Arsenic sulfide inhibits the progression of gastric cancer through regulating the circRNA_ASAP2/Wnt/ß-catenin pathway.

In our paper, the effects of As4S4 treatments on the growth and migration of gastric cancer (GC) cells were explored, and the potential underlying molecular mechanisms were also identified. Cell viability was evaluated by cell counting kit 8 assay. The expression of Ki-67 was examined using immunofluorescence staining. Cell apoptosis was assessed by flow cytometry. The migratory and invasion abilities of cells were determined using Transwell assay. The mRNA and protein levels of related gene were examined by RT-qPCR and western blotting, respectively. CircRNAs chip was performed to identify the differentiated expression of circRNAs in GC cells following the treatment with As4S4. Our results revealed that the proliferation, migration and invasion of GC cells were remarkably suppressed by the treatment with As4S4, while cell apoptosis was promoted. Furthermore, circRNA_ASAP2 was a novel target of As4S4 in GC, and it is involved in As4S4-modulated biological behavior alterations in GC cells. In addition, the activities of the Wnt/ß-catenin signaling in GC cells were affected by the overexpression circRNA_ASAP2 and the treatment with As4S4. Moreover, the behavior changes in GC cells caused by the knockdown of circRNA_ASAP2 were reversed by the treatment with Wnt agonist SKL2001. In summary, As4S4 could function as an antitumor agent in GC through regulating the circRNA_ASAP2/Wnt/ß-catenin pathway, which in turn influences the growth and metastasis of GC cells.

Recombinant Slit2 attenuates neuronal apoptosis via the Robo1-srGAP1 pathway in a rat model of neonatal HIE.

Apoptosis following hypoxic-ischemic injury to the brain plays a major role in neuronal cell death. The neonatal brain is more susceptible to injury as the cortical neurons are immature and there are lower levels of antioxidants. Slit2, an extracellular matrix protein, has been shown to be neuroprotective in various models of neurological diseases. However, there is no information about the role of Slit2 in neonatal hypoxia-ischemia. In this study, we evaluated the effect of Slit2 and its receptor Robo1 in a rat model with neonatal HIE. 10-day old rat pups were used to create the neonatal HIE model. The right common carotid artery was ligated followed by 2.5 h of hypoxia. Recombinant Slit2 was administered intranasally 1 h post HI, recombinant Robo1 was used as a decoy receptor and administered intranasally 1h before HI and srGAP1-siRNA was administered intracerebroventricularly 24 h before HI. Brain infarct area measurement, short-term and long-term neurological function tests, Western blot, immunofluorescence staining, Fluoro-Jade C staining, Nissl staining and TUNEL staining were the assessments done following drug administration. Recombinant Slit2 administration reduced neuronal apoptosis and neurological deficits after neonatal HIE which were reversed by co-administration of recombinant Robo1 and srGAP1-siRNA administration. Recombinant Slit2 showed improved outcomes possibly via the robo1-srGAP1 pathway which mediated the inhibition of RhoA. In this study, the results suggest that Slit2 may help in attenuation of apoptosis and could be a therapeutic agent for treatment of neonatal hypoxic ischemic encephalopathy.

RPEL-family rhoGAPs link Rac/Cdc42 GTP loading to G-actin availability.

RPEL proteins, which contain the G-actin-binding RPEL motif, coordinate cytoskeletal processes with actin dynamics. We show that the ArhGAP12- and ArhGAP32-family GTPase-activating proteins (GAPs) are RPEL proteins. We determine the structure of the ArhGAP12/G-actin complex, and show that G-actin contacts the RPEL motif and GAP domain sequences. G-actin inhibits ArhGAP12 GAP activity, and this requires the G-actin contacts identified in the structure. In B16 melanoma cells, ArhGAP12 suppresses basal Rac and Cdc42 activity, F-actin assembly, invadopodia formation and experimental metastasis. In this setting, ArhGAP12 mutants defective for G-actin binding exhibit more effective downregulation of Rac GTP loading following HGF stimulation and enhanced inhibition of Rac-dependent processes, including invadopodia formation. Potentiation or disruption of the G-actin/ArhGAP12 interaction, by treatment with the actin-binding drugs latrunculin B or cytochalasin D, has corresponding effects on Rac GTP loading. The interaction of G-actin with RPEL-family rhoGAPs thus provides a negative feedback loop that couples Rac activity to actin dynamics.

