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PubMed2005

Assays and properties of arfaptin 2 binding to Rac1 and ADP-ribosylation factors (Arfs).

Arfaptin 1 and 2 were identified as targets for GTP bound ADP-ribosylation factors (Arfs). Arfaptin 1 had no significant effects on guanine nucleotide binding to Arfs, nor enzymatic activities of guanine nucleotide exchange factor (GEF) and GTPase activating protein (GAP) acting on Arfs. However, arfaptin 1 inhibited Arf activation of cholera toxin and phospholipase D (PLD) in a dose-dependent manner. Only GTP-bound forms of Arf1, 5, and 6 interacted with arfaptin 1 and 2, but GTP-Arf1 showed the strongest binding to the arfaptins. In contrast to the binding of Arfs to arfaptins, GDP-Rac1 or dominant negative Rac1-N17N bound to arfaptin 2, whereas GTP-Rac1 or dominant active Rac1-Q61L did not bind to arfaptin 2. Neither GTP-Rac1 nor GDP-Rac1 bound to arfaptin 1. Based on our observation, we propose that arfaptin 2 is a target for GDP-Rac1 and for GTP-Arf1, and is involved in interactions between the Rac1 and Arfs signaling pathways. This chapter describes methods for investigating the interactions of arfaptins 1 and 2 with GTP- or GDP-liganded Arfs and Rac1.

ADP-Ribosylation Factors↗
PubMed2001

Cloning and expression analyses of cDNA encoding an ADP-ribosylation factor from wheat: tissue-specific expression of wheat ARF.

We isolated and characterized a cDNA clone encoding a small GTP-binding protein, which has a high similarity to mammalian ADP-ribosylation factor (ARF), from a cDNA library prepared from immature spikes of wheat (Triticum aestivum L.). The cDNA contained an open reading frame that encodes a polypeptide of 181 amino acids with a calculated molecular mass of 20.7 kDa. The deduced amino acid sequence showed a quite high homology to known ARFs from other organisms. In particular, the wheat ARF was completely identical to the rice ARF1. Genomic Southern hybridization suggested that wheat ARF is encoded by at least two or three copies of ARF genes. Northern analyses showed that the accumulation of the ARF transcripts was nearly constant throughout various environmental stresses in both shoots and roots. However the RNA transcript was preferentially expressed in roots rather than in shoots. A similar expression pattern was also observed at the protein level by Western analysis. The relative abundance of the ARF proteins in root and flower tissues may indicate a high level of vesicular transporting activity in the roots and flowers of wheat plants.

Journal Article↗
PubMed2003

Reasons for non-enrollment in a cohort study of ARF: the Program to Improve Care in Acute Renal Disease (PICARD) experience and implications for a clinical trials network.

BACKGROUND: Acute renal failure (ARF) is associated strongly with in-hospital mortality and morbidity. Previous clinical trials of ARF have been hampered by the heterogeneous population affected, difficulty defining ARF, delays in identification of ARF, and significant comorbid conditions, among other factors. METHODS: The Program to Improve Care in Acute Renal Disease (PICARD) phase I was a multicenter cohort study aimed to identify clinical characteristics and practice patterns associated with adverse and favorable outcomes in patients with ARF in intensive care units. Although PICARD used no interventions, signed informed consent was required of all study subjects or their proxies. RESULTS: Signed informed consent was obtained in 645 of 1,243 ARF episodes (52%). The fraction of patients not enrolled and reasons for non-enrollment varied widely across the 5 PICARD centers. Refusal by potential study subjects was infrequent, although the absence of family or proxy (15%) and refusal by family or proxy (18%) accounted for large fractions of non-enrolled subjects. Death (23%) and discharge (11%) before study personnel could evaluate patients were additional important reasons for non-enrollment. CONCLUSION: Understanding reasons for non-enrollment may help rationalize mortality and other outcome differences seen in clinical trials and cohort studies that require informed consent compared with historic reports of "all comers" with ARF.

