Abstract 810: FBXO15 is an F-box protein in E3 ligase complex for P-glycoprotein

Abstract

Abstract P-glycoprotein (P-gp), which is encoded by the multidrug resistance gene 1 (MDR1, also known as ABCB1), is one of the ATP-binding cassette (ABC) transporter superfamily that consists of two symmetrical halves each containing an ATP-binding domain and a transmembrane domain. P-gp pumps out a variety of physiological substances and anticancer agents, including anthracyclines, Vinca alkaloids and taxanes, from the cell. P-gp is widely expressed in normal human tissues, including the adrenal gland, colon, kidney, liver, angioendothelial cells and hematopoietic precursor cells and protects such tissues and cells from toxicity and biological effects of extraneous and physiological substances. In some cancers, the expression of P-gp confers resistance to the substrate anticancer agents. To reverse the resistance, control of the P-gp activity is important for cancer chemotherapy in patients, and it is therefore needed to understand the molecular mechanisms underlying P-gp expression and function. P-gp is expressed on cell surface after several modifications including glycosylation and phosphorylation. We have previously reported that inhibition of the mitogen-activated protein kinase (MAPK) pathway by specific inhibitors or siRNAs down-regulates P-gp expression. Here, we aimed to clarify the molecular mechanism underlying P-gp degradation. A MEK inhibitor U0126 decreased P-gp expression as well as the previous report, but a proteasome inhibitor MG132 restored the low P-gp expression in a dose-dependent manner. MG132 simultaneously recovered the MEK activity inhibited by U0126, and the restoration of P-gp expression was well correlated with the recovery of MEK activity. Treatment of cells with MG132 or lactacystin (another proteasome inhibitor) alone increased intracellular P-gp expression within 8 hours and cell surface P-gp at 24 hours compared with vehicle control, but bafilomycin A1, a specific inhibitor of autolysosome, could not until 12 hours. P-gp was co-immunoprecipitated with ubiquitin, and MG132 enhanced the polyubiquitinated P-gp in immunoprecipitation-western blot analyses. To identify the molecules regulating the ubiquitination of P-gp, binding proteins to the C-terminal intracellular domain of P-gp were searched by IP-MALDI-TOF/MS technique, and 22 candidates were found. Among the candidates, we focused on FBXO15 because it had been reported to be a subunit of E3 ligase complex with Skp1, Cul1 and Rbx1. FBXO15 knockdown by the siRNA enhanced cell surface P-gp expression in HCT-15, HEK293 and A498 cells which express endogenous P-gp. The FBXO15 cDNA transfection enhanced the ubiquitination of P-gp in HEK293 cells, and the co-transfection with FBXO15 siRNA suppressed the ubiquitination. These results suggest that P-gp expression is partly regulated by the ubiquitin proteasome system mediated by FBXO15. Involvement of the MAPK pathway in this system is to be explored. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 810. doi:1538-7445.AM2012-810