Fiber type-selective exercise effects on AS160 phosphorylation.

Earlier research using muscle tissue demonstrated that postexercise elevation in insulin-stimulated glucose uptake (ISGU) occurs concomitant with greater insulin-stimulated Akt substrate of 160 kDa (AS160) phosphorylation (pAS160) on sites that regulate ISGU. Because skeletal muscle is a heterogeneous tissue, we previously isolated myofibers from rat epitrochlearis to assess fiber type-selective ISGU. Exercise induced greater ISGU in type I, IIA, IIB, and IIBX but not IIX fibers. This study tested if exercise effects on pAS160 correspond with previously published fiber type-selective exercise effects on ISGU. Rats were studied immediately postexercise (IPEX) or 3.5 h postexercise (3.5hPEX) with time-matched sedentary controls. Myofibers dissected from the IPEX experiment were analyzed for fiber type (myosin heavy chain isoform expression) and key phosphoproteins. Isolated muscles from the 3.5hPEX experiment were incubated with or without insulin. Myofibers (3.5hPEX) were analyzed for fiber type, key phosphoproteins, and GLUT4 protein abundance. We hypothesized that insulin-stimulated pAS160 at 3.5hPEX would exceed sedentary controls only in fiber types characterized by greater ISGU postexercise. Values for phosphorylation of AMP-activated kinase substrates (acetyl CoA carboxylaseSer79 and AS160Ser704) from IPEX muscles exceeded sedentary values in each fiber type, suggesting exercise recruitment of all fiber types. Values for pAS160Thr642 and pAS160Ser704 from insulin-stimulated muscles 3.5hPEX exceeded sedentary values for type I, IIA, IIB, and IIBX but not IIX fibers. GLUT4 abundance was unaltered 3.5hPEX in any fiber type. These results advanced understanding of exercise-induced insulin sensitization by providing compelling support for the hypothesis that enhanced insulin-stimulated phosphorylation of AS160 is linked to elevated ISGU postexercise at a fiber type-specific level independent of altered GLUT4 expression.

Heat shock protein 90 inhibitor regulates necroptotic cell death via down-regulation of receptor interacting proteins.

17-(Dimethylaminoethylamino)-17-demethoxygeldanamycin (DMAG) acts as an inhibitor of heat shock protein 90 (HSP 90), which serves as a nodal protein of diverse signaling networks leading to a variety of biological implications. HSP90 plays the role of a chaperone for a variety of client proteins including receptor interacting protein 1 (RIP1). Since RIP1 and RIP3 are, respectively, required for zVAD- and tumor necrosis factor alpha (TNFα)-mediated necrotic cell death, we pursued to address the effects of DMAG on receptor-and nonreceptor-mediated necroptotic cell death. DMAG facilitated the degradation of receptor interacting protein 3 (RIP3) as well as RIP1, a known client protein of HSP90, in L929 cells. Consequently, DMAG rendered cells more sensitive to TNFα stimulation while it rescued cells from necrotic cell death caused by zVAD. From this study, we propose that DMAG-downregulated RIP1 can shift cell death typing from necroptosis to apoptosis. In contrast, the protective effect of DMAG on zVAD-induced cytotoxicity could be partly explained by the fact that zVAD mediates cytotoxicity via a RIP1 -dependent route. In summary, functional disruption of HSP90 by DMAG destabilized necroptosis proteins RIP1 and RIP3, which in turn regulated zVAD- and TNFα-induced necroptosis. Therefore, pharmacological modulation of necroptotic cell death through HSP90 could be a promising strategy for overcoming cancer drug resistance or protecting ischemic cell death.

General aspects and recent advances on bacterial protein toxins.

