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• W2012927537 abstract "To assess the role of phosphorylation of the human multidrug resistance MDR1 gene product P-glycoprotein for its drug transport activity, phosphorylation sites within its linker region were subjected to mutational analysis. We constructed a 5A mutant, in which serines at positions 661, 667, 671, 675, and 683 were replaced by nonphosphorylatable alanine residues, and a 5D mutant carrying aspartic acid residues at the respective positions to mimic permanently phosphorylated serine residues. Transfection studies revealed that both mutants were targeted properly to the cell surface and conferred multidrug resistance by diminishing drug accumulation. In contrast to wild-type P-glycoprotein, the overexpressed 5A and the 5D mutants exhibited no detectable levels of phosphorylation, either in vivo following metabolic labeling of cells with [32P]orthophosphate or in vitro in phosphorylation assays with protein kinase C, cAMP-dependent protein kinase, or a P-glycoprotein-specific protein kinase purified from multidrug-resistant KB-V1 cells. These results reconfirm that the major P-glycoprotein phosphorylation sites are located within the linker region. Furthermore, the first direct evidence is provided that phosphorylation/dephosphorylation mechanisms do not play an essential role in the establishment of the multidrug resistance phenotype mediated by human P-glycoprotein. To assess the role of phosphorylation of the human multidrug resistance MDR1 gene product P-glycoprotein for its drug transport activity, phosphorylation sites within its linker region were subjected to mutational analysis. We constructed a 5A mutant, in which serines at positions 661, 667, 671, 675, and 683 were replaced by nonphosphorylatable alanine residues, and a 5D mutant carrying aspartic acid residues at the respective positions to mimic permanently phosphorylated serine residues. Transfection studies revealed that both mutants were targeted properly to the cell surface and conferred multidrug resistance by diminishing drug accumulation. In contrast to wild-type P-glycoprotein, the overexpressed 5A and the 5D mutants exhibited no detectable levels of phosphorylation, either in vivo following metabolic labeling of cells with [32P]orthophosphate or in vitro in phosphorylation assays with protein kinase C, cAMP-dependent protein kinase, or a P-glycoprotein-specific protein kinase purified from multidrug-resistant KB-V1 cells. These results reconfirm that the major P-glycoprotein phosphorylation sites are located within the linker region. Furthermore, the first direct evidence is provided that phosphorylation/dephosphorylation mechanisms do not play an essential role in the establishment of the multidrug resistance phenotype mediated by human P-glycoprotein. Multidrug resistance (MDR) ( 1The abbreviations used are: MDRmultidrug resistanceFACSfluorescence-activated cell sortingPBSphosphate-buffered salineBSAbovine serum albuminFITCfluorescein isothiocyanate.) is a major impediment to effective cancer chemotherapy. In many human cancer cells, cross-resistance to a variety of natural product cytotoxic drugs is associated with the overexpression of the multidrug resistance MDR1 gene that encodes P-glycoprotein (reviewed in (1.Childs S. Ling V. Adv. Oncol. 1994; 1994: 21-36Google Scholar, 2.Germann U.A. Cytotechnology. 1993; 12: 33-62Crossref PubMed Scopus (32) Google Scholar, 3.Gottesman M.M. Pastan I. Annu. Rev. Biochem. 1993; 62: 385-427Crossref PubMed Scopus (3562) Google Scholar)). Highly homologous mdr or pgp genes have also been identified in rodents (reviewed in (1.Childs S. Ling V. Adv. Oncol. 1994; 1994: 21-36Google Scholar, 2.Germann U.A. Cytotechnology. 