Novel Function of Human RLIP76:  ATP-Dependent Transport of Glutathione Conjugates and Doxorubicin

Abstract

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.

The RAS-RAL axis in cancer: evidence for mutation-specific selectivity in non-small cell lung cancer

“…The involvement of RALBP1 in receptor endocytosis points to the prominence of RAL in signal transduction by these receptors, which regulate various cellular processes such as survival and proliferation [32,33] . RALBP1 has also been identified as a non-ABC transporter that causes resistance to chemotherapeutic drugs [34] . RALBP1 is known to cause resistance to anthracycline derivatives such as doxorubicin and glutathione conjugates by increasing their efflux from cells in an ATP-www.chinaphar.com Guin S et al Acta Pharmacologica Sinica npg dependent manner [34][35][36] .…”

“…RALBP1 has also been identified as a non-ABC transporter that causes resistance to chemotherapeutic drugs [34] . RALBP1 is known to cause resistance to anthracycline derivatives such as doxorubicin and glutathione conjugates by increasing their efflux from cells in an ATP-www.chinaphar.com Guin S et al Acta Pharmacologica Sinica npg dependent manner [34][35][36] . This transporter activity of RALBP1 helps to protect cancer cells from cytotoxic drugs and cellular stress [34] .…”

“…RALBP1 is known to cause resistance to anthracycline derivatives such as doxorubicin and glutathione conjugates by increasing their efflux from cells in an ATP-www.chinaphar.com Guin S et al Acta Pharmacologica Sinica npg dependent manner [34][35][36] . This transporter activity of RALBP1 helps to protect cancer cells from cytotoxic drugs and cellular stress [34] . RALBP1 also induces mitochondrial fission by promoting the phosphorylation of the GTPase DRP1 by CyclinB/CDK1 and facilitating the recruitment of DRP1 to mitochondrial membranes [37] .…”

Mitogenic and drug-resistance mediating effects of PKCα require 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].…”

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

“…Ral proteins mediate cellular functions via a repertoire of effectors such as RalBP1, a GTPase-activating protein for Rac1 and Cdc42 (Jullien-Flores et al, 1995), which also plays a role in the transport of glutathione conjugates (Awasthi et al, 2000), Sec5 and exo84, components of the exocyst involved in regulated secretion (Moskalenko et al, 2003), and the actin regulatory protein filamin (Ohta et al, 1999). 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).…”