Presence Of Phenylarsine Oxide Research Articles

How Lipids Regulate the Cell Surface Localization of HSPA1A, a Stress‐inducible 70‐kDa Heat Shock Protein

HSPA1A is a molecular chaperone that regulates survival stressed and cancer cells. In addition to its cytosolic pro‐survival functions, HSPA1A also localizes at the plasma membrane (PM) of stressed and tumor cells. In cancer cells, the presence of HSPA1A at the cell surface allows the latter cells to develop resistance to radiation therapy, show increased invasiveness, and develop distant metastasis. Therefore, abolishing HSPA1A from the surface of tumor cells is a promising therapeutic. However, HSPA1A’s pathway to the cell surface remains enigmatic because this protein lacks membrane localization signals. Considering that HSPA1A binds with high selectivity to negatively charged lipids, like phosphatidylserine (PS) and mono‐phosphorylated phosphoinositides (PIPs), we hypothesized that the interaction of HSPA1A with these lipids allows the chaperone to translocate and anchor to the PM. To test this hypothesis, we subjected human cell lines to heat shock, depleted specific lipid targets, and quantified HSPA1A’s PM localization using confocal microscopy and cell surface biotinylation. These experiments revealed that depletion of PS, PI(4)P, and PI(3)P by co‐transfecting HSPA1A with known lipid‐biosensors, significantly reduced HSPA1A’s surface presentation. In contrast, reducing PI(4,5)P2 availability by overexpression of the PLCδ‐PH biosensor had minimal effects on HSPA1A’s PM‐localization. Next, we manipulated the cellular lipid content using ionomycin (a PLC activator), phenylarsine oxide (PAO) and GSK‐A1 (general and a selective PI4‐Kinase inhibitors, respectively), wortmannin (a PI3‐Kinase inhibitor), and fendiline (a repurposed FDA approved drug that reduces PS) using lipid‐biosensors as positive and negative controls. These experiments revealed that HSPA1A’s PM localization was unaffected by ionomycin but was greatly reduced in the presence of PAO, GSK‐A1, wortmannin, and fendiline, corroborating the findings obtained by the co‐transfection experiments. We further verified these results by selectively depleting PI(4,5)P2 and PI(4)P using a well‐established rapamycin‐induced phosphatase system. Our findings strongly support the notion that HSPA1A’s surface presentation is a multifaceted lipid‐driven phenomenon controlled by the binding of the chaperone to specific endosomal and plasma membrane lipids. These findings provide the basis for future interventions to render tumor cells sensitive to radiation therapy.

Salicylic acid induces the proton conductance in the inner mitochondrial membrane of lupine cotyledons

The influence of salicylic acid (SA) on generation of membrane potential (Δψ) at the inner membrane of isolated mitochondria from cotyledons of lupine seedlings (Lupinus angustifolius L.) was investigated. The mitochondrial preparations conformed to all criteria of the intactness: the organelles were characterized by the integrity of their membranes and by tight coupling of oxidation and phosphorylation. High functional activity of mitochondria was also evident from their ability to generate Δψ during succinate oxidation and from the long-term maintenance of steady-state transmembrane potential by virtue of electrontransport chain (ETC) operation or ATP hydrolysis after the inhibition of respiratory ETC. The addition of SA to the incubation medium (0.5–1.0 mM) induced a fast and complete dissipation of Δψ after a distinct lag period. The Δψ was not restored by subsequent ATP hydrolysis, indicating that the phytohormone SA induced the proton conductance of the inner membrane. The SA-induced collapse of Δψ was observed under suppression of ETC by anaerobiosis, cyanide, or inhibitory concentrations of the phytohormone. The SAinduced dissipation of Δψ was not reversed by cyclosporine A but was prevented in the presence of dithiothreitol (DTT). Conversely, the incubation of mitochondria in the presence of phenylarsine oxide (PAO) known to oxidize the protein thiol groups also elevated the proton conductance and eliminated Δψ at the inner membrane of lupine mitochondria. The PAO-induced Δψ collapse was not reversed in the presence of ATP, but Δψ was restored after the addition of DTT. These results and the literature data suggest that, under suppressed ETC activity, salicylic acid permeabilizes the inner membrane of mitochondria from cotyledons of lupine seedlings due to opening of a specialized mitochondrial uncoupling channel (MUC) that is permeable to protons and, possibly, to other small cations (K+, Ca2+). An important role in the induction of MUC belongs apparently to oxidative stress resulting in oxidation of thiol groups in protein molecules that constitute this channel or regulate the channel activity.