1993; 12: 33-62Crossref PubMed Scopus (32) Google Scholar, 3.Gottesman M.M. Pastan I. Annu. Rev. Biochem. 1993; 62: 385-427Crossref PubMed Scopus (3562) Google Scholar)). Based on the amino acid sequence deduced from the MDR1 cDNA sequence, P-glycoprotein is predicted to consist of two similar halves, each of which contains a transmembrane domain and a nucleotide binding fold(4.Chen C. Chin J.E. Ueda K. Clark D.P. Pastan I. Gottesman M.M. Roninson I.B. Cell. 1986; 47: 381-389Abstract Full Text PDF PubMed Scopus (1717) Google Scholar, 5.Gros P. Croop J. Housman D. Cell. 1986; 47: 371-380Abstract Full Text PDF PubMed Scopus (856) Google Scholar). These structural elements identify the MDR1 gene product as a member of the superfamily of ATP-binding cassette transporters, which includes the cystic fibrosis transmembrane conductance regulator, and many other membrane-associated proteins from eukaryotic and prokaryotic origin (reviewed in (6.Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3371) Google Scholar)). Gene transfer experiments involving MDR1 cDNA have corroborated that expression of P-glycoprotein is sufficient to endow drug-sensitive cells with multidrug resistance (reviewed in 2, 3). P-glycoprotein is an integral plasma membrane protein that functions as an energy-dependent drug efflux pump to reduce the intracellular accumulation of cytotoxic agents (reviewed in (1.Childs S. Ling V. Adv. Oncol. 1994; 1994: 21-36Google Scholar, 2.Germann U.A. Cytotechnology. 1993; 12: 33-62Crossref PubMed Scopus (32) Google Scholar, 3.Gottesman M.M. Pastan I. Annu. Rev. Biochem. 1993; 62: 385-427Crossref PubMed Scopus (3562) Google Scholar)). P-glycoprotein interacts directly with a variety of anticancer drugs and transports them across the plasma membrane lipid bilayer. P-glycoprotein exhibits a substrate-stimulated ATPase activity(7.Ambudkar S.V. Lelong I.H. Zhang J.P. Cardarelli C.O. Gottesman M.M. Pastan I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8472-8476Crossref PubMed Scopus (380) Google Scholar, 8.Sarkadi B. Price E.M. Boucher R.C. Germann U.A. Scarborough G.A. J. Biol. Chem. 1992; 267: 4854-4858Abstract Full Text PDF PubMed Google Scholar), suggesting that ATP hydrolysis may provide the energy required for the drug transport mechanism (reviewed in (1.Childs S. Ling V. Adv. Oncol. 1994; 1994: 21-36Google Scholar, 2.Germann U.A. Cytotechnology. 1993; 12: 33-62Crossref PubMed Scopus (32) Google Scholar, 3.Gottesman M.M. Pastan I. Annu. Rev. Biochem. 1993; 62: 385-427Crossref PubMed Scopus (3562) Google Scholar)). multidrug resistance fluorescence-activated cell sorting phosphate-buffered saline bovine serum albumin fluorescein isothiocyanate. P-glycoprotein was described as a phosphoglycoprotein(9.Carlsen S.V. Till J.E. Ling V. Biochim. Biophys. Acta. 1977; 467: 238-250Crossref PubMed Scopus (78) Google Scholar, 10.Juliano R.L. Ling V. Biochim. Biophys. Acta. 1976; 455: 152-162Crossref PubMed Scopus (2851) Google Scholar), and several studies have corroborated that both native and recombinant P-glycoproteins are phosphorylated in vivo(11.Bates S.E. Currier S.J. Alvarez M. Fojo A.T. Biochemistry. 1992; 31: 6366-6372Crossref PubMed Scopus (78) Google Scholar, 12.Center M.S. Biochem. Pharmacol. 1985; 34: 1471-1476Crossref PubMed Scopus (65) Google Scholar, 13.Chambers T.C. Chalikonda I. Eilon G. Biochem. Biophys. Res. Commun. 1990; 169: 253-259Crossref PubMed Scopus (69) Google Scholar, 14.Germann U.A. Willingham M.C. Pastan I. Gottesman M.M. Biochemistry. 1990; 29: 2295-2303Crossref PubMed Scopus (116) Google Scholar, 15.Hamada H. Hagiwara K.-I. Nakajima T. Tsuruo T. Cancer Res. 1987; 47: 2860-2865PubMed Google Scholar, 16.Ma L. Marquardt D. Takemoto L. Center M.S. J. Biol. Chem. 1991; 266: 5593-5599Abstract Full Text PDF PubMed Google Scholar, 17.Richert N.D. Aldwin L. Nitecki D. Gottesman M.M. Pastan I. Biochemistry. 