Bacterial pathogens produce protein toxins to influence host-pathogen interactions and tip the outcome of these encounters toward the benefit of the pathogen. Protein toxins modify host-specific targets through posttranslational modifications (PTMs) or noncovalent interactions that may inhibit or activate host cell physiology to benefit the pathogen. Recent advances have identified new PTMs and host targets for toxin action. Understanding the mechanisms of toxin action provides a basis to develop vaccines and therapies to combat bacterial pathogens and to develop new strategies to use toxin derivatives for the treatment of human disease.

Arsenite exposure altered the expression of NMDA receptor and postsynaptic signaling proteins in rat hippocampus.

Chronic arsenic exposure has an adverse effect on neurobehavioral function. Our previous study demonstrated an elevated arsenic level, ultra-structure changes and reduced NR2A gene expression in hippocampus, and impaired spatial learning in arsenite-exposed rats. The NMDA receptor and the postsynaptic signaling proteins CaMKII, postsynaptic density protein 95 (PSD-95), synaptic Ras GTPase-activating protein (SynGAP) and nuclear activated extracellular-signal regulated kinase (ERK1/2) play important roles in synaptic plasticity, learning and memory. We hypothesized that the above molecular expression changes may contribute to arsenic neurotoxicity. In present study, the expression of NMDA receptor and postsynaptic signaling proteins in hippocampus were evaluated in rats exposed to 0, 2.72, 13.6 and 68 mg/L sodium arsenite for 3 months. Decreased protein expression of NR2A, PSD-95 and p-CaMKII α in the hippocampus of arsenite-exposed rats was observed, while the expression of SynGAP, a negative regulator of Ras-MAPK activity, was increased when compared with the controls. Additionally, decreased p-ERK1/2 activity was found in the hippocampus of arsenite-exposed rats. These data suggest that altered expression of NMDA receptor complex and postsynaptic signaling proteins may explain arsenic-induced neurotoxicity.

Effects of prenatal ethanol exposure on the lungs of postnatal lambs.

Prenatal ethanol exposure increases collagen deposition and alters surfactant protein (SP) expression and immune status in lungs of near-term fetal sheep. Our objectives were to determine 1) whether these prenatal effects of repeated gestational ethanol exposure persist after birth and 2) whether surfactant phospholipid composition is altered following prenatal ethanol exposure. Pregnant ewes were chronically catheterized at 90 days of gestational age (DGA) and given a 1-h daily infusion of ethanol (0.75 g/kg, n = 9) or saline (n = 7) from 95 to 135 DGA; ethanol administration ceased after 135 DGA. Lambs were born naturally at full term (146 ± 0.5 DGA). Lung tissue was examined at 9 wk postnatal age for alterations in structure, SP expression, and inflammation; bronchoalveolar lavage fluid was examined for alterations in surfactant phospholipid composition. At 134 DGA, surfactant phospholipid concentration in amniotic fluid was significantly reduced (P < 0.05) by ethanol exposure, and the composition was altered. In postnatal lambs, there were no significant differences between treatment groups in birth weight, postnatal growth, blood gas parameters, and lung weight, volume, tissue fraction, mean linear intercept, collagen content, proinflammatory cytokine gene expression, and bronchoalveolar lavage fluid surfactant phospholipid composition. Although SP-A, SP-B, and SP-C mRNA levels were not significantly different between treatment groups, SP-D mRNA levels were significantly greater (P < 0.05) in ethanol-treated animals; as SP-D has immunomodulatory roles, innate immunity may be altered. The adverse effects of daily ethanol exposure during late gestation on the fetal lung do not persist to 2 mo after birth, indicating that the developing lung is capable of repair.

AS160 associates with the Na+,K+-ATPase and mediates the adenosine monophosphate-stimulated protein kinase-dependent regulation of sodium pump surface expression.

