Accelerated metabolism and exclusion of 4-hydroxynonenal through induction of RLIP76 and hGST5.8 is an early adaptive response of cells to heat and oxidative stress

To explore the role of lipid peroxidation (LPO) products in the initial phase of stress mediated signaling, we studied the effect of mild, transient oxidative or heat stress on parameters that regulate the cellular concentration of 4-hydroxynonenal (4-HNE). When K562 cells were exposed to mild heat shock (42 degrees C, 30 min) or oxidative stress (50 microM H2O2, 20 min) and allowed to recover for 2 h, there was a severalfold induction of hGST5.8, which catalyzes the formation of glutathione-4-HNE conjugate (GS-HNE), and RLIP76, which mediates the transport of GS-HNE from cells (Awasthi, S., Cheng, J., Singhal, S. S., Saini, M. K., Pandya, U., Pikula, S., Bandorowicz-Pikula, J., Singh, S. V., Zimniak, P., and Awasthi, Y. C. (2000) Biochemistry 39, 9327-9334). Enhanced LPO was observed in stressed cells, but the major antioxidant enzymes and HSP70 remained unaffected. The stressed cells showed higher GS-HNE-conjugating activity and increased efflux of GS-HNE. Stress-pre-conditioned cells with induced hGST5.8 and RLIP76 acquired resistance to 4-HNE and H2O2-mediated apoptosis by suppressing a sustained activation of c-Jun N-terminal kinase and caspase 3. The protective effect of stress pre-conditioning against apoptosis was abrogated by coating the cells with anti-RLIP76 IgG, which inhibited the efflux of GS-HNE from cells, indicating that the cells acquired resistance to apoptosis by metabolizing and excluding 4-HNE at a higher rate. Induction of hGST5.8 and RLIP76 by mild, transient stress and the resulting resistance of stress-pre-conditioned cells to apoptosis appears to be a general phenomenon since it was not limited to K562 cells but was also evident in lung cancer cells, H-69, H-226, human leukemia cells, HL-60, and human retinal pigmented epithelial cells. These results strongly suggest a role of LPO products, particularly 4-HNE, in the initial phase of stress mediated signaling.     

Lipid peroxidation and cell cycle signaling: 4-hydroxynonenal,a key molecule in stress mediated signaling

Role of lipid peroxidation products, particularly 4-hydroxynonenal (4-HNE) in cell cycle signaling is becoming increasingly clear. In this article, recent studies suggesting an important role of 4-HNE in stress mediated signaling for apoptosis are critically evaluated. Evidence demonstrating the modulation of UV, oxidative stress, and chemical stress mediated apoptosis by blocking lipid peroxidation by the alpha-class glutathione S-transferases (GSTs) is presented which suggest an important role of these enzymes in protection against oxidative stress and a role of lipid peroxidation products in stress mediated signaling. Overexpression of 4-HNE metabolizing GSTs (mGSTA4-4, hGSTA4-4, or hGST5.8) protects cells against 4-HNE, oxidative stress (H(2)O(2) or xanthine/xanthine oxidase), and UV-A mediated apoptosis by blocking JNK and caspase activation suggesting a role of 4-HNE in the mechanisms of apoptosis caused by these stress factors. The intracellular concentration of 4-HNE appears to be crucial for the nature of cell cycle signaling and may be a determinant for the signaling for differentiation, proliferation, transformation, or apoptosis. The intracellular concentrations of 4-HNE are regulated through a coordinated action of GSTs (GSTA4-4 and hGST5.8) which conjugate 4-HNE to GSH to form the conjugate (GS-HNE) and the transporter 76 kDa Ral-binding GTPase activating protein (RLIP76), which catalyze ATP-dependent transport of GS-HNE. A mild stress caused by heat, UV-A, or H(2)O(2)with no apparent effect on the cells in culture causes a rapid, transient induction of hGST5.8 and RLIP76. These stress preconditioned cells acquire ability to metabolize and exclude 4-HNE at an accelerated pace and acquire relative resistance to apoptosis by UV and oxidative stress as compared to unconditioned control cells. This resistance of stress preconditioned cells can be abrogated by coating the cells with anti-RLIP76 antibodies which block the transport of GS-HNE. These studies and previous reports discussed in this article strongly suggest a key role of 4-HNE in stress mediated signaling.     

