Transport of glutathione conjugates and chemotherapeutic drugs by RLIP76 (RALBP1): A novel link between G‐protein and tyrosine kinase signaling and drug resistance

Abstract

Our studies have shown that RLIP76 (RALBP1), a 76 kDa Ral-binding, Rho/Rac-GAP and Ral effector protein, is a novel multispecific transporter of xenobiotics as well as GS-Es. Like previously characterized ABC transporters, it mediates ATP-dependent transport of structurally unrelated amphiphilic xenobiotics and displays inherent ATPase activity, which is stimulated by its substrate allocrites. It does not have significant sequence homology with ABC transporters and differs from the ABC transporters in several other important aspects, including (i) lack of any close homologs in humans, (ii) lack of a classical Walker domain, (iii) integral membrane association without clearly defined transmembrane domains and (iv) its role as a direct link to Ras/Ral/Rho and EGF-R signaling through its multifunctional nature, including GAP activity, regulation of exocytosis as well as clathrin-coated pit-mediated receptor endocytosis. Its multifunctional nature derives from the presence of multiple motifs, including a Rho/Rac GAP domain, a Ral effector domain binding motif, 2 distinct ATP-binding domains, a H ؉ -ATPase domain, PKC and tyrosine kinase phosphorylation sites and the ability to undergo fragmentation into multiple smaller peptides which participate as components of macromolecular functional complexes. One of the physiologic functions of RLIP76 is regulation of intracellular concentration of the electrophilic intermediates of oxidative lipid metabolism by mediating efflux of GS-E formed from oxidative degradation of arachidonic acid, including leukotrienes and the 4HNE-GSH conjugate. RLIP76-mediated transport of amphiphilic chemotherapeutic agents such as anthracyclines and vinca alkaloids as well as GS-E produced during oxidative metabolism places this multifunctional protein in a central role as a resistance mechanism for preventing apoptosis caused by chemotherapeutic agents and a variety of external/internal stressors, including oxidative stress, heat shock and radiation. GSH 1 is a ubiquitous nucleophilic sulfhydryl tripeptide, which protects cells from damage caused by endogenously generated (endobiotic) as well as exogenous (xenobiotic) electrophilic and oxidant chemicals. GSH can serve as an electron donor for reduction of oxidant species, including highly toxic lipid hydroperoxides generated as a common toxic intermediate during any oxidative stress (chemical, heat, radiation) of sufficient magnitude. Reduction of these highly reactive species terminates lipid peroxidation, a chain reaction that can markedly amplify the damaging effects of oxidative injury. Through formation of thioether conjugates, GSH also functions as a scavenger of the exogenous alkylating agents, as well as of less reactive but still quite toxic alkylating electrophiles generated as downstream products of lipid peroxidation. Formation of GS-Es occurs spontaneously, as well as through glutathione S-transferase-catalyzed reactions, and is the first step in the metabolism of electrophiles to mercapturates. In addition to being precursors of ...

Mitogenic and drug-resistance mediating effects of PKCα require RLIP76

“…RLIP76 protein levels were measured by specific ELISA [15] and normalized to total protein in crude cell homogenate measured by Bradford's assay (A). The normalized values, showing RLIP76 protein as a percentage of total protein, are presented for RLIP76 +/+ and RLIP76 −/− MEFs treated with RLIP76-proteoliposomes (10,20,40 or 60 μg purified RLIP76 protein/ml). Control liposomes contained an equal amount of heat-inactivated purified RLIP76.…”

“…Increased PKCα-mediated ☆ Abbreviations: RLIP76 (RALBP1), Ral-interacting protein; DOX, doxorubicin; SCLC, small cell lung cancer; NSCLC, non-small cell lung cancer; GSH, glutathione; GS-E, glutathione-electrophile conjugate; DNP-SG, dinitrophenyl S-glutathione; MRP, multidrug-resistance associated protein; PMA, phorbol ester (phorbol 12-myristate 13-acetate); PKC, protein-kinase-C. We have recently identified a new target of PKCα, RLIP76 (RALBP1). RLIP76 is a Ralbinding Rho-GAP protein (inhibitor of Rho-signaling) which we have shown to be the predominant cellular mechanism for ATP-dependent effux of glutathione-conjugate (GS-E) and chemotherapy drugs (such as DOX) [6][7][8][9][10][11][12][13][14][15][16][17][18]. Although it can function as a drugresistance transporter, the major physiological role of RLIP76 appears to be the regulation of intracellular levels of lipid-alkenals and alkenal-glutathione conjugates, the formation of which is an early and obligatory event in course of oxidant/radiant stress or signaling [8][9][10]19] The transport activity of RLIP76 functions to regulate cellular levels of glutathionyladducts of lipidoxidation derived reactive oxygen species which are known to exert direct effects in cell proliferation, differentiation, and apoptosis [8][9][10]19].…”

“…RLIP76 is a Ralbinding Rho-GAP protein (inhibitor of Rho-signaling) which we have shown to be the predominant cellular mechanism for ATP-dependent effux of glutathione-conjugate (GS-E) and chemotherapy drugs (such as DOX) [6][7][8][9][10][11][12][13][14][15][16][17][18]. Although it can function as a drugresistance transporter, the major physiological role of RLIP76 appears to be the regulation of intracellular levels of lipid-alkenals and alkenal-glutathione conjugates, the formation of which is an early and obligatory event in course of oxidant/radiant stress or signaling [8][9][10]19] The transport activity of RLIP76 functions to regulate cellular levels of glutathionyladducts of lipidoxidation derived reactive oxygen species which are known to exert direct effects in cell proliferation, differentiation, and apoptosis [8][9][10]19].RLIP76 is activated to increase its GS-E and DOX transporting activity through phosphorylation by PKCα at T297 [16,17]. The greater transporter activity of the phosphorylated form of RLIP76, which predominates in NSCLC, is the primary determinant of intrinsic DOX-resistance of NSCLC [16,17].…”

Expression profiling of Ral-depleted bladder cancer cells identifies RREB-1 as a novel transcriptional Ral effector

“…Through these, RalA and/or RalB regulate endocytosis of membrane receptors, exocytosis and delivery of polarized membrane proteins (Shipitsin and Feig, 2004), cytoskeletal dynamics (Lebreton et al, 2004), drug resistance (Awasthi et al, 2003), motility (Gildea et al, 2002) and both anchorage-dependent and anchorage-independent proliferation (Chien and White, 2003).…”

Activation of Ras-Ral Pathway Attenuates p53-independent DNA Damage G2 Checkpoint

“…Through the RalGEFs Ras stimulates the Ral proteins that can result in activation of the RLIP76/ RalBP1 protein functioning as Cdc42/Rac-GTPase-activating protein and a transporter of amphilitic molecules, activation of phospholipase D1, and alteration of the activity of a group of transcription factors (4,17,18). These Ral activities are involved in the regulation of cell morphology and proliferation, receptor endocytosis, and transport of xenobiotics (17,18,34). Noteworthy, unlike in mouse fibroblasts, the activation of RalGEFs in human cells rather than the activation of Raf or PI3K plays a central role in the Ras-induced oncogenic transformation (19,20).…”