Acute Kidney Injury↗
PubMed2002

ARAP1: a point of convergence for Arf and Rho signaling.

We have identified ARAP1 and ARAP2 and examined ARAP1 as a possible link between phosphoinositide-, Arf-, and Rho-mediated cell signaling. ARAP1 contains Arf GAP, Rho GAP, Ankyrin repeat, Ras-associating, and five PH domains. In vitro, ARAP1 had Rho GAP and phosphatidylinositol (3,4,5) trisphosphate (PIP3)-dependent Arf GAP activity. ARAP1 associated with the Golgi. The Rho GAP activity mediated cell rounding and loss of stress fibers when ARAP1 was overexpressed. The Arf GAP activity mediated changes in the Golgi apparatus and the formation of filopodia, the latter a consequence of increased cellular activity of Cdc42. The Arf GAP and Rho GAP activities both contributed to inhibiting cell spreading. Thus, ARAP1 is a PIP3-dependent Arf GAP that regulates Arf-, Rho-, and Cdc42-dependent cell activities.

3T3 Cells↗
PubMed1992

Inhibition by brefeldin A of a Golgi membrane enzyme that catalyses exchange of guanine nucleotide bound to ARF.

A wide variety of membrane transformations important in intracellular transport are inhibited by the fungal metabolite brefeldin A (refs 1-4), implying that the target for this drug is central to the formation and maintenance of subcellular compartments. Brefeldin A added to cells causes the rapid and reversible dissociation of a Golgi-associated peripheral membrane protein (M(r) 110,000) which was found to be identical to one of the subunits of the coat of Golgi-derived (non-clathrin) coated vesicles, beta-COP, implying that brefeldin A prevents transport by blocking the assembly of coats and thus the budding of enclosed vesicles. In addition to the coatomer (a cytosol-derived complex of seven polypeptide chains, one of which is beta-COP), the non-clathrin (COP) coat of Golgi-derived vesicles contains stoichiometric amounts of a small (M(r) approximately 20,000) GTP-binding protein, the ADP-ribosylation factor (ARF). Binding of ARF to Golgi membranes is necessary before coatomer/beta-COP can bind these membranes (ref. 12; and D. J. Palmer et al., manuscript submitted), so the primary effect of brefeldin A seems to be on the reaction responsible for ARF binding. Indeed, like beta-COP, ARF is dissociated from the Golgi complex by treatment with brefeldin A and brefeldin A prevents ARF from associating in vitro, but the mechanism of this action by brefeldin A has been unclear. Here we report the discovery of an enzyme in a Golgi-enriched fraction that catalyses guanine nucleotide (GDP-GTP) exchange on ARF-1 protein, and which is inhibited by brefeldin A. We suggest that activation of ARF proteins for membrane localization by compartmentalized exchange enzymes is in general the first committed step in membrane transformation pathways.

ADP-Ribosylation Factors↗
PubMed2004

Germline mutations of the INK4a-ARF gene in patients with suspected genetic predisposition to melanoma.

Germline anomalies of the INK4a-ARF and Cdk4 genes were sought in a series of 89 patients suspected of having a genetic predisposition to melanoma. Patients were selected based on the following criteria: (a) familial melanoma (23 cases), (b) multiple primary melanoma (MPM; 18 cases), (c) melanoma and additional unrelated cancers (13 cases), (d) age at diagnosis less than 25 years (21 cases), and (e) nonphoto-induced melanoma (NPIM; 14 cases). Mutations of INK4a-ARF and Cdk4 were characterised by automated sequencing, and germline deletions of INK4a-ARF were also examined by real-time quantitative PCR. Seven germline changes of INK4a-ARF, five of which were novel, were found in seven patients (8%). Four were very likely to be pathogenic mutations and were found in three high-risk melanoma families and in a patient who had a pancreatic carcinoma in addition to melanoma. Three variants of uncertain significance were detected in one MPM patient, one patient <25 years, and one NPIM patient. No germline deletion of INK4a-ARF was found in 71 patients, and no Cdk4 mutation was observed in the 89 patients. This study confirms that INK4a-ARF mutations are infrequent outside stringent familial criteria, and that germline INK4a-ARF deletions are rarely involved in genetic predisposition to melanoma.