Functional reconstitution of Ral-binding GTPase activating protein,RLIP76, in proteoliposomes catalyzing ATP-dependent transport of glutathione conjugate of 4-hydroxynonenal

Earlier studies from our laboratories have shown that RLIP76, a previously described Ral-binding GTPase activating protein (Jullien-Flores et al., 1995, J. Biol. Chem. 270: 22473), is identical with the xenobiotic transporter DNP-SG ATPase, and can catalyze ATP-dependent transport of glutathione-conjugates as well as doxorubin (Awasthi et al., 2000, Biochemistry, 39: 9327). We have now reconstituted purified bacterially expressed RLIP76 in proteoliposomes, and have studied ATP-dependent uptake of the glutathione conjugate of 4-hydroxynonenal (GS-HNE) by these vesicles. Results of these studies show that RLIP76 reconstituted in proteoliposomes catalyzes ATP-dependent transport of GS-HNE against a concentration gradient. The transport of GS-HNE is saturable with respect to ATP as well as GS-HNE with K(m) values of 1.4mM and 2.5 microM, respectively. These studies demonstrate that RLIP76 mediates active transport of GS-HNE, and are consistent with our previous work showing that RLIP76-mediated efflux of GS-HNE regulates the intracellular concentration of 4-HNE and thereby affects 4-HNE mediated signaling.     

Role of 4-hydroxynonenal in stress-mediated apoptosis signaling

In this mini review we summarize recent studies from our laboratory, which show the involvement of 4-hydroxynonenal (4-HNE) in cell cycle signaling. We demonstrate 4-HNE induced apoptosis in various cell lines is accompanied with c-Jun-N-terminal kinase and caspase-3 activation. Cells exposed to mild, transient, heat or oxidative stress acquire capacity to exclude intracellular 4-HNE at a faster rate by inducing hGST5.8 which conjugate 4-HNE to GSH, and RLIP76 which mediates the ATP-dependent transport of the GSH-conjugate of 4-HNE. The cells preconditioned with mild transient stress acquire resistance to H(2)O(2) and 4-HNE induced apoptosis by excluding intracellular 4-HNE at an accelerated pace. Furthermore, a decrease in intracellular concentration of 4-HNE achieved by transfecting cells with mGSTA4-4 or hGSTA4-4 results in a faster growth rate. These studies strongly suggest a role of 4-HNE in stress mediated signaling.     

Transport functions and physiological significance of 76 kDa Ral-binding GTPase activating protein (RLIP76)

We have recently demonstrated that a previously known Ral-binding GTPase activating protein, RLIP76, can also catalyze ATP-dependent transport of various structurally unrelated xeno- and endobiotics irrespective of their net charge (Awasthi et al., 2000, Biochemistry, 39: 9327). RLIP76 is a non-ATP binding cassette (ABC) protein but it has two ATP-binding sites and shows basal ATPase activity which is stimulated in the presence of its transport substrates (allocrites) such as doxorubicin (DOX) and S-(2,4-dinitrophenyl) glutathione (DNP-SG). Proteoliposomes reconstituted with purified RLIP76 catalyze ATP-dependent, saturable transport of DOX, as well as of glutathione-conjugates including leukotrienes (LTC4) and the GSH-conjugate of 4-hydroxynonenal (GS-HNE). In erythrocytes the majority of transport activity for DOX, GS-HNE, and LTC4 is accounted for by RLIP76. Cells exposed to mild oxidative stress show a rapid and transient induction of RLIP76 resulting in an increased efflux of GS-HNE and acquire resistance to oxidative stress mediated toxicity and apoptosis. Cells transfected with RLIP76 acquire resistance to DOX through increased efflux of the drug suggesting its possible role in the mechanisms of drug-resistance. In this article, we discuss the significance of transport functions of RLIP76 highlighting its role in the defense mechanisms against oxidative injury, and modulation of signaling mechanisms.     