1988; 27: 7607-7613Crossref PubMed Scopus (106) Google Scholar, 18.Roy S.N. Horwitz S.B. Cancer Res. 1985; 45: 3856-3863PubMed Google Scholar, 19.Schurr E. Raymond M. Bell J.C. Gros P. Cancer Res. 1989; 49: 2729-2734PubMed Google Scholar, 20.Yu G. Ahmad S. Aquino A. Fairchild C.R. Trepel J.B. Ohno S. Suzuki K. Tsuruo T. Cowan K.H. Glazer R.I. Cancer Commun. 1991; 3: 181-188Crossref PubMed Scopus (142) Google Scholar). Numerous studies have been conducted to address the importance of phosphorylation for the multidrug transporter activity of P-glycoprotein. Many multidrug-resistant cell lines were shown to express elevated levels of protein kinases, in particular protein kinase C (21.Aquino A. Warren B. Omichinski J. Hartman K.D. Glazer R.I. Biochim. Biophys. Res. Commun. 1990; 166: 723-728Crossref PubMed Scopus (39) Google Scholar, 22.Fine R.L. Patel J. Chabner B.A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 582-586Crossref PubMed Scopus (243) Google Scholar, 23.Palayoor S.T. Stein J.M. Hait W.N. Biochem. Biophys. Res. Commun. 1987; 148: 718-725Crossref PubMed Scopus (57) Google Scholar, 24.Posada J.A. McKeegan E.M. Worthington K.F. Morin M.J. Jaken S. Tritton T.R. Cancer Commun. 1989; 1: 285-292Crossref PubMed Scopus (70) Google Scholar) and changes in levels and or activities of protein kinases, such as protein kinase C or cAMP-dependent protein kinase (protein kinase A) have been suggested to play a role in modulating levels of multidrug resistance mediated by P-glycoprotein. Attempts have been made to correlate the degree of phosphorylation of P-glycoprotein with its drug efflux activity (Refs. 11, 13, 16, 22, 25, and 26; reviewed in (27.Germann U.A. Chambers T.C. Ambudkar S.V. Pastan I. Gottesman M.M. J. Bioenerg. Biomembr. 1995; 27: 53-61Crossref PubMed Scopus (73) Google Scholar)). Generally an increase in protein kinase activity and/or phosphorylation of P-glycoprotein has been associated with increased levels of multidrug resistance. Many of these studies involved the use of activators and/or inhibitors of protein kinases to modulate the state of phosphorylation of P-glycoprotein, but these regulatory molecules are not very specific and often cause multiple cellular effects. For example, several protein kinase inhibitors including staurosporine and derivatives thereof, calphostin C, or certain isoquinolinesulfonamide derivatives may directly interact with P-glycoprotein and affect its drug efflux activity independent of, or in addition to, their effects on P-glycoprotein phosphorylation (28.Miyamoto K.I. Wakusawa S. Inoko K. Takagi K. Koyama M. Cancer Lett. 1992; 64: 177-183Crossref PubMed Scopus (17) Google Scholar, 29.Sato W. Yusa K. Naito M. Tsuruo T. Biochem. Biophys. Res. Commun. 1990; 173: 1252-1257Crossref PubMed Scopus (98) Google Scholar, 30.Wakusawa S. Inoko K. Miyamoto K. Kajita S. Hasegawa T. Harimaya K. Koyama M. J. Antibiot. (Tokyo). 1993; 46: 353-355Crossref PubMed Scopus (34) Google Scholar, 31.Wakusawa S. Nakamura S. Tajima K. Miyamoto K.I. Hagiwara M. Hidaka H. Mol. Pharmacol. 1992; 41: 1034-1038PubMed Google Scholar). Various protein kinase agonists (e.g. 12-O-tetradecanoylphorbol-13-acetate or diacylglycerol) and antagonists (e.g. staurosporine, H-87) may also affect MDR1 gene expression(32.Chaudhary P.M. Roninson I.B. Oncol. Res. 1992; 4: 281-290PubMed Google Scholar, 33.Kim S.-H. Park J.-I. Chung B.-S. Kang C.