Adolescent↗
PubMed2006

ARF-BP1 as a potential therapeutic target.

In this review, we discuss the recent identification of ARF-BP1 (also known as Mule, UREB1, E3(histone), LASU1, and HectH9). ARF-BP1, a HECT domain-containing E3 ubiquitin ligase, interacts with ARF and p53. Its ubiquitin ligase activity is inhibited by ARF. Inactivation of ARF-BP1 stabilised p53 and induced apoptosis. Notably, inactivation of ARF-BP1 also caused cell growth repression in p53-null cells and breast cancer cells with mutant p53. Thus, ARF-BP1 emerges as a novel therapeutic target against cancer regardless of p53 status.

Cell Division↗
PubMed2004

Inactivation of the ARF-MDM-2-p53 pathway in sporadic Burkitt's lymphoma in children.

Burkitt's lymphomas (BLs) are characterized by an activated MYC gene that provides a constitutive proliferative signal. However, activated myc can initiate ARF-dependent activation of p53 and apoptosis as well. Data derived from cell culture and animal models suggest that the inactivation of the ARF-MDM-2-p53 apoptotic signaling pathway may be a necessary secondary event for the development of BL. This has not been tested in freshly excised BL tissue. We investigated the ARF-MDM-2-p53 pathway in tumor specimen from 24 children with sporadic BL/B-ALL. Direct sequencing revealed a point mutation in the p53 gene in four BL. Overexpression of MDM-2 was evident in 10 of the BL samples analyzed by real-time quantitative PCR. Deletion of the CDKN2A locus that encodes ARF or reduced expression of ARF could not be detected in any BL by fluorescence in situ hybridization analysis or real-time quantitative PCR, respectively. Our results indicate that the ARF-MDM-2-p53 apoptotic pathway is disrupted in about 55% of the cases of childhood sporadic BL. We suggest that in addition to the inactivation of the ARF-MDM-2-p53 protective checkpoint function other antiapoptotic mutations may occur in a substantial part of children with sporadic BL.

Adolescent↗
PubMed2003

MDM2-ARF complex regulates p53 sumoylation.

The p53 tumor suppressor is regulated by MDM2-mediated ubiquitination and degradation. Ubiquitination of p53 is regulated by ARF, which binds to MDM2 and inhibits its E3 ligase function. P53 is also subjected to modification by conjugation of SUMO-1. We found that a p53 mutant deficient for MDM2 binding (p53(14Q19S)) is poorly sumoylated in vivo compared to wild-type p53. Overexpression of MDM2 increases the level of p53 sumoylation, which is further stimulated by expression of ARF. Stimulation of p53 sumoylation requires a highly conserved region (102-116) encoded by exon 2 of ARF and correlates with the ability of ARF to target p53 to the nucleolus. An MDM2 deletion mutant (MDM2(Delta222-437)) with activated cryptic nucleolar localization signal also targets p53 to the nucleolus and efficiently promotes p53 sumoylation in the absence of ARF. Direct targeting of p53 to the nucleolus enhances its sumoylation in an MDM2- and ARF-dependent fashion. These results show that p53 sumoylation is regulated by MDM2- and ARF-mediated nucleolar targeting.

ADP-Ribosylation Factors↗
PubMed2006

A functional link between the tumour suppressors ARF and p33ING1.

The ARF tumour suppressor protein plays a critical role in the activation of p53 in response to oncogenic stress. ARF can activate p53 through nucleolar sequestration of Mdm2. However, several lines of evidence indicate that this is not the only way of action of ARF, and alternative mechanisms must exist. p33ING1 is a putative tumour suppresor, which induces cell-cycle arrest and apoptosis in a p53-dependent manner. Here, we describe that ARF and p33ING1 can interact in vivo. We also show that the subcellular localization of ING1 can be modulated by ARF protein levels, causing a displacement from nuclear to nucleolar localization. Finally, the ability of p33ING1 to cause cell-cycle arrest and induction of p21CIP1, or Mdm2, is impaired in ARF-deficient primary mouse fibroblasts. Based on these observations, we propose that the interaction with p33ING1 represents a novel mechanism for the tumour suppression function of ARF.