Mitogenic responses of vascular smooth muscle cells to lipid peroxidation-derived aldehyde 4-hydroxy-trans-2-nonenal (HNE): role of aldose reductase-catalyzed reduction of the HNE-glutathione conjugates in regulating cell growth

Products of lipid peroxidation such as 4-hydroxy-trans-2-nonenal (HNE) trigger multiple signaling cascades that variably affect cell growth, differentiation, and apoptosis. Because glutathiolation is a significant metabolic fate of these aldehydes, we tested the possibility that the bioactivity of HNE depends upon its conjugation with glutathione. Addition of HNE or the cell-permeable esters of glutathionyl-4-hydroxynonenal (GS-HNE) or glutathionyl-1,4-dihydroxynonene (GS-DHN) to cultures of rat aortic smooth muscle cells stimulated protein kinase C, NF-kappaB, and AP-1, and increased cell growth. The mitogenic effects of HNE, but not GS-HNE or GS-DHN, were abolished by glutathione depletion. Pharmacological inhibition or antisense ablation of aldose reductase (which catalyzes the reduction of GS-HNE to GS-DHN) prevented protein kinase C, NF-kappaB, and AP-1 stimulation and the increase in cell growth caused by HNE and GS-HNE, but not GS-DHN. The growth stimulating effect of GS-DHN was enhanced in cells treated with antibodies directed against the glutathione conjugate transporters RLIP76 (Ral-binding protein) or the multidrug resistance protein-2. Overexpression of RLIP76 abolished the mitogenic effects of HNE and its glutathione conjugates, whereas ablation of RLIP76 using RNA interference promoted the mitogenic effects. Collectively, our findings suggest that the mitogenic effects of HNE are mediated by its glutathione conjugate, which has to be reduced by aldose reductase to stimulate cell growth. These results raise the possibility that the glutathione conjugates of lipid peroxidation products are novel mediators of cell signaling and growth.     

Mitogenic and drug-resistance mediating effects of PKCalpha require RLIP76

PKCalpha-activation is a key signaling event governing cell growth, stress-resistance, and drug-resistance. Our recent studies demonstrated that DOX-resistance mediating effects of PKCalpha require the presence of RLIP76, and their concerted action is sufficient to explain intrinsic DOX-resistance of NSCLC [S.S. Singhal, D. Wickramarachchi, J. Singhal, S. Yadav, Y.C. Awasthi, et al., Determinants of differential doxorubicin sensitivity between SCLC and NSCLC. FEBS Lett. 580 (2006) 2258-2264]. Present studies were carried out to further explore the suggestion from the previous studies that the mitogenic effects of PKCalpha also require RLIP76. RLIP76-/- MEFs were resistant to PKCalpha-depletion mediated growth inhibition, as well as to the PKCalpha-dependent mitogen, phorbol 12-myristate 13-acetate (PMA). Augmenting cellular levels of RLIP76 using purified recombinant RLIP76 increased growth rate in all cells, and restored the sensitivity of RLIP76-/- MEFs to both inhibition through PKCalpha-depletion and stimulation through PMA. These results show that RLIP76 is a necessary down-stream effector for PKCalpha-mediated mitogenesis.     

Effects of mGST A4 transfection on 4-hydroxynonenal-mediated apoptosis and differentiation of K562 human erythroleukemia cells

Cellular levels of downstream products of membrane lipid oxidation appear to regulate differentiation in K562 human erythroleukemia cells. 4-Hydroxynonenal (4-HNE) is a diffusible and relatively stable product of peroxidation of arachidonic and linoleic acids, cellular levels of which are regulated through metabolism to glutathione (GSH) conjugate by glutathione S-transferases (GSTs). A group of immunologically related alpha-class mammalian GSTs expressed in mice (mGST A4-4), rat (rGST A4-4), human (hGST A5.8), and other species, as well as the more distantly related human hGST A4-4, preferentially utilize 4-HNE as a substrate and are suggested to be major determinants of intracellular levels of 4-HNE. Present studies were designed to examine the effects of 4-HNE on K562 cells and to study the effect of transfection of mGSTA4-4 in these cells. Exposure of K562 cells to 20 microM 4-HNE for 2 h resulted in a rapid erythroid differentiation of K562 cells, as well as apoptosis evidenced by characteristic DNA laddering. Stable transfection of cells with mGST A4-4 resulted in a fivefold increase in GST-specific activity toward 4-HNE compared with wild-type or vector-only transfected cells. The mGST A4-4-transfected cells were resistant to the cytotoxic, apoptotic, and differentiating effects of 4-HNE. The mGST A4 transfection also conferred resistance to direct oxidative stress (IC(50) of H(2)O(2) 22, 23, and 35 microM for wild-type, vector-transfected, and mGST A4-transfected cells, respectively). mGST A4-4-transfected cells also showed a higher rate of proliferation compared with wild-type or vector-transfected K562 cells (doubling time 22.1 +/- 0.7, 31 +/- 1.2, and 29 +/- 0.6 h, respectively). Cellular 4-HNE levels determined by mass spectrometry were lower in mGST A4-4-transfected cells compared to cells transfected with vector alone (5.9 pmol/5 x 10(7) cells and 62.9 pmol/5 x 10(7) cells, respectively). Our studies show that 4-HNE can induce erythroid differentiation in K562 cells and that overexpression of mGST A4 suppresses 4-HNE levels and inhibits erythroid differentiation and apoptosis.     