-D. Hidaka H. Cancer Lett. 1993; 74: 37-41Crossref PubMed Scopus (28) Google Scholar, 34.Sampson K.E. Wolf C.L. Abraham I. Cancer Lett. 1993; 68: 7-14Crossref PubMed Scopus (38) Google Scholar, 35.Uchiumi T. Kohno K. Tanimura H. Hidaka K. Asakuno K. Abe H. Uchida Y. Kuwano M. FEBS Lett. 1993; 326: 11-16Crossref PubMed Scopus (55) Google Scholar). Thus, the role of phosphorylation of P-glycoprotein has not been clearly established. Recent approaches have focused on the identification of the phosphorylation sites within the primary structure of P-glycoprotein. In human P-glycoprotein, phosphoserine, but not phosphothreonine or phosphotyrosine, has been detected by phosphoamino acid analysis(15.Hamada H. Hagiwara K.-I. Nakajima T. Tsuruo T. Cancer Res. 1987; 47: 2860-2865PubMed Google Scholar, 16.Ma L. Marquardt D. Takemoto L. Center M.S. J. Biol. Chem. 1991; 266: 5593-5599Abstract Full Text PDF PubMed Google Scholar, 19.Schurr E. Raymond M. Bell J.C. Gros P. Cancer Res. 1989; 49: 2729-2734PubMed Google Scholar). Chambers and co-workers (36.Chambers T.C. Pohl J. Raynor R.L. Kuo J.F. J. Biol. Chem. 1993; 268: 4592-4595Abstract Full Text PDF PubMed Google Scholar, 37.Chambers T.C. Pohl J. Glass D.B. Kuo J.F. Biochem. J. 1994; 299: 309-315Crossref PubMed Scopus (69) Google Scholar) demonstrated that the major phosphorylation sites of the human MDR1 gene product are confined to a central cytosolic segment of approximately 60 amino acids that connects the two homologous halves of P-glycoprotein. This region, commonly referred to as the linker region, is characterized by a high content of charged amino acids (approximately 30-40%) and contains several consensus sequences for phosphorylation by protein kinases requiring basic amino acid residues near the phosphoacceptor group (e.g. protein kinase C, protein kinase A). A cluster of four serine residues was shown to be phosphorylated in vitro by protein kinase C (Ser-661, Ser-667, Ser-671) and/or protein kinase A (Ser-667, Ser-671, and Ser-683)(36.Chambers T.C. Pohl J. Raynor R.L. Kuo J.F. J. Biol. Chem. 1993; 268: 4592-4595Abstract Full Text PDF PubMed Google Scholar, 37.Chambers T.C. Pohl J. Glass D.B. Kuo J.F. Biochem. J. 1994; 299: 309-315Crossref PubMed Scopus (69) Google Scholar). Three of these four serine residues appear to be phosphorylated in vivo, namely Ser-661, Ser-667, and Ser-671(26.Chambers T.C. Zheng B. Kuo J.F. Mol. Pharmacol. 1992; 41: 1008-1015PubMed Google Scholar, 36.Chambers T.C. Pohl J. Raynor R.L. Kuo J.F. J. Biol. Chem. 1993; 268: 4592-4595Abstract Full Text PDF PubMed Google Scholar, 37.Chambers T.C. Pohl J. Glass D.B. Kuo J.F. Biochem. J. 1994; 299: 309-315Crossref PubMed Scopus (69) Google Scholar). Similarly, the linker region of the mouse mdr1 P-glycoprotein has been demonstrated to be phosphorylated in vitro at analogous serine residues, namely Ser-669 by protein kinase C and at Ser-681 by protein kinase A(38.Orr G.A. Han E.K.-H. Browne P.C. Nieves E. O'Connor B.M. Yang C.-P.H. Horwitz S.B. J. Biol. Chem. 1993; 268: 25054-25062Abstract Full Text PDF PubMed Google Scholar). Several protein kinase C and/or protein kinase A consensus phosphorylation sites are also present in the linker region of the mouse mdr3 and the hamster pgp1 P-glycoproteins, but the actual sites of phosphorylation have not yet been described. In analogy with the R domain between the two halves of the cystic fibrosis transmembrane conductance regulator, a target for multisite phosphorylation by protein kinase A believed to regulate the cAMP-dependent cystic fibrosis transmembrane conductance regulator chloride channel activity, it has been hypothesized that the phosphorylatable linker region of P-glycoprotein may be a regulatory domain that controls its drug transport function (36.Chambers T.C. Pohl J. Raynor R.L. Kuo J.F. J. Biol. Chem. 1993; 268: 4592-4595Abstract Full Text PDF PubMed Google Scholar, 38.Orr G.A. Han E.K.-H. Browne P.C. Nieves E. O'Connor B.M. Yang C.