To involvement the conformation of the adenine nucleotide translocase in opening the Tl+-induced permeability transition pore in Ca2+-loaded rat liver mitochondria

The conformation of adenine nucleotide translocase (ANT) has a profound impact in opening the mitochondrial permeability transition pore (MPTP) in the inner membrane. Fixing the ANT in ‘c’ conformation by phenylarsine oxide (PAO), tert-butylhydroperoxide (tBHP), and carboxyatractyloside as well as the interaction of 4,4′-diisothiocyanostilbene-2,2′-disulfonate (DIDS) with mitochondrial thiols markedly attenuated the ability of ADP to inhibit the MPTP opening. We earlier found (Korotkov and Saris, 2011) that calcium load of rat liver mitochondria in medium containing TlNO3 and KNO3 stimulated the Tl+-induced MPTP opening in the inner mitochondrial membrane. The MPTP opening as well as followed increase in swelling, a drop in membrane potential (ΔΨmito), and a decrease in state 3, state 4, and 2,4-dinitrophenol-uncoupled respiration were visibly enhanced in the presence of PAO, tBHP, DIDS, and carboxyatractyloside. However, these effects were markedly inhibited by ADP and membrane-penetrant hydrophobic thiol reagent, N-ethylmaleimide (NEM) which fix the ANT in ‘m’ conformation. Cyclosporine A additionally potentiated these effects of ADP and NEM. Our data suggest that conformational changes of the ANT may be directly involved in the opening of the Tl+-induced MPTP in the inner membrane of Ca2+-loaded rat liver mitochondria. Using the Tl+-induced MPTP model is discussed in terms finding new transition pore inhibitors and inducers among different chemical and natural compounds.

Thiol dependent isomerization of bovine albumin

Albumin isomerizes to the aged form in the presence of cysteine at pH 8.9 and low ionic strength. Albumins with a high fatty acid and Cu(II) content do not produce isomers, and recover this capacity after an acid expansion. Isomers have the free thiol group fully oxidized (non-mercaptalbumin) and have been isolated for the first time from aged albumins by anion and cation exchange chromatography. Isomers have a higher susceptibility to limited tryptic digestion and show a decrease in the fluorescence of bound dansylamide, a typical marker of site I. Aging in the presence of phenylarsine oxide, which complexes vicinal thiols, impairs the formation of isomers and increases the free –SH groups of albumin.

Constructing a reporter for the activated conformation of carbamoyl phosphate synthetase

Whereas the primary role of most carbamoyl phosphate synthetases (CPSs) is catalysis of the entry step of arginine and/or pyrimidine biosynthesis, using glutamine as a nitrogen source, CPSI has evolved to function in the urea cycle and to use only free ammonia. Additional features unique to CPSI are a requirement for the allosteric activator N-acetylglutamate (AGA) and conserved cysteine residues at positions 1327 and 1337 in human CPSI (hCPSI). Upon AGA activation, the cysteine residues assume a proximate arrangement and can form a disulfide bond in the presence of phenylarsine oxide. Since the only available crystal structure is for the Escherichia coli CPS (eCPS), we are carrying out reciprocal mutation engineering of that enzyme with the goal of using disulfide formation in eCPS as a reporter of the activated conformation. eCPS residues Pro909 and Gly919 correspond, respectively, to hCPSI residues Cys1327 and Cys1337. The individual mutations Pro909Cys and Gly919Cys are tolerated by eCPS. Gly919Cys is able to catalyze carbamoyl phosphate formation nearly as well as wild type eCPS, with respective specific activities of 0.22 and 0.86 μmoles citrulline/min/mg. Pro909Cys has a much lower specific activity of 0.0083 μmoles citrulline/min/mg. Both mutants have significantly decreased ATP Km values, with a 25 fold decrease for Pro909Cys and a 100 fold decrease for Gly919Cys. Analysis of the double mutant Pro909Cys Gly919Cys is underway.