Purification and functional reconstitution of intact ral-binding Gtpase activating protein, RLIP76, in artificial liposomes.

We have recently shown that RLIP76, a ral-binding GTPase activating protein, mediates ATP-dependent transport of glutathione-conjugates (GS-E) and doxorubicin (DOX) (S. Awasthi et al., Biochemistry 39,9327,2000). Transport function of RLIP76 was found to be intact despite considerable proteolytic fragmentation in preparations used for those studies, suggesting either that the residual intact RLIP76 was responsible for transport activity, or that the transport activity could be reconstituted by fragments of RLIP76. If the former were true, intact RLIP76 would have a much higher specific activity for ATP-hydrolysis than the fragmented protein. We have addressed this question by comparing transport properties of recombinant RLIP76 and human erythrocyte membrane RLIP76 purified in buffers treated with either 100 or 500 microM serine protease inhibitor, PMSF. The purity and identity of recombinant and human erythrocyte RLIP76 was established by SDS/PAGE and Western-blot analysis. These studies confirmed the origin of the 38 kDa protein, previously referred to as DNP-SG ATPase, from RLIP76. Higher PMSF concentration resulted in lower yield of the 38 kDa band and higher yield of intact RLIP76 from both human and recombinant source. In contrast, the substrate-stimulated ATPase activity in presence of DNP-SG, doxorubicin, daunorubicin, or colchicine were unaffected by increased PMSF; similarly, ATP-dependent transport of doxorubicin in proteoliposomes reconstituted with RLIP76 was unaffected by higher PMSF. These results indicated that limited proteolysis by serine proteases does not abrogate the transport function of RLIP76. Comparison of transport kinetics for daunorubicin between recombinant vs human erythrocyte RLIP76 revealed higher specific activity of transport for tissue purified RLIP76, indicating that additional factors present in tissue purified RLIP76 can modulate its transport activity.     

POB1 over-expression inhibits RLIP76-mediated transport of glutathione-conjugates, drugs and promotes apoptosis

RLIP76 (RALBP1) is a Ral-binding nucleotidase which functions as an energy-dependent transporter for glutathione (GSH)-conjugates as well as structurally unrelated xenobiotics. Partner of RALBP1 (POB1), also referred to as REPS2, was identified as the human RLIP76-binding protein, which contains a coiled-coil C-terminal region that binds with the RLIP76. Recent studies show that over-expression of POB1 in prostate cancer cells induces apoptosis. In present studies, we have purified POB1 and one of its deletion mutants POB1(1-512) (lacking the RLIP76-binding domain), and examined their effect on the transport activity of RLIP76. Both doxorubicin and a model GSH-conjugate, dinitrophenyl-S-glutathione (DNP-SG), transport were inhibited by POB1 in a concentration-dependent manner but not by POB1(1-512), lacking RLIP76-binding site. Liposomal delivery of recombinant POB1 to H358 (NSCLC) cancer cells caused apoptosis in a concentration-dependent manner, whereas the POB1 mutant deficient in RLIP76-binding site did not exert this effect. Augmentation of cellular POB1 resulted in increased intracellular DOX-accumulation as well as decreased rate of efflux from cells. These results show for the first time that POB1 can regulate the transport function of RLIP76 and are consistent with our previous studies showing that inhibition of RLIP76 induces apoptosis in cancer cells through the accumulation of endogenously formed GSH-conjugates.     