-P.H. Horwitz S.B. J. Biol. Chem. 1993; 268: 25054-25062Abstract Full Text PDF PubMed Google Scholar). The identification of the major sites of phosphorylation provides an opportunity to use site-directed mutagenesis to address the role of phosphorylation of P-glycoprotein for its drug efflux activity. Our approach was to substitute five consensus sites for phosphorylation by protein kinase C (Ser-661, Ser-667, Ser-671, Ser-675, Ser-683) within the linker region of P-glycoprotein by nonphosphorylatable alanine residues (5A mutant), or by aspartic acid residues to mimic permanently phosphorylated serine-like residues (5D mutant). The 5A and 5D mutants of P-glycoprotein were tested for their ability to confer multidrug resistance to drug-sensitive cells and were characterized for drug-binding capacity and state of phosphorylation. To facilitate the construction of P-glycoprotein phosphorylation mutants, two unique restriction sites, ClaI and XbaI, were introduced into the nucleotide sequence of the human MDR1 cDNA open reading frame at positions 1941 and 2060, flanking the sequences encoding the linker region. The ClaI and XbaI sites were created by a T → C transition at position 1943 and a G → A transition at position 2063. These changes in the nucleotide did not affect the encoded amino acid sequence. Three MDR1 cDNA fragments, an ApaI-ClaI fragment encompassing nucleotides 1586-1946, a ClaI-XbaI fragment encompassing nucleotides 1941-2065, and an XbaI-Asp718 fragment encompassing nucleotides 2060-2780 were generated by the polymerase chain reaction using pMDR2000XS (39.Ueda K. Cardarelli C. Gottesman M.M. Pastan I. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 3004-3008Crossref PubMed Scopus (1046) Google Scholar) as a template. Each of these three fragments was subcloned individually into the appropriate restriction sites of a pGem2-derived vector pEACXA, ( 2U. A. Germann, unpublished observations.) and the correctness of their sequence was confirmed. Then the three subcloned PCR fragments were ligated together and inserted to replace the native ApaI-Asp718 MDR1 cDNA fragment in pSX-MDR1/A-wt, a pGem2-derived plasmid carrying the full-length MDR1 cDNA isolated from the multidrug-resistant KB-V1 cell line (40.Kioka N. Tsubota J. Kakehi Y. Komano T. Gottesman M.M. Pastan I. Ueda K. Biochem. Biophys. Res. Commun. 1989; 162: 224-231Crossref PubMed Scopus (172) Google Scholar) as an SstII-XhoI fragment.2 This control plasmid was named pSX-MDR1/A-wt-CX and contains novel ClaI and XbaI restriction sites. For constructing P-glycoprotein mutants in which five serine residues at positions Ser-661, Ser-667, Ser-671, Ser-675, and Ser-683 were substituted with either alanine or aspartic acid, modified ClaI-XbaI fragments were generated by chemical synthesis of a series of eight complementary and overlapping oligodeoxynucleotides. Oligodeoxynucleotides UAG-114 (5′-CGATGCCTTGGAAAT-3′), UAG-115 (5′-TCATTTGAAGACATTTCCAAGGCAT-3′), UAG-92 (5′-GTCTTCAAATGATTCAAGATCCGCTCTA-3′), UAG-93 (5′-TCTTTTTCTTATTAGAGCGGATCTTGAA-3′), UAG-94 (5′-ATAAGAAAAAGAGCAACTCGTAGGGCTGTCCGTGGAGCA-3), UAG-95 (5′-GTCTTGGGCTTGTGCTCCACGGACAGCCCTACGAGTTGC-3′), UAG-122 (5′-CAAGCCCAAGACAGAAAGCTTGCTACCAAAGAGGCT-3′), and UAG-123 (5′-CTAGAGCCTCTTTGGTAGCAAGTTCTCT-3′) were designed for the 5A mutant, and UAG-114, UAG-115, UAG-116 (5′-GTCTTCAAATGATTCAAGATCCGATCTA-3′), UAG-117 (TCTTTTTCTTATTAGATCGGATCTTGAA-3′), UAG-118 (5′-ATAAGAAAAAGAGATACTCGTAGGGATGTCCGTGGAGAC-3′), UAG-119 (5′-GTCTTGGGCTTGGTCTCCACGGACATCCCTACGAGTATC-3′), UAG-120 (5′-CAAGCCCAAGACAGAAAGCTTGATACCAAAGAGGCT-3′), and UAG-121 (5′-CTAGAGCCTCTTTGGTATCAAGCTTTCT-3′) for the 5D mutant. All oligodeoxynucleotides were gel-purified and (except for UAG-114, UAG-121, and UAG-123) phosphorylated at the 5′ end using T4 polynucleotide kinase according to standard procedures(41.