Inhibition of protein-tyrosine phosphatase stimulates the dynamin-dependent endocytosis of ROMK1.

We have previously shown that inhibiting protein-tyrosine kinase increased whereas inhibiting protein-tyrosine phosphatase (PTP) decreased renal outer medullary potassium channel 1 (ROMK1) channel activity (1). We have now used confocal microscopy, the patch clamp technique, and biotin labeling to further examine the role of tyrosine phosphorylation in regulating ROMK1 trafficking. Human embryonic kidney 293 cells were cotransfected with c-Src and green fluorescent protein-ROMK1, which has the same biophysical properties as those of ROMK1. Patch clamp studies have shown that phenylarsine oxide (PAO), an inhibitor of PTP, decreased the activity of ROMK1. Moreover, addition of PAO reduced the cell surface localization of green fluorescent protein-ROMK1 detected by confocal microscopy and diminished the surface ROMK1 density by 65% measured by biotin labeling. Also, PAO treatment significantly increased the phosphorylation of ROMK1. The notion that the effect of PAO is mediated by stimulating tyrosine phosphorylation-induced endocytosis of ROMK1 has also been supported by findings that mutating the tyrosine residue 337 of ROMK1 to alanine abolished the effect of PAO. Finally, the inhibitory effect of PAO on ROMK1 was completely blocked in the cells co-transfected with dominant negative dynamin (dynaminK44A). This indicates that the tyrosine phosphorylation-induced endocytosis of ROMK1 is dynamin-dependent. We conclude that inhibiting PTP increases ROMK1 phosphorylation and results in a dynamin-dependent internalization of the channel.

CAMP Does Not Inhibit Convulxin-Induced Tyrosyl-Phosphorylation of Human Platelet Proteins, Including PLCγ2, But Completely Blocks the Integrin αIIbβ3-Dependent Dephosphorylation Step: Comparisons with RGDS Peptide, Cytochalasin D, and Phenylarsine Oxide

Convulxin (Cvx) isolated fromCrotalus durissus terrificusvenom, induces platelet aggregation, phospholipase C (PLC) activation, and tyrosyl-phosphorylation (PTP) of multiple proteins, including PLCγ2 by a mechanism independent of integrin αIIbβ3. However, PTP induced by Cvx is followed by a dephosphorylation step in a platelet aggregation-dependent manner. Here we show that increasing intraplatelet content of cAMP with forskolin is associated with the inhibition of Cvx-induced platelet aggregation, ATP secretion, and inositol-phosphates production. However, the early onset of Cvx-induced PTP is not sensitive to cAMP (including PLCγ2), and it also occurs in the presence of integrin αIIbβ3-antagonist (RGDS peptide, RGDS) or inhibitors of actin polymerization (cytochalasin D, CD) and tyrosine-phosphatases (phenylarsine oxide, PAO). However, forskolin, RGDS, and CD prevented the dephosphorylation step together with inhibition of platelet aggregation, whereas in the presence of phenylarsine oxide (PAO) the dephosphorylation step was replaced by an increase in the number and intensity of tyrosyl-phosphorylated proteins. Our data provide evidence to conclude that (i) cAMP inhibits platelet aggregation at a downstream site to PLCγ2 tyrosyl-phosphorylation; (ii) Cvx-induced PTP is independent on integrin αIIbβ3engagement, actin polymerization, and tyrosine-phosphatases activation; (iii) integrin αIIbβ3mediates the dephosphorylation step in a platelet aggregation-dependent manner; and (iv) Cvx and collagen stimulate platelets by a similar signal transduction pathway.