Novel function of human RLIP76: ATP-dependent transport of glutathione conjugates and doxorubicin

Active transport of conjugated and unconjugated electrophiles out of cells is essential for cellular homeostasis. We have previously identified in human tissues a transporter, DNP-SG [S-(2, 4-dinitrophenyl)glutathione] ATPase, capable of carrying out this function [Awasthi et al. (1998) Biochemistry 37, 5231-5238, 5239-5248]. We now report the cloning of DNP-SG ATPase. The sequence of the cDNA clone was identical to that of human RLIP76, a known Ral-binding protein. RLIP76 expressed in E. coli was purified by DNP-SG affinity chromatography. Purified recombinant RLIP76: (1) had ATPase activity stimulated by DNP-SG or doxorubicin (DOX), and the K(m) values of RLIP76 for ATP, DOX, and DNP-SG were similar to those reported for DNP-SG ATPase; (2) upon reconstitution with asolectin as well as with defined lipids, catalyzed ATP-dependent transport of DNP-SG and DOX with kinetic parameters similar to those of DNP-SG ATPase; (3) when transfected into K562 cells, resulted in increased resistance to DOX, and increased ATP-dependent transport of DNP-SG and DOX by inside-out membrane vesicles from transfected cells; (4) direct uptake of purified RLIP76 protein into mammalian cells from donor proteoliposomes confers DOX resistance. These results indicate that RLIP76, in addition to its role in signal transduction, can catalyze transport of glutathione conjugates and xenobiotics, and may contribute to the multidrug resistance phenomenon.     

RLIP76 Depletion Enhances Autophagic Flux in U251 Cells

Our previous study showed that RalA-binding protein 1 (RLIP76) is overexpressed in gliomas and is associated with higher tumour grade and decreased patient survival. Furthermore, RLIP76 downregulation increases chemosensitivity of glioma cells to temozolomide by inducing apoptosis. However, other mechanisms underlying RLIP76-associated chemoresistance are unknown. In this study, we investigated the effect of RLIP76 depletion on autophagy. RLIP76 was knocked down in U251 glioma cells using shRNA and autophagy-related proteins, and PI3K/Akt signalling components were evaluated. RLIP76 depletion significantly increased cell autophagy as demonstrated by a significant increase in LC3 II, autophagy protein 5 (ATG-5), and Beclin1, and a decrease in p62 expression levels. Furthermore, RLIP76 knockdown increased autophagic flux in U251 cells as autolysosome numbers increased relative to autophagosome numbers. Autophagy induced by RLIP76 knockdown resulted in increased apoptosis that was independent of temozolomide treatment. Moreover, RLIP76 knockdown decreased PI3K and Akt activation. RLIP76 depletion also resulted in decreased levels of the anti-apoptotic protein Bcl2. LY294002, a PI3K/Akt pathway inhibitor, led to increased autophagy and apoptosis in U251 RLIP76-depleted cells. Therefore, RLIP76 knockdown increased autophagic flux and apoptosis in U251 glioma cells, possibly through inhibition of the PI3K/Akt pathway. Thus, this study provides a novel mechanism for the role of RLIP76 in glioma pathogenesis and chemoresistance.     

Identification of membrane-anchoring domains of RLIP76 using deletion mutant analyses