The Na(+),K(+)-ATPase is the major active transport protein found in the plasma membranes of most epithelial cell types. The regulation of Na(+),K(+)-ATPase activity involves a variety of mechanisms, including regulated endocytosis and recycling. Our efforts to identify novel Na(+),K(+)-ATPase binding partners revealed a direct association between the Na(+),K(+)-ATPase and AS160, a Rab-GTPase-activating protein. In COS cells, coexpression of AS160 and Na(+),K(+)-ATPase led to the intracellular retention of the sodium pump. We find that AS160 interacts with the large cytoplasmic NP domain of the α-subunit of the Na(+),K(+)-ATPase. Inhibition of the activity of the adenosine monophosphate-stimulated protein kinase (AMPK) in Madin-Darby canine kidney cells through treatment with Compound C induces Na(+),K(+)-ATPase endocytosis. This effect of Compound C is prevented through the short hairpin RNA-mediated knockdown of AS160, demonstrating that AMPK and AS160 participate in a common pathway to modulate the cell surface expression of the Na(+),K(+)-ATPase.

p190RhoGAP negatively regulates Rho activity at the cleavage furrow of mitotic cells.

Previous studies demonstrated that p190RhoGAP (p190) negatively affects cytokinesis in a RhoGAP-dependent manner, suggesting that regulation of Rho may be a critical mechanism of p190 action during cytokinesis. P190 localizes to the cleavage furrow (CF) of dividing cells, and its levels decrease during late mitosis by an ubiquitin-mediated mechanism, consistent with the hypothesis that high RhoGTP levels are required for completion of cytokinesis. To determine whether RhoGTP levels in the CF are affected by p190 and to define the phase(s) of cytokinesis in which p190 is involved, we used FRET analysis alone or in combination with time-lapse microscopy. In normal cell division activated Rho accumulated at the cell equator in early anaphase and in the contractile ring, where it co-localized with p190. Real-time movies revealed that cells expressing elevated levels of p190 exhibited multiple cycles of abnormal CF site selection and ingression/regression, which resulted in failed or prolonged cytokinesis. This was accompanied by mislocalization of active Rho at the aberrant CF sites. Quantified data revealed that in contrast to ECT2 and dominate negative p190 (Y1283Ap190), which resulted in hyper-activated Rho, Rho activity in the CF was reduced by wild type p190 in a dose-dependent manner. These results suggest that p190 regulates cytokinesis through modulation of RhoGTP levels, thereby affecting CF specification site selection and subsequent ring contraction.

Inhibition of contraction-stimulated AMP-activated protein kinase inhibits contraction-stimulated increases in PAS-TBC1D1 and glucose transport without altering PAS-AS160 in rat skeletal muscle.

OBJECTIVE: Phosphorylation of two members of the TBC1 domain family of proteins, Akt substrate of 160 kDa (AS160, also known as TBC1D4) and TBC1D1, has been implicated in the regulation of glucose transport in skeletal muscle. Insulin-stimulated phosphorylation (measured using the phospho-Akt substrate [PAS] antibody) of AS160 and TBC1D1 appears to occur in an Akt-dependent manner, but the kinases responsible for contraction-stimulated PAS-AS160 and PAS-TBC1D1 remain unclear. AMP-activated protein kinase (AMPK) and Akt, both activated by contraction, can each phosphorylate AS160 and TBC1D1 in cell-free assays. RESEARCH DESIGN AND METHODS: To evaluate the roles of AMPK and Akt on insulin- or contraction-stimulated PAS-AS160, PAS-TBC1D1, and glucose transport, rat epitrochlearis was incubated with and without compound C (inhibitor of AMPK) or Wortmannin (inhibitor of phosphatidylinositol [PI] 3-kinase, which is upstream of Akt) before and during insulin stimulation or contraction. RESULTS: Insulin-stimulated glucose transport and phosphorylation of both AS160 and TBC1D1 were completely inhibited by Wortmannin. Wortmannin eliminated contraction stimulation of phospho-Ser(21/9)glycogen synthase kinase 3alpha/beta (pGSK3; Akt substrate) and PAS-AS160 but did not significantly alter pAMPK, phospho-Ser79acetyl CoA carboxylase (pACC; AMPK substrate), PAS-TBC1D1, or glucose transport in contraction-stimulated muscle. Compound C completely inhibited contraction-stimulated pACC and PAS-TBC1D1 and partially blocked glucose transport, but it did not significantly alter pAkt, pGSK3, or PAS-AS160. CONCLUSIONS: These data suggest that 1) insulin stimulates glucose transport and phosphorylation of AS160 and TBC1D1 in a PI 3-kinase/Akt-dependent manner, 2) contraction stimulates PAS-AS160 (but not PAS-TBC1D1 or glucose transport) in a PI 3-kinase/Akt-dependent manner, and 3) contraction stimulates PAS-TBC1D1 and glucose transport (but not PAS-AS160) in an AMPK-dependent manner.

