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Our recent studies have shown that RLIP76 (RalBP1) is a major doxorubicin and glutathione-conjugate transporter in lung cancer cells and that its transport activity is greater in NSCLC as compared with SCLC. Because differences in PKC activity have been implicated as a major determinant of differential doxorubicin accumulation and sensitivity in lung cancer cells, we examined the effect of PKC-α on the transport function of RLIP76. Present studies demonstrate that RLIP76 is phosphorylated in-vitro by PKC-α. Through deletion mutant analyses of 4 potential phosphorylation sites, we identified T297 and S509 as targets for phosphorylation by PKC-α. PKC-α- mediated phosphorylation caused a 76% increase in doxorubicin and glutathione-conjugate transport activity. in the presence of ATP resulted in a significant increase in transport activity (1.76 fold activation) of wild-type RLIP76. Effect of PKC-α mediated phosphorylation on DOX-transport by lung cancer RLIP76 was examined using purified RLIP76 from H1618 SCLC and H226 NSCLC. As was the case for recombinant RLIP76, DOX-transport activity of protein purified from SCLC or NSCLC cell line was increased approximately 70% when pre-incubated with PKC-α and ATP. These findings indicate that RLIP76 is phosphorylated by PKC-α, and that this phosphorylation results in increased transport activity of RLIP76 from both SCLC and NSCLC. We conclude that RLIP76 plays a critical role in lung cancer doxorubicin resistance, and that it is regulated by PKC-α. Key Words: Doxorubicin, Drug-resistance, Deletion mutants, RLIP76, RALBP1, PKC-α.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]