RLIP76 (RALBP1) is a multifunctional transporter involved in signaling and transmembrane movement of solute allocrites, which include glutathione conjugates and several natural product antineoplastic agents [Awasthi, S., et al. (2000) Biochemistry 39, 9327-9334; (2001) Biochemistry 40, 4159-4168]. Our previous studies suggested that the membrane-anchoring domain resides in the N-terminus of RLIP76, despite the lack of identifiable membrane-spanning domains. Amino acid sequence analysis indicated that this region of RLIP76 contains sequences that are similar to those of vector peptides. We, therefore, have studied the effect of a series of deletion mutant proteins on hydrophobicity and transport activity. RLIP76 or one of its derived deletion mutants was expressed in Escherichia coli, and bacteria were lysed and extracted in buffer without or with the nonionic detergent polidocanol. The ratio of RLIP76 in the detergent/aqueous extracts was found to be 2.5 for the wild-type protein, but decreased to 0.7 in the mutant in which amino acids 154-219 were deleted. Deletion of only one segment of this region (amino acids 171-185) alone resulted in a significant decrease in this ratio to 1.0. For the mutants with deletions within the region from amino acid 154 to 219, loss of hydrophobicity correlated with less incorporation of mutants into artificial liposomes, and decreased transport activity toward doxorubicin and dinitrophenyl-S-glutathione. In contrast, deletion of one of the two ATP-binding sites (at amino acids 65-80 or 415-448) or both sites did not affect hydrophobicity but reduced or abrogated transport activity. NSCLC (H358) stably transfected with del171-185 and del154-219 showed that loss of these regions results in a decrease in the extent of membrane association of RLIP76. Confocal laser immunohistochemistry colocalized amino acids 171-185 with her2/neu on the cell surface. Depletion of wild-type RLIP76 using si-RNA directed to this region in cells transfected with del171-185 resulted in the loss of cell surface expression. These finding demonstrate that amino acids 171-185 constitute a cell surface epitope which is necessary for optimal transport of anthracycline and glutathione conjugates by RLIP76, and that this peptide could be a novel target for antineoplastic therapy.     

RLIP76 is the major ATP-dependent transporter of glutathione-conjugates and doxorubicin in human erythrocytes.

We have recently demonstrated that RLIP76, a Ral-binding GTPase activating protein mediates ATP-dependent transport of glutathione (GSH) conjugates of electrophiles (GS-E) as well as doxorubicin (DOX), and that it is identical with DNP-SG ATPase, a GS-E transporter previously characterized by us in erythrocyte membranes (Awasthi et al. Biochemistry 39, 9327-9334). Multidrug resistance-associated protein (MRP1) belonging to the family of the ABC-transporters has also been suggested to be a GS-E transporter in human erythrocytes. Using immunological approaches, the present studies were designed to elucidate the relative contributions of RLIP76, MRP1, and P-glycoprotein (Pgp), in the ATP-dependent transport of GS-E and DOX in human erythrocytes. In Western blot analyses using antibodies against RLIP76, a strong expression of RLIP76 was observed in erythrocytes. Immunohistochemical studies using a fluorescent probe showed association of RLIP76 with erythrocyte membrane, which was consistent with its transport function. Neither MRP1 nor Pgp were detected in erythrocytes when the antibodies against MRP1 or Pgp were used. In erythrocyte inside-out vesicles (IOVs) coated with antibodies against RLIP76, a dose-dependent inhibition of the ATP-dependent transport of DOX and GS-E, including S-(dinitrophenyl)glutathione (DNP-SG), leukotriene C(4), and the GSH conjugate of 4-hydroxynonenal, was observed with a maximal inhibition of about 70%. On the contrary, in the IOVs coated with the antibodies against MRP1 or Pgp no significant inhibition of the ATP-dependent transport of these compounds was observed. These findings suggest that RLIP76 is the major ATP-dependent transporter of GS-E and DOX in human erythrocytes.     

Functional reassembly of ATP-dependent xenobiotic transport by the N- and C-terminal domains of RLIP76 and identification of ATP binding sequences

We have recently shown that RLIP76, a Ral-binding, GTPase-activating protein, is an ATP-dependent transporter of doxorubicin (DOX) as well as glutathione conjugates [Awasthi, S., et al. (2000) Biochemistry 39, 9327-9334]. RLIP76 overexpressed in human cells or transformed E. coli undergoes proteolysis to yield several fragments, including two prominent peptides, N-RLIP76(1-367) and C-RLIP76(410-655), from the N- and C-terminal domains, respectively. To investigate whether the fragmentation of RLIP76 has any relevance to its transport function, we have studied the characteristics of these two peptide fragments. Recombinant N-RLIP76(1-367) and C-RLIP76(410-655) were purified from overexpressing transformed E. coli. While N-RLIP76(1-367) readily underwent proteolysis, showing SDS-gel patterns similar to those of RLIP76, C-RLIP76(410-655) was resistant to such degradation. Both N-RLIP76(1-367) and C-RLIP76(410-655) had ATPase activity (K(m) for ATP, 2.5 and 2.0 mM, respectively) which was stimulated by DNP-SG, DOX, and colchicine (COL). ATP binding to both peptides was confirmed by photoaffinity labeling with 8-azido-ATP that was increased in the presence of compounds that stimulated their ATPase activity. Photoaffinity labeling was also increased in the presence of vanadate, indicating trapping of a reaction intermediate in the ATP binding site. The ATP binding sites in N-RLIP76(1-367) and C-RLIP76(410-655) were identified to be (69)GKKKGK(74) and (418)GGIKDLSK(425), respectively. Mutation of K(74) and K(425) to M residues, in N-RLIP76(1-367) and C-RLIP76(410-655), respectively, abrogated their ATPase activity as well as azido-ATP labeling. Proteoliposomes reconstituted with either N-RLIP76(1-367) or C-RLIP76(410-655) alone did not catalyze ATP-dependent transport of DOX or COL. However, proteoliposomes reconstituted with a mixture of N-RLIP76(1-367) and C-RLIP76(410-655) mediated such transport. Proteoliposomes reconstituted with the mixture of mutant peptides lacking ATPase activity did not exhibit transport activity. Present studies have identified the ATP binding sites in RLIP76, and show that DOX and COL transport can be reconstituted by two fragments of RLIP76.     