3T3 Cells↗
PubMed2004

Arf-1 (ADP-ribosylation factor-1) is involved in the activation of a mammalian Na+-selective current.

Stimulation of mammalian cells often results in an increase in the intracellular Na(+) concentration, brought about by Na(+) influx into the cell via Na(+)-permeable ion channels. In some cell types, particularly renal epithelia and mast cells, non-hydrolysable analogues of GTP, such as GTP[S] (guanosine 5'-[gamma-thio]triphosphate), activate a non-voltage-activated Na(+)-selective current. We have carried out whole-cell patch-clamp experiments to examine how GTP[S] activates the Na(+) current in a rat mast cell line. The ability of GTP[S] to activate Na(+) influx was prevented by including GTP in the pipette solution, indicating the involvement of small G-proteins. Brefeldin A and Arf-1-(2-17), inhibitors of Arf-1 (ADP-ribosylation factor-1) proteins, suppressed the activation of Na(+) entry by GTP[S]. However, non-active succinylated Arf-1-(2-17) or an N-terminal myristoylated peptide directed towards Arf-5 were ineffective. Arf proteins modulate the cytoskeleton, and disruption of the cytoskeleton with cytochalasin D or its stabilization with phalloidin impaired the development of the Na(+) current. Disaggregation of microtubules was without effect. Dialysis with cAMP or inhibition of cAMP phosphodiesterase with caffeine both decreased the extent of Na(+) entry, and this was not prevented by pre-treatment with broad-spectrum protein kinase inhibitors. Collectively, our results suggest that the mechanism of activation of a mammalian non-voltage-activated Na(+)-selective current requires an Arf small G-protein, most probably Arf-1.

ADP-Ribosylation Factor 1↗
PubMed2005

Localization and function of Arf family GTPases.

Arfs are a family of Ras-related GTP-binding proteins that function in the regulation of membrane trafficking and structure. The six mammalian Arf proteins are expressed ubiquitously and so it is anticipated that each will have a distinct localization and function within the cell. It has been assumed that much of this specificity will be defined by determining which regulators of Arfs, the GEFs (guanine nucleotide-exchange factors) and GAPs (GTPase-activating proteins) function with which Arf proteins. Although in vitro assays may indicate Arf preferences for the numerous Arf GEFs and GAPs that have been identified, in the cell the different Arfs, GEFs and GAPs are targeted to specific compartments where they carry out their functions. We have embarked on studies to define regions of the Arf1 and Arf6 proteins that determine their sites of action and specific activities at the Golgi and plasma membrane respectively. Chimaeras were made between Arf1 and Arf6 in order to identify regions of the protein that contributed to targeting and function. Whereas Arf6 is targeted to the plasma membrane through multiple regions along the protein, we have found a Golgi-targeting region in Arf1 that is sufficient to target Arf6 to the Golgi complex.

ADP-Ribosylation Factor 1↗
PubMed2005

Arf, Sec7 and Brefeldin A: a model towards the therapeutic inhibition of guanine nucleotide-exchange factors.