Rapid desensitization of B-cell receptor by a dithiol-reactive protein tyrosine phosphatase inhibitor: uncoupling of membrane IgM from syk inhibits signals leading to Ca 2+ mobilization

B-cell antigen receptor (BCR)-mediated calcium response can be blocked by phenylarsine oxide (PAO), a dithiol group-reactive protein tyrosine phosphatase inhibitor. We have examined the mechanism of this inhibition in BL41 Burkitt lymphoma cells. PAO-dependent inhibition is not restricted to the BCR-mediated functions, as evidenced by the failure of the same cells to mobilize Ca 2+ in response to CD19 cross-linking. In contrast, calcium response induced by a putative syk activator, H 2O 2, exhibited only a moderate sensitivity to PAO, demonstrating that PAO did not cause general suppression of all the functions leading to Ca 2+ mobilization. BCR cross-linking or H 2O 2 treatment leads to the induction of almost complete non-responsiveness for the reciprocal stimulation. Since BCR cross-linking did not generate non-responsiveness to H 2O 2 in the presence of PAO, and PAO-treated cells remained responsive to syk activation by H 2O 2, we suppose that PAO may inhibit BCR-mediated signal transduction events upstream of syk activation. This assumption was supported by additional data, indicating that PAO was able to modulate functions of at least 2 different protein tyrosine kinase enzymes involved in BCR-mediated signaling. PAO induced rapid and dose-dependent tyrosine phosphorylation of lyn and selectively inhibited BCR-mediated tyrosine phosphorylation of syk. The results presented in this paper demonstrate that PAO may provoke cellular desensitization process by alteration of the signal transducer functions of lyn and syk tyrosine kinase enzymes.

Stage-specific tyrosine phosphorylation of actin in Dictyostelium discoideum cells.

A 45 kDa protein in Dictyostelium discoideum cells that was recognized by a phosphotyrosine-specific antibody was identified by its binding activity to DNase I and its 2D-electrophoretic behavior as actin. The reactivity of actin with the antibody was transiently enhanced for about 30 minutes shortly after starving cells were reintroduced into nutrient medium. This effect indicates a modification of actin that is regulated under physiological conditions. A similar effect was obtained when growing cells were treated with phenylarsine oxide (PAO), an inhibitor of phosphotyrosine phosphatases. This effect was reversed and the cells fully recovered upon addition of the PAO antagonist 2,3-dimercaptopropanol. Starved cells did not show this enhancement of antibody labelling, which indicates that the response to PAO depends on the developmental stage. Phosphorylated amino acid residues were identified after in vivo labelling with [32P]phosphate in the presence of PAO. Part of the radioactivity in the actin band was recovered as phosphotyrosine, another part as phosphoserine. PAO caused the cells to form elongated blebs, to round up and finally to become immobilized. Fluorescence labelling with phalloidin of cells that were fixed at different times of PAO treatment revealed a progressive decrease in the staining for actin filaments and showed that these alterations in cytoskeleton organization were readily reversible, in accordance with the reversal of tyrosine phosphorylation at actin.

Multiple pathways for ligand internalization in rat hepatocytes I: Effects of anoxia, phenylarsine oxide and monensin

It has been suggested that there are multiple pathways for the cellular internalization of insulin. To investigate these pathways we have examined the effects of three perturbations of endocytosis on the insulin internalization process and have compared these effects with those obtained using an asialoglycoprotein, asialofetuin (Afet), and epidermal growth factor (EGF). Freshly isolated hepatocytes were incubated with radiolabeled ligands and internalization measured under conditions of anoxia to deplete cellular ATP, in the presence of phenylarsine oxide (PAO) to inhibit endocytosis, and in the presence of monensin to interfere with endosomal acidification. Afet internalization essentially was blocked by all three treatment processes, while insulin internalization was inhibited approximately 40% in the presence of anoxia, and 54% in the presence of PAO. Monensin exhibited differential effects on internalization of high and low insulin concentrations. The effects of the treatment processes on EGF internalization were intermediate to those seen with Afet and insulin. These results suggest that insulin and EGF utilize routes of internalization exhibiting different energy requirements that may correspond to coated pit, non-coated pit, and fluid-phase internalization pathways. The observations with Afet internalization remain consistent with utilization of the coated pit pathway.