Transfection of mGSTA4 in HL-60 cells protects against 4-hydroxynonenal-induced apoptosis by inhibiting JNK-mediated signaling

The mammalian alpha-class glutathione S-transferase (GST) isozymes mGSTA4-4, rGSTA4-4, and hGSTA4-4 are known to utilize 4-hydroxynonenal (4HNE) as a preferred substrate. During the present studies, we have examined the effect of transfecting human myeloid HL-60 cells with mGSTA4, on 4-HNE-induced apoptosis and the associated signaling mechanisms. Results of these studies show that treatment of the wild-type or vector-only-transfected HL-60 cells with 20 microM 4-HNE caused apoptosis within 2 h. The cells transfected with mGSTA4 did not undergo apoptosis under these conditions even after 4 h. In the wild-type and vector-transfected cells, apoptosis was preceded by JNK activation and c-Jun phosphorylation within 30 min, and an increase in AP-1 binding within 2 h of treatment with 20 microM 4-HNE. In mGSTA4-transfected cells, JNK activation and c-Jun phosphorylation were observed after 1 h, and increased AP-1 binding was observed after 8 h under these conditions. In the control cells, 20 microM 4-HNE caused caspase 3 activation and poly(ADP-ribose) polymerase cleavage within 2 h, while in mGSTA4-transfected cells, a lesser degree of these effects was observed even after 8 h. Transfection with mGSTA4 also provided protection to the cells from 4-HNE and doxorubicin cytotoxicity (1.6- and 2.6-fold, respectively). These results show that 4-HNE mediates apoptosis through its effects on JNK and caspase 3, and that 4-HNE metabolizing GST isozyme(s) may be important in the regulation of this pathway of oxidative-stress-induced apoptosis.     

The RLIP76 N-terminus binds ARNO to regulate PI 3-kinase,Arf6 and Rac signaling,cell spreading and migration

RLIP76 is a multifunctional protein involved in tumor growth and angiogenesis, and a promising therapeutic target in many cancers. RLIP76 harbors docking sites for many proteins, and we have found that it interacts with ARNO, a guanine nucleotide exchange factor for Arf6, and that RLIP76 regulates activation of Rac1 via Arf6, and regulates cell spreading and migration in an ARNO and Arf6-dependent manner. Here we show that ARNO interacts with the RLIP76 N-terminal domain, and this domain was required for RLIP76-dependent cell spreading and migration. We identified two sites in the RLIP76 N-terminus with differential effects on ARNO binding and downstream signaling: Ser29/Ser30 and Ser62. Ser29/30 mutation to Alanine inhibited ARNO interaction and was sufficient to block RLIP76-dependent cell spreading and migration, as well as RLIP76-dependent Arf6 activation. In contrast, RLIP76(S62A) interacted with ARNO and supported Arf6 activation. However, both sets of mutations blocked Rac1 activation. RLIP76-mediated Rac and Arf6 activation required PI3K activity. S29/30A mutations inhibited RLIP76-dependent PI3K activation, but S62A mutation did not. Together these results show that ARNO interaction with the RLIP76 N-terminus regulates cell spreading and motility via PI3K and Arf6, independent of RLIP76 control of Rac.