An Arf1 GTPase mutant with different responses to GEF inhibitors.

Guanine nucleotide exchange factors (GEFs) stimulate the activation of small GTP-binding proteins (GTPases). Establishing their specificity is a challenging issue, in which chemical genetics are rapidly gaining interest. We report a mutation in the Arf1 GTPase, K38A, which differentially alters its sensitivity to GEF inhibitors. The mutation renders Arf1 insensitive to LM11, a GEF inhibitor that we previously discovered by structure-based screening. In contrast, full inhibition by the natural compound Brefeldin A (BFA) is retained. We show that the mutation is otherwise silent towards the biochemical and cellular properties of Arf1, notably its binding to effectors as measured by a novel GEF-protection assay. This is thus the first GTPase mutant with different responses to two classes of inhibitors, and a novel tool to analyze Arf and ArfGEF specificity and functions in vitro and in cells.

Endothelial cell barrier protection by simvastatin: GTPase regulation and NADPH oxidase inhibition.

The statins, hydroxy-3-methylglutaryl-CoA reductase inhibitors that lower serum cholesterol, exhibit myriad clinical benefits, including enhanced vascular integrity. One potential mechanism underlying increased endothelial cell (EC) barrier function is inhibition of geranylgeranylation, a covalent modification enabling translocation of the small GTPases Rho and Rac to the cell membrane. While RhoA inhibition attenuates actin stress fiber formation and promotes EC barrier function, Rac1 inhibition at the cell membrane potentially prevents activation of NADPH oxidase and subsequent generation of superoxides known to induce barrier disruption. We examined the relative regulatory effects of simvastatin on RhoA, Rac1, and NADPH oxidase activities in the context of human pulmonary artery EC barrier protection. Confluent EC treated with simvastatin demonstrated significantly decreased thrombin-induced FITC-dextran permeability, a reflection of vascular integrity, which was linked temporally to simvastatin-mediated actin cytoskeletal rearrangement. Compared with Rho inhibition alone (Y-27632), simvastatin afforded additional protection against thrombin-mediated barrier dysfunction and attenuated LPS-induced EC permeability and superoxide generation. Statin-mediated inhibition of both Rac translocation to the cell membrane and superoxide production were attenuated by geranylgeranyl pyrophosphate (GGPP), indicating that these effects are due to geranylgeranylation inhibition. Finally, thrombin-induced EC permeability was modestly attenuated by reduced Rac1 expression (small interfering RNA), whereas these effects were made more pronounced by simvastatin pretreatment. Together, these data suggest EC barrier protection by simvastatin is due to dual inhibitory effects on RhoA and Rac1 as well as the attenuation of superoxide generation by EC NADPH oxidase and contribute to the molecular mechanistic understanding of the modulation of EC barrier properties by simvastatin.

Impaired insulin-stimulated phosphorylation of Akt and AS160 in skeletal muscle of women with polycystic ovary syndrome is reversed by pioglitazone treatment.

