RESUMO
The multidrug resistant associated protein 1 (or MRP1, ABCC1) encodes two cytoplasmic linker domains (L0 and L1) composed of highly charged sequences with multiple protein kinase A/C phosphorylation sites. In this report we made use of the scanning peptide approach to identify MRP1 linker L1 (L1MRP1) interacting proteins. Scanning heptapeptides covering L1MRP1 126 amino acids (Ile846- Leu972) were synthesized and used in pull-down assays to isolate proteins from cell extracts (human multidrug resistant SCLC cell line; H69/AR). The results show four high affinity binding sequences in L1MRP1 domain [866FLRTYAST867; 906SAGKQLQRQLSSS912; 925ISRHHNSTA927 and 954AQTGQVKLSVYW959] that bound â¼55 kDa and 110 kDa polypeptides. The latter polypeptides were identified by mass spectrometry as α- and ß-tubulin monomers and dimers. Western blotting with monoclonal antibodies to α- and ß-tubulin proteins confirmed the mass-spectrometry results. Moreover, using recombinant full-length GST-Linker 1 fusion polypeptide (GST-L1MRP1), we confirmed the peptide scanning approach demonstrating specific binding of tubulin to GST-L1MRP1. Intriguingly, substitutions of serine residues in L1MRP1 by aspartic acid, but not alanine, abolished its binding to tubulin, suggesting that phosphorylation of Ser871, 915, 930, and 961 within L1MRP1 may modulate its binding to tubulin. Taken together, the results of this study suggest possible interaction between MRP1 and tubulin that is modulated by phosphorylation of specific sequences in the L1MRP1 domain.
Assuntos
Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Tubulina (Proteína)/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular Tumoral , Humanos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/química , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapas de Interação de Proteínas , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Tubulina (Proteína)/químicaRESUMO
The overexpression of P-glycoprotein (P-gp, ABCB1) in cancer cells often leads to multidrug resistance (MDR) through reduced drug accumulation. However, certain P-gp-positive cells display hypersensitivity, or collateral sensitivity, to certain compounds that are believed to induce Pgp-dependent oxidative stress. We have previously reported that MDR P-gp-positive CHO cells are collaterally sensitive to verapamil (VRP; Laberge et al. (2009) [1]). In this report we extend our previous findings and show that drug resistant CHO cells are also collaterally sensitive to physiologic levels of progesterone (PRO) and deoxycorticosterone (DOC). Both PRO and DOC collateral sensitivities in CH(R)C5 cells are dependent on P-gp-expression and ATPase, as knockdown of P-gp expression with siRNA or inhibition of P-gp-ATPase with PSC833 reverses PRO- and DOC-induced collateral sensitivity. Moreover, the mitochondrial complexes I and III inhibitors (antimycin-A and rotenone, respectively) synergize with PRO and DOC-induced collateral sensitivity. We also show that VRP inhibits PRO and DOC collateral sensitivity, consistent with earlier findings relating to the VRP's modulation of PRO and DOC-stimulation of P-gp ATPase. The findings of this study demonstrate a P-gp-dependent collateral sensitivity of MDR cells in the presence of physiologically achievable concentrations of progesterone and deoxycorticosterone.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Desoxicorticosterona/farmacologia , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistência a Múltiplos Medicamentos/fisiologia , Progesterona/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/metabolismo , Animais , Antimicina A/administração & dosagem , Antimicina A/farmacologia , Células CHO , Cricetinae , Cricetulus , Ciclosporinas/farmacologia , Desoxicorticosterona/administração & dosagem , Sinergismo Farmacológico , Complexo de Proteínas da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Técnicas de Silenciamento de Genes , Progesterona/administração & dosagem , RNA Interferente Pequeno/genética , Espécies Reativas de Oxigênio/metabolismo , Rotenona/administração & dosagem , Rotenona/farmacologia , Verapamil/farmacologiaRESUMO
Human ABCC1 is a member of the ATP-binding cassette (ABC) transporter superfamily, and its overexpression has been shown to cause multidrug resistance by active efflux of a wide variety of anticancer drugs. ABCC1 has been shown to exist and possibly function as a homodimer. However, a possible heterocomplex involving ABCC1 has been indicated. In this study, we performed an interactive proteomics study to examine proteins that bind to and form heterocomplexes with ABCC1 using coimmunoprecipitation and tandem mass spectrometry (MS/MS) analyses. We found that ATP synthase α binds to ABCC1 in plasma membranes with a ratio of 2:1. The ATP synthase α binding site in ABCC1 is located in the linker domain at the carboxyl core of ABCC1, and phosphorylation of the linker domain at the protein kinase A site enhances ATP synthase α binding. The interaction between ABCC1 and ATP synthase α in a heterocomplex may indicate a novel function of ABCC1 in regulating extracellular ATP level and purinergic signaling cascade.
Assuntos
ATPases Mitocondriais Próton-Translocadoras/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Células HEK293 , Humanos , Imunoprecipitação , ATPases Mitocondriais Próton-Translocadoras/química , Modelos Biológicos , Dados de Sequência Molecular , Proteínas Associadas à Resistência a Múltiplos Medicamentos/química , Fosforilação , Ligação Proteica , Mapeamento de Interação de Proteínas/métodos , Proteômica/métodos , Reprodutibilidade dos TestesRESUMO
P-glycoprotein (or P-gp1, ABCB1) expression in tumor cells is causative of multidrug resistance through the active efflux of drugs across the cell membrane. However, the over-expression of P-glycoprotein in some tumor cells has been associated with increased sensitivity, or "collateral sensitivity", of multidrug resistant cells to specific drugs, including the calcium channel blocker verapamil. We previously demonstrated that collateral sensitivity to verapamil correlates with the effect of this drug on P-gp1 ATPase, and is reversed by inhibitors of P-gp1 ATPase (e.g., PSC 833 and Ivermectin). In this report, we expand on our earlier study and demonstrate that P-gp1 expression in drug-resistant cells modulates collateral sensitivity. Using P-gp1-specific siRNA, P-gp1 expression in the multidrug resistant CH(R)C5 cells was significantly down-regulated beginning on day 2 post-transfection of siRNA. Furthermore, down-regulation of P-gp1 led to increased sensitivity of CH(R)C5 cells to paclitaxel and doxorubicin, but not to cis-platinum, due to inhibition of P-gp1 drug efflux pump. Down-regulation of P-gp1 expression completely reversed collateral sensitivity to verapamil. Moreover, known inhibitors of ETC, rotenone and antimycin A which cause an increase in reactive oxygen species, synergized with verapamil-induced collateral sensitivity leading to increased cell death as determined by MTT cell survival assay. Similarly, the addition of hydrogen peroxide also synergized with verapamil. Taken together, the results of this study demonstrate a direct link between P-gp1 expression and collateral sensitivity of drug-resistant cells, possibly due to an increase in reactive oxygen species.