GEFs (guanine nucleotide-exchange factors), which stimulate GDP dissociation from small G-proteins, are pivotal regulators of signalling pathways activated by small G-proteins. In the case of Arf proteins, which are major regulators of membrane traffic in the cell and have recently been found to be involved in an increasing number of human diseases, GDP/GTP exchange is stimulated by GEFs that carry a catalytic Sec7 domain. Recent structural results captured snapshots of the exchange reaction, revealing that Sec7 domains secure Arf-GDP to membranes before nucleotide exchange takes place, taking advantage of a built-in structural device in Arf proteins that couples their affinity for membranes to the nature of the bound nucleotide. One of the Arf-Sec7 intermediates was trapped by BFA (Brefeldin A), an uncompetitive inhibitor of Arf activation that has been instrumental in deciphering the molecular principles of membrane traffic at the Golgi. BFA targets a low-affinity Arf-Sec7 intermediate of the exchange reaction. It binds at the Arf-GDP/Sec7 interface, thus freezing the complex in an abortive conformation that cannot proceed to nucleotide dissociation. In the cell, this results in the specific inhibition of Arf1 by a subset of its GEFs, and the efficient and reversible block of membrane traffic at the Golgi. The mechanism of BFA leads to the concept of 'interfacial inhibition', in which a protein-protein interaction of therapeutic interest is stabilized, rather than impaired, by a drug. Up-regulated activity of small G-proteins is involved in various human diseases, making their GEFs attractive candidates to interrupt specifically the corresponding signalling pathway. Interfacial inhibitors are proposed as an alternative to competitive inhibitors that may be explored for their inhibition.

ADP-Ribosylation Factors↗
PubMed2005

Nonsteroidal anti-inflammatory drugs and risk of ARF in the general population.

BACKGROUND: Most epidemiological studies evaluating the association between nonsteroidal anti-inflammatory drugs (NSAIDs) and acute renal failure (ARF) found an increased risk for developing ARF while taking NSAIDs. Despite these studies, little is known about the effect of dose and duration of therapy, risk of individual NSAIDs, comorbidity, or concomitant use of other nephrotoxic drugs. METHODS: This is a nested case-control study using the General Practice Research Database from the United Kingdom. Participants were 386,916 patients aged 50 to 84 years on January 1, 1997, and free of known cancer, renal disorder, cirrhosis, or systemic connective tissue disease. After validation of cases identified from this cohort, 103 patients were confirmed as idiopathic cases of ARF and compared with 5,000 controls frequency matched by age and sex. RESULTS: Current users of NSAIDs had a relative risk (RR) for ARF of 3.2 (95% confidence interval [CI], 1.8 to 5.8), and the risk declined after treatment was discontinued. Increased risk was present with both short- and long-term therapy and was slightly greater among users of high doses. History of heart failure (HF), hypertension, diabetes, and hospitalizations and consultant visits in the previous year were all associated with a greater risk for ARF. There was a suggestion of a modification of the effect of NSAIDs in patients with hypertension and those with HF. Use of selected cardiovascular drugs was associated with a 5-fold increase in risk for ARF. Diuretics presented the greatest risk. Risk increased with concomitant use of NSAIDs and diuretics (RR, 11.6; 95% CI, 4.2 to 32.2) and NSAIDs and calcium channel blockers (RR, 7.8; 95% CI, 3.0 to 20.5). CONCLUSION: NSAID users had a 3-fold greater risk for developing a first-ever diagnosis of clinical ARF compared with non-NSAID users in the general population. NSAIDs should be used with special caution in patients with hypertension and/or HF.

Acute Kidney Injury↗
PubMed2004

Role for PP2A in ARF signaling to p53.

Activation of the ARF-p53 tumor suppressor pathway is one of the cell's major defense mechanisms against cancer induced by oncogenes. The ARF-p53 pathway is dysfunctional in a high proportion of human cancers. The regulation of the ARF-p53 signaling pathway has not yet been well characterized. In this study polyoma virus (Py) is used as a tool to better define the ARF-p53 signaling pathway. Py middle T-antigen (PyMT) induces ARF, which consequently up-regulates p53. We show that Py small T-antigen (PyST) blocks ARF-mediated activation of p53. This inhibition requires the small T-antigen PP2A-interacting domain. Our results reveal a previously unrecognized role of PP2A in the modulation of the ARF-p53 tumor suppressor pathway.

Amino Acid Sequence↗
PubMed1996

Activation of phospholipase D and phosphatidylinositol 4-phosphate 5-kinase in HL60 membranes is mediated by endogenous Arf but not Rho.

