in pMSC cell lines with TZAP overexpression or knockout. The effects of TZAP overexpression or knockout on the differentiation potential of pMSCs were assessed by alizarin red S staining after osteogenic induction or by oil red O staining after adipogenic induction. The effect of TZAP overexpression and the involvement of the p53 signaling pathway were evaluated by detecting changes in ARF, MDM2, P53 and P21 protein levels in pMSCs.ResultsTZAP levels were significantly elevated in late-passage pMSCs compared to those in early-passage pMSCs. We also observed significantly increased levels of the senescence markers p16INK4A and p21. Overexpression of TZAP reduced the differentiation potential of the cells, leading to premature senescence in early-passage pMSCs, while knockout of TZAP led to the opposite phenotype in late-passage pMSCs. Furthermore, overexpression of TZAP activated the P53 pathway (ARF-MDM2-P53-P21WAF/CDKN1A) in vitro. TZAP also downregulated the expression levels of PPARγ and Cebpα, two key modulators of adipogenesis.ConclusionsThis study demonstrates that the level of TZAP is closely related to differentiation potential in pMSCs and affects cellular senescence outcomes via the p53 pathway. Therefore, attenuation of intracellular TZAP levels could be a new strategy for improving the efficiency of pMSCs in cell therapy and tissue engineering applications.

Dual Role of the Alternative Reading Frame ARF Protein in Cancer.

The CDKN2a/ARF locus expresses two partially overlapping transcripts that encode two distinct proteins, namely p14ARF (p19Arf in mouse) and p16INK4a, which present no sequence identity. Initial data obtained in mice showed that both proteins are potent tumor suppressors. In line with a tumor-suppressive role, ARF-deficient mice develop lymphomas, sarcomas, and adenocarcinomas, with a median survival rate of one year of age. In humans, the importance of ARF inactivation in cancer is less clear whereas a more obvious role has been documented for p16INK4a. Indeed, many alterations in human tumors result in the elimination of the entire locus, while the majority of point mutations affect p16INK4a. Nevertheless, specific mutations of p14ARF have been described in different types of human cancers such as colorectal and gastric carcinomas, melanoma and glioblastoma. The activity of the tumor suppressor ARF has been shown to rely on both p53-dependent and independent functions. However, novel data collected in the last years has challenged the traditional and established role of this protein as a tumor suppressor. In particular, tumors retaining ARF expression evolve to metastatic and invasive phenotypes and in humans are associated with a poor prognosis. In this review, the recent evidence and the molecular mechanisms of a novel role played by ARF will be presented and discussed, both in pathological and physiological contexts.

Sexually dimorphic tumor suppression by small mitochondrial Arf.

Internal translational initiation of the mRNA encoding the Arf tumor suppressor yields an N-terminally truncated small Arf protein (smArf) that lacks amino acid residues required for Mdm2 binding and p53 activation. Here, we report that female, but not male, mice engineered to produce only smArf in lieu of the full-length Arf protein retain residual, sexually dimorphic tumor suppressive activity.

Tumor suppressor activity of miR-451: Identification of CARF as a new target

microRNAs (miRs) have recently emerged as small non-coding regulators of gene expression. We performed a loss-of-function screening by recruiting retrovirus mediated arbitrary manipulation of genome coupled with escape of cells from 5-Aza-2′-deoxycytidine (5-Aza-dC)-induced senescence. miRNA pool from cells that emerged from 5-Aza-dC-induced senescence was subjected to miR-microarray analysis with respect to the untreated control. We identified miR-451 as one of the upregulated miRs and characterized its functional relevance to drug resistance, cell growth, tumor suppressor proteins p53 and pRb, and stress response. We report that miR-451 caused growth arrest in cells leading to their resistance to 5-Aza-dC-induced senescence. Decrease in cyclin D1, CDK4 and phosphorylated pRB supported the growth arrest in miR-451 transfected cells. We demonstrate that Collaborator of ARF (CARF) protein is a new target of miR-451 that intermediates its function in tumor suppressor and stress signaling.

High prevalence of antibodies to core+1/ARF protein in HCV-infected patients with advanced cirrhosis.

Hepatitis C virus (HCV) possesses a second open reading frame (ORF) within the core gene encoding an additional protein, known as the alternative reading frame protein (ARFP), F or core+1. The biological significance of the core+1/ARF protein remains elusive. However, several independent studies have shown the presence of core+1/ARFP antibodies in chronically HCV-infected patients. Furthermore, a higher prevalence of core+1/ARFP antibodies was detected in patients with HCV-associated hepatocellular carcinoma (HCC). Here, we investigated the incidence of core+1/ARFPantibodies in chronically HCV-infected patients at different stages of cirrhosis in comparison to chronically HCV-infected patients at earlier stages of disease. Using ELISA, we assessed the prevalence of anti-core+1 antibodies in 30 patients with advanced cirrhosis [model for end-stage liver disease (MELD) ≥15] in comparison with 50 patients with mild cirrhosis (MELD <15) and 164 chronic HCV patients without cirrhosis. 28.7 % of HCV patients with cirrhosis were positive for anti-core+1 antibodies, in contrast with 16.5 % of non-cirrhotic HCV patients. Moreover, there was significantly higher positivity for anti-core+1 antibodies in HCV patients with advanced cirrhosis (36.7 %) compared to those with early cirrhosis (24 %) (P<0.05). These findings, together with the high prevalence of anti-core+1 antibodies in HCV patients with HCC, suggest that core+1 protein may have a role in virus-associated pathogenesis, and provide evidence to suggest that the levels of anti-core+1 antibodies may serve as a marker for disease progression.