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 229-246Google Scholar). Equimolar amounts of eight oligodeoxynucleotides were annealed in the presence of 10 mM MgCl2 by heating to 80°C and slow cooling to room temperature. Subsequently, the annealed oligodeoxynucleotides were introduced into ClaI and XbaI double-digested pSX-MDR1/A-wt-CX, and their DNA sequences were confirmed. Finally, the wild type and two mutant MDR1 cDNAs were isolated as SstII-XhoI fragments and placed under control of Harvey murine sarcoma virus long terminal repeats in the pCO1 retroviral vector(42.Pastan I. Gottesman M.M. Ueda K. Lovelace E. Rutherford A.V. Willingham M.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4486-4490Crossref PubMed Scopus (476) Google Scholar), to give the expression vectors pHaMDR1/A-wt-CX (wild-type control), pHaMDR1/A-wt-5A (encoding mutant carrying five alanine residues at positions 661, 667, 671, 675, and 683), and pHaMDR1/A-wt-5D (encoding mutant carrying five aspartic acid residues at positions 661, 667, 671, 675, and 683). Murine NIH 3T3 fibroblasts and human KB-3-1 carcinoma cells were maintained as monolayer cultures at 37°C in 5% CO2 using Dulbecco's modified Eagle's medium supplemented with 4.5 g/liter glucose, 2 mML-glutamine, 50 units/ml penicillin, 50 μg/ml streptomycin, and 10% (v/v) calf serum (NIH 3T3 cells) or 10% (v/v) fetal bovine serum (KB-3-1 cells). 250,000 NIH 3T3 or 500,000 KB-3-1 cells each were transfected with the pHaMDR1/A-wt-CX, pHaMDR1/A-wt-5A, pHaMDR1/A-wt-5D expression plamids (10 μg each) as described previously(43.Germann U.A. Gottesman M.M. Pastan I. J. Biol. Chem. 1989; 264: 7418-7424Abstract Full Text PDF PubMed Google Scholar). Stable transfectants were selected for drug resistance in the presence of vincristine (NIH 3T3 cells at 30 ng/ml, KB-3-1 cells at 3 ng/ml) or colchicine (NIH 3T3 cells at 60 ng/ml, KB-3-1 cells at 6 ng/ml). Mass populations of highly drug-resistant transfectants were selected in stepwise increasing concentrations of vincristine as follows. Approximately 200,000 cells were seeded per 10-cm dish, and increasing amounts of vincristine were added. Cells were grown for 7-14 days until colonies were visible to the eye. The highest vincristine concentration survived by all three different transfectants was chosen to adapt cell populations during two passages. Then the next step of selection was initiated as described above. Retrospectively, adaptation concentrations of vincristine for drug selection were 180 ng/ml, 600 ng/ml, and 2400 ng/ml for NIH 3T3 sublines, and 18 ng/ml, 48 ng/ml, and 300 ng/ml for KB-3-1 sublines. Drug resistance profiles of the NIH 3T3 and KB-3-1 parental cell lines and vincristine-selected transfectants were determined by measuring cell survival in colony formation assays as described(44.Shen D. Cardarelli C. Hwang J. Cornwell M. Richert N. Ishii S. Pastan I. Gottesman M.M. J. Biol. Chem. 1986; 261: 7762-7770Abstract Full Text PDF PubMed Google Scholar). Average cloning efficiencies for these assays were 10-20% for NIH 3T3 sublines and approximately 50% for KB sublines. Drug accumulation assays were performed as described previously(26.Chambers T.C. Zheng B. Kuo J.F. Mol. Pharmacol. 