OBJECTIVE: Insulin resistance in skeletal muscle is a major risk factor for type 2 diabetes in women with polycystic ovary syndrome (PCOS). However, the molecular mechanisms underlying skeletal muscle insulin resistance and the insulin-sensitizing effect of thiazolidinediones in PCOS in vivo are less well characterized. RESEARCH DESIGN AND METHODS: We determined molecular mediators of insulin signaling to glucose transport in skeletal muscle biopsies of 24 PCOS patients and 14 matched control subjects metabolically characterized by euglycemic-hyperinsulinemic clamps and indirect calorimetry, and we examined the effect of 16 weeks of treatment with pioglitazone in PCOS patients. RESULTS: Impaired insulin-mediated total (R(d)) oxidative and nonoxidative glucose disposal (NOGD) was paralleled by reduced insulin-stimulated Akt phosphorylation at Ser473 and Thr308 and AS160 phosphorylation in muscle of PCOS patients. Akt phosphorylation at Ser473 and Thr308 correlated positively with R(d) and NOGD in the insulin-stimulated state. Serum free testosterone was inversely related to insulin-stimulated R(d) and NOGD in PCOS. Importantly, the pioglitazone-mediated improvement in insulin-stimulated glucose metabolism, which did not fully reach normal levels, was accompanied by normalization of insulin-mediated Akt phosphorylation at Ser473 and Thr308 and AS160 phosphorylation. AMPK activity and phosphorylation were similar in the two groups and did not respond to pioglitazone in PCOS patients. CONCLUSIONS: Impaired insulin signaling through Akt and AS160 in part explains insulin resistance at the molecular level in skeletal muscle in PCOS, and the ability of pioglitazone to enhance insulin sensitivity involves improved signaling through Akt and AS160. Moreover, our data provide correlative evidence that hyperandrogenism in PCOS may contribute to insulin resistance.

Geldanamycin anisimycins activate Rho and stimulate Rho- and ROCK-dependent actin stress fiber formation.

Heat shock protein 90 (Hsp90) is a member of the heat shock family of molecular chaperones that regulate protein conformation and activity. Hsp90 regulates multiple cell signaling pathways by controlling the abundance and activity of several important protein kinases and cell cycle-related proteins. In this report, we show that inhibition of Hsp90 by geldanamycin or its derivative, 17-allylamino-17-desmethoxygeldamycin, leads to activation of the Rho GTPase and a dramatic increase in actin stress fiber formation in human tumor cell lines. Inactivation of Rho prevents geldanamycin-induced actin reorganization. Hsp90 inactivation does not alter the appearance of filopodia or lamellipodia and tubulin architecture is not visibly perturbed. Our observations suggest that Hsp90 has an important and specific role in regulating Rho activity and Rho-dependent actin cytoskeleton remodeling.

Rac1 links integrin-mediated adhesion to the control of lactational differentiation in mammary epithelia.

The expression of tissue-specific genes during mammary gland differentiation relies on the coincidence of two distinct signaling events: the continued engagement of beta1 integrins with the extracellular matrix (ECM) and a hormonal stimulus from prolactin (Prl). How the integrin and Prl receptor (PrlR) systems integrate to regulate milk protein gene synthesis is unknown. In this study, we identify Rac1 as a key link. Dominant-negative Rac1 prevents Prl-induced synthesis of the milk protein beta-casein in primary mammary epithelial cells cultured as three-dimensional acini on basement membrane. Conversely, activated Rac1 rescues the defective beta-casein synthesis that occurs under conditions not normally permissive for mammary differentiation, either in beta1 integrin-null cells or in wild-type cells cultured on collagen. Rac1 is required downstream of integrins for activation of the PrlR/Stat5 signaling cascade. Cdc42 is also necessary for milk protein synthesis but functions via a distinct mechanism to Rac1. This study identifies the integration of signals provided by ECM and hormones as a novel role for Rho family guanosine triphosphatases.

Distinct signals regulate AS160 phosphorylation in response to insulin, AICAR, and contraction in mouse skeletal muscle.