Abstract 3175: Context-dependent role of ARF for response to chemotherapy in muscle invasive bladder cancer

Abstract Muscle invasive bladder cancer (MIBC) is a significant cause of cancer death, in part because there are relatively few treatments and these have limited efficacy. Our studies show that expression of 14ARF (aka ARF), one of the major transcripts encoded by the CDKN2A tumor suppressor locus, is frequently up-regulated in MIBC, particularly in more aggressive disease subtypes. Our results further show that accumulation of ARF protein in the nucleolus is associated with poor outcome and reduced response to chemotherapy. Using both genetically-engineered mouse (GEM) models and human xenograft models of MIBC, we demonstrate that tumors expressing ARF display poor response to treatment with the platinum-based chemotherapy agent cisplatin. This is mediated in part by expression of the integrin-binding protein ITGB3BP (CENPR) and reflects ARF-dependent inhibition of protein translation, which is attenuated by drug treatment. Our findings reveal an unexpected context-dependent role for ARF in modulating drug response in bladder cancer through its ability to regulate protein translation. Citation Format: Lijie Rong, Tomasz Owczarek, Takashi Kobayashi, Cory Abate-Shen. Context-dependent role of ARF for response to chemotherapy in muscle invasive bladder cancer [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 3175. doi:10.1158/1538-7445.AM2017-3175

Small mitochondrial Arf (smArf) protein corrects p53-independent developmental defects of Arf tumor suppressor-deficient mice

The mouse p19Arf (human p14ARF) tumor suppressor protein, encoded in part from an alternative reading frame of the Ink4a (Cdkn2a) gene, inhibits the Mdm2 E3 ubiquitin ligase to activate p53. Arf is not expressed in most normal tissues of young mice but is induced by high thresholds of aberrant hyperproliferative signals, thereby activating p53 in incipient tumor cells that have experienced oncogene activation. The single Arf mRNA encodes two distinct polypeptides, including full-length p19Arf and N-terminally truncated and unstable p15smArf ("small mitochondrial Arf") initiated from an internal in-frame AUG codon specifying methionine-45. Interactions of p19Arf with Mdm2, or separately with nucleophosmin (NPM, B23) that localizes and stabilizes p19Arf within the nucleolus, require p19Arf N-terminal amino acids that are not present within p15smArf We have generated mice that produce either smARF alone or M45A-mutated (smArf-deficient) full-length p19Arf proteins. BCR-ABL-expressing pro/pre-B cells producing smArf alone are as oncogenic as their Arf-null counterparts in generating acute lymphoblastic leukemia when infused into unconditioned syngeneic mice. In contrast, smArf-deficient cells from mice of the ArfM45A strain are as resistant as wild-type Arf+/+ cells to comparable oncogenic challenge and do not produce tumors. Apart from being prone to tumor development, Arf-null mice are blind, and their male germ cells exhibit defects in meiotic maturation and sperm production. Although ArfM45A mice manifest the latter defects, smArf alone remarkably rescues both of these p53-independent developmental phenotypes.

P14ARF interacts with the focal adhesion kinase and protects cells from anoikis

The ARF protein functions as an important sensor of hyper-proliferative stimuli restricting cell proliferation through both p53-dependent and -independent pathways. Although to date the majority of studies on ARF have focused on its anti-proliferative role, few studies have addressed whether ARF may also have pro-survival functions. Here we show for the first time that during the process of adhesion and spreading ARF re-localizes to sites of active actin polymerization and to focal adhesion points where it interacts with the phosphorylated focal adhesion kinase. In line with its recruitment to focal adhesions, we observe that hampering ARF function in cancer cells leads to gross defects in cytoskeleton organization resulting in apoptosis through a mechanism dependent on the Death-Associated Protein Kinase. Our data uncover a novel function for p14ARF in protecting cells from anoikis that may reflect its role in anchorage independence, a hallmark of malignant tumor cells.

Characterization of ADP ribosylation factor 1 gene from Exopalaemon carinicauda and its immune response to pathogens challenge and ammonia-N stress

ADP ribosylation factors (Arf), as highly conserved small guanosine triphosphate (GTP)-binding proteins, participates in intracellular trafficking and organelle structure. In this study, a full-length cDNA of Arf1 (designated EcArf1) was cloned from Exopalaemon carinicauda by using rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of EcArf1 was 1428bp, which contains an open reading frame (ORF) of 549bp, encoding a 182 amino-acid polypeptide with the predicted molecular weight of 20.69kDa and estimated isoelectric point was 7.24. Sequence analysis revealed that the conserved Arf protein family signatures were identified in EcArf1. The deduced amino acid sequence of EcArf1 shared high identity (95%-98%) with that of other species and clustered together with Arf1 of other shrimp in the NJ phylogenetic tree, indicating that EcArf1 should be a member of the Arf1 family. Quantitative real-time RT-qPCR analysis indicated that EcArf1 was expressed in hemocytes, hepatopancreas, gills, muscle, ovary, intestine, stomach and heart, and the most abundant level was in hemocytes and gills, which were also the two main target tissues of pathogen infection and environmental stress. After Vibrio parahaemolyticus challenge, EcArf1 transcripts level significantly increased in hemocytes and hepatopancreas at 3h and 6h, respectively. The expression of EcArf1 in hemocytes and hepatopancreas significantly up-regulated at 12h and 6h respectively, and down-regulated at 72h and 48h, respectively. EcArf1 expression in hepatopancreas and gills both significantly increased at 6h and decreased at 24h under ammonia-N stress. The results suggested that EcArf1 might be involved in immune responses to pathogens (V.parahaemolyticus and WSSV) challenge and ammonia-N stress in E.carinicauda.