1992; 41: 1008-1015PubMed Google Scholar). Subconfluent cells from a 10-cm dish were harvested by trypsinization into phosphate-buffered saline (PBS), washed twice with PBS containing 1% bovine serum albumin (PBS-BSA), and resuspended in PBS-BSA at a concentration of approximately 106 cells/ml. 2.5 × 105 cells were incubated for 30 min at room temperature with 5 μg of human P-glycoprotein-specific MRK16 monoclonal antibody (a gift of Hoechst Japan Ltd.) or 5 μg IgG2a isotype control antibody (Pharmingen), washed twice with PBS-BSA, and reacted with fluorescein-conjugated (FITC-labeled) goat-anti-mouse IgG2a antibody (1:10 diluted; Jackson Immunoresearch Laboratories, West Grove, PA) for 30 min at room temperature. The cells were again washed twice as described above, and the levels of FL1 fluorescence were analyzed using a FACSort flow cytometer with LYSIS II software (Beckton-Dickinson FACS System, San Jose, CA). Each sample was individually compared with the respective isotype control. For analysis of total cell extracts, cells were scraped into ice-cold PBS containing 1% (v/v) aprotinin (PBSAp), washed twice with PBSAp, extracted with radioimmune precipitation buffer (10 mM Tris HCl, pH 7.2, 0.15 M NaCl, 1% sodium deoxycholate, 0.1% SDS, 1% Triton X-100, 1% (v/v) aprotinin) and centrifuged at 100,000 × g for 30 min. Extracts were diluted with an equal volume of 2 × SDS-polyacrylamide gel electrophoresis sample buffer (NOVEX) and heated at 37°C for 10 min before loading on 8% SDS-polyacrylamide gels. For preparation of crude membranes, cells were scraped as described above and washed once with PBSAp and once with lysis buffer (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 1 mM MgCl2, 1% (v/v) aprotinin). Cells were resuspended in lysis buffer, incubated on ice for 45 min, and Dounce-homogenized (30 times with pestles A and B). An equal volume of TSNa (10 mM Tris-HCl, pH 7.5, 250 mM sucrose, 50 mM NaCl, 1% (v/v) aprotinin) was added to the lysate, followed by centrifugation at 500 × g for 10 min. The low speed supernatant was centrifuged at 100,000 × g for 1 h. The high speed pellet was washed once with TSNa, resuspended in TSNa, and stored at −80°C until use. Proteins were resolved by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose or polyvinylidine difluoride membranes as described previously(45.Tanaka S. Currier S. Bruggemann E.P. Ueda K. Germann U.A. Pastan I. Gottesman M.M. Biochem. Biophys. Res. Commun. 1990; 166: 180-186Crossref PubMed Scopus (50) Google Scholar). Immunostaining was performed using the monoclonal anti-P-glycoprotein antibody C219 (0.1 or 0.2 μg/ml; a gift from Centocor) or several different polyclonal antisera raised against the human multidrug transporter including 4007 antiserum (1:500 dilution) (45.Tanaka S. Currier S. Bruggemann E.P. Ueda K. Germann U.A. Pastan I. Gottesman M.M. Biochem. Biophys. Res. Commun. 1990; 166: 180-186Crossref PubMed Scopus (50) Google Scholar), PEPG2 (1:100 dilution)(46.Bruggemann E.P. Chaudhary V. Gottesman M.M. Pastan I. BioTechniques. 1991; 10: 202-209PubMed Google Scholar), and PEPG13 (1:100 dilution) (46.Bruggemann E.P. Chaudhary V. Gottesman M.M. Pastan I. BioTechniques. 1991; 10: 202-209PubMed Google Scholar) as primary antibodies, the appropriate horseradish peroxide-linked anti-IgG secondary antibody (1:1000 or 1:5000 dilution; Amersham Corp.), and detection by enhanced chemiluminescence (ECL) as described by the manufacturer (Amersham). Photoaffinity labeling of 100 μg of crude membranes was performed with 0.4 μM [3H]azidopine (20 μCi/ml) in the absence or presence of 100 μM vinblastine, as described previously(47.Bruggemann E.P. Germann U.A. Gottesman M.M. Pastan I. J. Biol. Chem. 1989; 264: 15483-15488Abstract Full Text PDF PubMed Google Scholar). Crude membranes (75 μg of protein) were incubated with 100 μl of 20 mM HEPES, pH 7.4, 10 mM MgCl2, 10 μM [γ-32P]ATP (2.2 × 104 cpm/pmol), and the indicated protein kinase for 20 min at 23°C. The reaction mixture was supplemented with 0.5 mM CaCl2 and 100 μg/ml phosphatidylserine for phosphorylation by protein kinase C (rat brain; Calbiochem; 0.2-0.25 units), with 0.5 mM EGTA and 5 mM cAMP for phosphorylation with protein kinase A (rabbit muscle; Sigma; 1.65 pM units), or with 5 mM MnCl2 for phosphorylation by “V-1 kinase” (partially