Insulin and contraction increase GLUT4 translocation in skeletal muscle via distinct signaling mechanisms. Akt substrate of 160 kDa (AS160) mediates insulin-stimulated GLUT4 translocation in L6 myotubes, presumably through activation of Akt. Using in vivo, in vitro, and in situ methods, insulin, contraction, and the AMP-activated protein kinase (AMPK) activator AICAR all increased AS160 phosphorylation in mouse skeletal muscle. Insulin-stimulated AS160 phosphorylation was fully blunted by wortmannin in vitro and in Akt2 knockout (KO) mice in vivo. In contrast, contraction-stimulated AS160 phosphorylation was only partially decreased by wortmannin and unaffected in Akt2 KO mice, suggesting additional regulatory mechanisms. To determine if AMPK mediates AS160 signaling, we used AMPK alpha2-inactive (alpha2i) transgenic mice. AICAR-stimulated AS160 phosphorylation was fully inhibited, whereas contraction-stimulated AS160 phosphorylation was partially reduced in the AMPK alpha2i transgenic mice. Combined AMPK alpha2 and Akt inhibition by wortmannin treatment of AMPK alpha2 transgenic mice did not fully ablate contraction-stimulated AS160 phosphorylation. Maximal insulin, together with either AICAR or contraction, increased AS160 phosphorylation in an additive manner. In conclusion, AS160 may be a point of convergence linking insulin, contraction, and AICAR signaling. While Akt and AMPK alpha2 activities are essential for AS160 phosphorylation by insulin and AICAR, respectively, neither kinase is indispensable for the entire effects of contraction on AS160 phosphorylation.

Regulation of RhoGAP specificity by phospholipids and prenylation.

Among the key protein regulators of the various and numerous small GTPases are the GTPase activating proteins (GAPs). Experimental studies of some of the approximately 170 GAPs predicted by the human genome indicate that their catalytic GAP activity is regulated by a variety of mechanisms, including phosphorylation, protein-protein interactions, proteolysis, and interactions with lipids. Most reported biochemical studies to address the specificity of GAPs for particular GTPases have been conducted in vitro with bacterially produced GTPases. Thus, the potential influence of these various regulatory mechanisms in the context of GAP-GTPase specificity may be overlooked in such assays. Here, we present experimental studies that highlight the role of lipids in modulating the GTPase specificity for some of the Rho GAPs. We find that particular phospholipids can substantially alter the substrate "preference" for the p190 GAPs. We find that C-terminal prenylation of GTPases can influence the specificity of GAP interactions as well. These observations emphasize the limitations of standard in vitro GAP assays in definitively establishing the physiologically relevant GTPase targets for particular GAPs.

AlFx affects the formation of focal complexes by stabilizing the Arf-GAP ASAP1 in a complex with Arf1.

Aluminum fluoride (AlFx) is known to activate directly the alpha subunit of G-proteins but not the homologous small GTP-binding proteins. However, AlFx can stabilize complexes formed between Ras, RhoA or Cdc42 and their corresponding GTPase-activating proteins (GAPs). Here, we demonstrate that Arf1GDP can be converted into an active conformation by AlFx to form a complex with the Arf-GAP ASAP1 in vitro and in vivo. Within this complex ASAP1, which GAP activity is inoperative, can still alter the recruitment of paxillin to the focal complexes, thus indicating that ASAP1 interferes with focal complexes independently of its GAP activity.

A genetic screen identifies PITX1 as a suppressor of RAS activity and tumorigenicity.

Activating mutations of RAS frequently occur in subsets of human cancers, indicating that RAS activation is important for tumorigenesis. However, a large proportion of these cancers still retain wild-type RAS alleles, suggesting that either the RAS pathway is activated in a distinct manner or another pathway is deregulated. To uncover novel tumor-suppressor genes, we screened an RNA-interference library for knockdown constructs that transform human primary cells in the absence of ectopically introduced oncogenic RAS. Here we report the identification of PITX1, whose inhibition induces the RAS pathway and tumorigenicity. Interestingly, we observed low expression of PITX1 in prostate and bladder tumors and in colon cancer cell lines containing wild-type RAS. Restoration of PITX1 in the colon cancer cells inhibited tumorigenicity in a wild-type RAS-dependent manner. Finally, we identified RASAL1, a RAS-GTPase-activating protein, as a transcription target through which PITX1 affects RAS function. Thus, PITX1 suppresses tumorigenicity by downregulating the RAS pathway through RASAL1.