Analysis of Angiotensin II Binding to Human Platelets: Differences in Young and Old Subjects

We examined the binding of radiolabeled angiotensin II (AII) to human platelets to characterize the apparent increase in AII receptors observed in older subjects. At 22 degrees C, the amount of radioactivity associated with platelets from older subjects increased continuously for more than 2 hours. The same amount of radioactivity was displaced by addition of unlabeled AII at 30 min and 60 min. In the presence of phenylarsine oxide, in the cold, or when labeled antagonist was the ligand, binding came to equilibrium by 30 min. High pressure liquid chromatography demonstrated that 125I-AII was the major radioactive compound in the supernatant and platelets after incubation, but the platelets also contained radiolabeled AII fragments. Thus, some degradation accompanied interaction of AII and platelets. Phenylarsine oxide did not prevent degradation of bound AII, suggesting that degradation precedes internalization. On average, maximum binding was greater in older subjects whether platelets were incubated with 125I-AII alone, with 125I-AII and phenylarsine oxide to prevent internalization, or when the competitive inhibitor 125I-sar1,ile8-AII was the radioligand. Variability of binding among subjects also increased with age. Thus, platelets bind, degrade, and internalize AII, and the three processes occur to a greater extent in platelets from some, but not all older subjects.

Insulin-stimulated Protein Tyrosine Phosphorylation in Intact Cells Evaluated by Giant Two-dimensional Gel Electrophoresis

We have studied the insulin-stimulated phosphorylation of proteins in NIH 3T3 cells expressing high numbers of human insulin receptors (HIR 3.5 cells) using the technique of giant two-dimensional gel electrophoresis. In serum-deprived cells, insulin stimulated the phosphorylation of more than 25 proteins; all but two of these were also phosphorylated in response to 15% (v/v) fetal bovine serum, which also stimulated the phosphorylation of additional proteins thought to be direct substrates for protein kinase C. In cells pretreated insulin specifically stimulated the phosphorylation insulin specifically stimulated the phosphorylation of at least 26 predominantly cytosolic proteins, only one of which was observed in insulin-treated cells not exposed to phenylarsine oxide. Serum was without effect in cells pretreated with phenylarsine oxide. In phenylarsine oxide-pretreated cells, phosphoamino acid analysis of 10 of the most highly labeled insulin-stimulated phosphoproteins showed that all 10 were labeled predominantly or exclusively on tyrosine residues. The phosphorylation of several of these could be stimulated in vitro by the addition of insulin to a detergent extract of cells in the presence of Mn2+ and ATP. In general, the insulin-stimulated phosphorylations observed in the presence of phenylarsine oxide were more rapid than those observed in its absence. Finally, a variety of other growth factors and mitogens did not stimulate any of the insulin-stimulated phosphorylations in the presence of phenylarsine oxide. Thus, the use of this inhibitor apparently unmasked a number of novel insulin-specific protein phosphorylations that were ordinarily undetectable. We suggest that at least some of these proteins may be direct substrates for the insulin receptor protein tyrosine kinase and may play significant roles in insulin action.

Glycosaminoglycan Free Chains: External Plasma Membrane Components Distinct from The Membrane Proteoglycans

Heparan sulfate and chondroitin sulfate glycosaminoglycans of BALB/c 3T3 fibroblasts, metabolically labeled with [3H]glucosamine and [35S]sulfate precursors, are resolved by preparative Sepharose CL-4B chromatography into distinct products, the proteoglycans and the glycosaminoglycan free chains, the latter resistant to appreciable molecular weight shift upon alkaline borohydride reduction. The in situ localization of these cell layer molecules was probed with glycosaminoglycan degrading enzymes (lyases) of bacterial origin, which were used to digest isotopically prelabeled monolayer cultures prior to extraction with a nonionic detergent in the presence of protease inhibitors. Most of the total cellular complement of glycosaminoglycan free chains, in addition to the proteoglycans, proved accessible to the lyases under conditions which did not appreciably affect cell viability or morphology. Because these results were also obtained under low temperature (4 degrees C) conditions and in the presence of phenylarsine oxide, a sulfhydryl reagent that irreversibly inhibits endocytosis, the effects of the lyases are not dependent upon internalization by the cells. The cellular production and cell surface expression of the glycosaminoglycan free chains were not materially altered when lysosomal function was pharmacologically inhibited, confirming that the free chains are not intracellular intermediates in the lysosomal degradation pathways of proteoglycans. Contrary to the prevailing model, our observations establish that, at least in the cell line under study, glycosaminoglycan free chains are located on the external leaflet of the plasma membrane, as such suggesting that these products are biologically active components of cell surfaces.

