An incomplete picture of transport mechanisms responsible for drug-resistance due to accumulation-defects has led to a wide array of opinions due to inconsistent or conflicting data regarding the relative significance of a multitude of transport mechanisms. Animal and human clinical data indicate that the ABC-transporters P-glycoprotein (Pgp), MRP1 and related transporters are clearly able to mediate drug-accumulation defects in cultured malignant cells, but correlations with pathology, clinical resistance and outcomes in lung cancer are poor, and attempts at improving therapeutic efficacy by targeting these have not been successful [1
]. Our findings have supplied a missing piece of the puzzle to the understanding of multi-specific transport mechanisms, a stress-responsive non-ABC, high capacity transporter, which must have had significant confounding effect in studies of ABC-transporters. Taken together with studies by others [3
] linking RLIP76 with key stress-response, cell-cycling, and endocytosis proteins, a novel integrated signaling model emerges in which RLIP76 plays a key effector role in different signaling pathways though its GS-E transport activity [7
RLIP76, is a protein cloned previously by others as a Ral-effector and Ral-GAP that bridged the Ras and Ral pathways, and displayed GAP activity towards Rho/Rac G-proteins [3
]. Concomitantly, recent studies by other investigators indicate that RLIP76 plays a crucial role in clathrin-coated pit mediated receptor/ligand pair endocytosis, particularly as related to TGF-β, EGF, and insulin [3
]. Other investigators have shown that binding of cdc2 to RLIP76 is essential to shut off endocytosis during mitosis [6
]. A direct implication of our findings is that the execution of endocytosis, mitosis, and apoptosis is regulated by signaling pathways such as Ral, Ras, Rho/Rac and cdc2 by regulation of the rate of efflux of physiological pro-apoptotic GS-E by RLIP76, which couples ATP-hydrolysis, with GS-E transport [6
]. Additional results by other investigators have also shown that dissociation of RLIP76 from membrane upon binding with cdc2 results in its translocation to the mitotic spindle where it is purported to function as a motor for spindle movement [6
]. These studies suggest a more general hypothesis that RLIP76 functions as a modular ATPase that provides energy to different cellular protein by binding to different adaptor proteins.
The role of RLIP76 in intracellular signaling, through removal of intracellular GS-E, appears to be as a signal terminating mechanism. Recently, we have shown that POB1 binds to and inhibits the transport activity of RLIP76, and enhances DOX accumulation and cytotoxicity as well as inhibition of endocytosis [12
]. The central importance of RLIP76 in critical cancer-related signaling pathways is emphasized by recent findings that the termination of endocytosis during mitosis occurs as a result of formation of a complex between RLIP76, cdc2 and cyclin B1. This complex results in dissociation of RLIP76 from the membrane and its translocation to the mitotic spindle where it may play a role as a molecular motor for spindle movement during anaphase [6
Cdc2 (cdk1), a catalytic subunit of protein kinase complex, M-phase promoting factor (MPF) has critical regulatory functions during mitosis as well as in apoptosis [6
]. These properties of cdc2 are of potential interest, because it has recently been shown to bind with RLIP76, regulate endocytosis, and translocate RLIP76 to the mitotic spindle, where it may bind to sites known to bind ATPase which provide energy for this process. Cdc2 is known to be phosphorylated by PKC as well as tyrosine-kinase, which is important for its catalytic activity [6
]. The RLIP76-binding domain of cdc2 has not been identified, but it is known to bind to RLIP76 at aa 481–625 [6
]. Our present studies have demonstrated that cdc2 inhibits RLIP76 transport activity in concentration dependent manner and enhances DOX accumulation and cytotoxicity. In summary, our findings indicate that RLIP76 is a common effector protein for regulating cellular GS-E levels, and that multiple signaling proteins may regulate cell proliferation, differentiation, motility, and apoptosis in part through modulating RLIP76 transport activity.