Membrane-associated phospholipase D (PLD) in HL60 cells can be activated by the small GTP-binding proteins Arf and RhoA, but polyphosphorylated inositol lipids were required for maximum activity. The intact lipid was required because neither inositol 1,4, 5-trisphosphate nor stearoyl-arachidonyl glycerol could substitute for phosphatidylinositol 4,5-bisphosphate (PIP2). Arf-stimulated but not Rho-stimulated PLD activity was increased by the inclusion of Mg2+ and ATP. ATP-dependent PLD activation occurred when phosphatidylinositol 4-phosphate (PIP), PIP2, or phosphatidylinositol 3,4,5-trisphosphate (PIP3) were included, but PIP2 formation was only detected with PIP; no PIP3 production was detected under any conditions. Therefore, the ATP-dependent increase in PLD activity cannot be explained by PIP2 or PIP3 formation. Association of endogenous Arf and RhoA with membranes was increased by incubation with GTPgammaS. This treatment increased membrane PLD and PIP kinase activities in the absence of exogenous p21 proteins. Reduction of Arf translocation suppressed the increase in PLD and PIP kinase activities, whereas complete removal of Rho but not Arf from membranes with RhoGDI was without effect on PLD activity but increased PIP kinase activity. Therefore, although recombinant Arf and Rho can activate PLD and PIP kinase in HL60 cells, it is the endogenous Arf but not Rho that regulates PLD, and thus a role for Rho in the physiological regulation of PLD in HL60 cells is unlikely.

ADP-Ribosylation Factors↗
PubMed2004

Different requirements for the cytostatic and apoptotic effects of type I interferons. Induction of apoptosis requires ARF but not p53 in osteosarcoma cell lines.

The regulation of cell growth is one of the most important effects of type I interferons (IFNs). This response may involve a cytostatic effect or the induction of apoptosis depending on the cell context. Often the growth-inhibitory response of type I IFNs is studied in tumor cell lines carrying mutations of tumor suppressor genes, and therefore, the growth-inhibitory effect can be influenced by inactivation of these important regulators of cell proliferation. In this report, we explored the role of the ARF-p53 pathway in the growth-inhibitory effect of type I IFNs. We found that p53 is only induced in cells that express p14(ARF) (p19(ARF) in mouse cells). Surprisingly, mouse embryonal fibroblasts that are null for p19(ARF) or P53, even after transformation with oncogenic RAS, respond as well as wild type to the growth-inhibitory effect of type I IFNs. Similarly, human ARF(-/-) U2OS and P53(-/-) SAOS-2 cells show a significant decrease in cell proliferation. However, only SAOS-2 or U2OS reconstituted with inducible p14(ARF) undergo apoptosis in response to IFN beta treatment, and this effect was not inhibited by expression of dominant negative p53. These data suggest that (i) at least in specific cell types, the induction of apoptosis by type I IFNs requires an ARF pathway that is p53-independent and (ii) the cytostatic and pro-apoptotic effects of type I IFNs employ different pathways.

Animals↗
PubMed2004

p14 Arf promotes small ubiquitin-like modifier conjugation of Werners helicase.

Here we demonstrate a novel p53-independent interaction between the nucleolar tumor suppressors, p14 Arf and Werners helicase (WRN). Binding of p14 Arf to WRN is multivalent and resembles the binding of p14 Arf to Mdm2. Residues 2-14 and 82-101 of p14 Arf and residues in the central region and C terminus of WRN have particular importance for binding. p14 Arf promotes small ubiquitin-like modifier (SUMO) modification of WRN in a synergistic manner with the SUMO-conjugating enzyme, UBCH9. p14 Arf causes redistribution of WRN within the nucleus, and this effect is reversed by expression of a SUMO-specific protease, thus implicating the SUMO conjugation pathway in WRN re-localization. We establish that the ability to promote SUMO conjugation is a general property of the p14 Arf tumor suppressor.

DNA Helicases↗