Phenylarsine oxide stimulates hexose transport in 3T3-L1 adipocytes by a mechanism other than an increase in surface transporters

Phenylarsine oxide (PAO) has been shown to exert a biphasic effect on glucose transport in 3T3-L1 adipocytes. At 10 μm, PAO activates transport threefold, but at higher concentrations an inhibition of transport is observed. In this paper we report a procedure for the subcellular fractionation of these cells which we use to examine the distribution of glucose transporters following PAO challenge. Quantitative immunoblotting showed that the glucose transporter content of the plasma membrane fraction increased with increasing PAO concentrations; a parallel increase in another insulin-responsive protein, the transferrin receptor, also occurred. However, cell-surface labeling procedures for the glucose transporter and transferrin receptor showed that PAO actually decreased the cell-surface concentrations of these proteins; the basis of this discrepancy may be that in the presence of PAO, intracellular vesicles containing these proteins associate with the plasma membrane, but do not fuse with it. The possibility that PAO modulated transport by direct interaction with the glucose transporter was investigated by examining the effects of PAO on transport in both erythrocytes and a reconstituted system of purified erythrocyte transporter in lipid vesicles. PAO was without effect on the rate of transport in these systems. The hypothesis that the stimulatory effect of PAO on transport might be due to the activation of the insulin receptor kinase activity was examined by assessing the phosphotyrosine content of the receptor and other proteins using anti-phosphotyrosine antibodies. PAO alone caused no detectable increase in receptor phosphotyrosine content. However, the combination of PAO and insulin led to the tyrosine phosphorylation of two proteins of M r 68,000 and 57,000 which were not detected in cells treated with either PAO or insulin, and an increased phosphotyrosine content of proteins of M r 95,000 and 165,000 when compared to cells treated with insulin alone.

Effect of vanadate on the cellular accumulation of pp15, an apparent product of insulin receptor tyrosine kinase action.

The possible involvement of a 15-kDa phosphotyrosyl protein, pp15, in insulin action was investigated by using the insulin-mimetic agent, vanadate. Vanadate, a phosphotyrosine phosphatase inhibitor, was found to mimic insulin in 3T3-L1 adipocytes by three criteria. First, kinetic and concentration-dependence studies verified the insulin-like effect of vanadate in activating 2-deoxyglucose uptake. Insulin had an additive activating effect at a submaximal vanadate concentration, but showed no further activation at a saturating vanadate concentration. The trivalent arsenical, phenylarsine oxide (PAO) which forms complexes with vicinal dithiols, markedly inhibited vanadate-activated hexose transport in agreement with our previous studies in which PAO abolished the insulin-activated component of sugar uptake. Second, in situ phosphorylation experiments showed that vanadate activated tyrosine phosphorylation of the insulin receptor's beta-subunit. Exposure of vanadate-treated cells to PAO further increased the level of beta-subunit phosphorylation. The increased level of phosphorylation in the presence of PAO occurred only on tyrosyl residues. Third, vanadate caused the accumulation of a phosphorylated 15-kDa protein in the presence of PAO, but not in its absence. The characteristics of this protein were identical to those of pp15: 1) both proteins behaved identically by two-dimensional gel electrophoresis, 2) digestion of both proteins with trypsin gave rise to apparently identical phosphopeptides, and 3) both proteins contained phosphotyrosine as the only phosphoamino acid. The results indicate that both vanadate and insulin stimulate the accumulation of pp15 in the presence of PAO. The dithiol,2,3-dimercaptopropanol, but not a monothiol, reversed the effects of PAO on the inhibition of vanadate-induced hexose transport and the accumulation of pp15, thus implicating a vicinal dithiol in these actions of vanadate and insulin. Our results support the hypothesis that turnover of the phosphoryl group of pp15, a product of insulin receptor tyrosine kinase action, is coupled to signal transmission to the glucose transport system.