purified from KB-V1 cells; 5 μg of protein). ( 3S. V. Ambudkar, unpublished data.) All reactions were terminated by the addition of 37.5 mM EDTA. Immunoprecipitations with P-glycoprotein-specific monoclonal antibody C219 (10 μg/sample) or polyclonal antiserum PEPG13 (6 μl/sample) were carried out as described previously(47.Bruggemann E.P. Germann U.A. Gottesman M.M. Pastan I. J. Biol. Chem. 1989; 264: 15483-15488Abstract Full Text PDF PubMed Google Scholar). After precipitation with ice-cold acetone (47.Bruggemann E.P. Germann U.A. Gottesman M.M. Pastan I. J. Biol. Chem. 1989; 264: 15483-15488Abstract Full Text PDF PubMed Google Scholar), the proteins were size-fractionated by 7.5% SDS-polyacrylamide gel electrophoresis and electrophoretically transferred onto nitrocellulose(7.Ambudkar S.V. Lelong I.H. Zhang J.P. Cardarelli C.O. Gottesman M.M. Pastan I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8472-8476Crossref PubMed Scopus (380) Google Scholar). The blots were exposed to NEF-496 (DuPont) film at −80°C for 12-24 h. Cultures were metabolically labeled with 0.1 mCi/ml [32P]orthophosphate for 4.5 h at 37°C. Cells were washed twice and harvested into ice-cold PBS. PBS and all solutions for immunoprecipitations were supplemented with 1 mM phenylmethylsulfonyl fluoride, 0.2 mg/ml aprotinin, 5 mM sodium fluoride, 0.2 mM sodium orthovanadate, and 20 nM okadaic acid. After centrifugation at 500 × g for 5 min the cell pellets were suspended in 0.5 ml of radioimmune precipitation buffer, incubated on ice for 30 min, and centrifuged at 100,000 × g for 30 min. Protein concentrations of the supernatants were determined by the method of Bradford(48.Bradford M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216068) Google Scholar). Equivalent amounts of extracts (0.5 mg of protein) were subjected to immunoprecipitation with 13 μl of P-glycoprotein-specific antibody PEPG13 (46.Bruggemann E.P. Chaudhary V. Gottesman M.M. Pastan I. BioTechniques. 1991; 10: 202-209PubMed Google Scholar) as described previously(47.Bruggemann E.P. Germann U.A. Gottesman M.M. Pastan I. J. Biol. Chem. 1989; 264: 15483-15488Abstract Full Text PDF PubMed Google Scholar). Proteins were precipitated with acetone (47.Bruggemann E.P. Germann U.A. Gottesman M.M. Pastan I. J. Biol. Chem. 1989; 264: 15483-15488Abstract Full Text PDF PubMed Google Scholar) and redissolved in 60 μl SDS-polyacrylamide gel electrophoresis sample buffer without boiling. Samples were divided in two, and each half was electrophoresed on an 8% SDS-polyacrylamide gel. One gel was stained with Coomassie Blue, destained, dried, and subjected to autoradiography with Kodak XAR5 film at −70°C. The duplicate gel was transferred to nitrocellulose, and P-glycoprotein was detected by immunostaining with C219 monoclonal antibody as described above. Oligodeoxynucleotide-mediated mutagenesis was used to alter the major putative phosphorylation sites within the linker region of the human MDR1 gene product (Fig. 1). Five clustered serine residues at positions 661, 667, 671, 675, and 683, all representing protein kinase C and/or protein kinase A consensus sequences, were targeted for substitution with either nonphosphorylatable alanine residues or aspartic residues to mimic permanently phosphorylated serine-like residues. Four of these five targeted serine residues (Ser-661, Ser-667, Ser-671, and Ser-683) have previously been demonstrated to be phosphorylated in vitro by protein kinase C and/or protein kinase A(36.Chambers T.C. Pohl J." @default.
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• W2012927537 title "Characterization of Phosphorylation-defective Mutants of Human P-glycoprotein Expressed in Mammalian Cells" @default.
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