Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.3.44 (P-glycoprotein)
13,344 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Okadaic acid (OA)-resistant variants of Chinese hamster ovary cells, clones CHO/OAR6-6 and CHO/OAR2-3, were isolated from a CHO-K1 culture. These variant cells were 17- to 26-fold more resistant to OA than the parental cells. The phosphorylase phosphatase activity of the variant cell extracts was 2- to 4-fold more resistant to OA than that of the parental cells in the presence of inhibitor 2, a specific inhibitor of type 1 protein serine/threonine phosphatase (PP1). Nucleotide sequencing of PP2A alpha (an isotype of PP2A catalytic subunit) cDNA demonstrated that both variants have a T-->G transversion at the first base of codon 269 (805 nt), which results in substitution of glycine for cysteine. We expressed in COS-1 cells a mutant PP2A alpha tagged with the influenza hemagglutinin epitope. The recombinant mutant PP2A alpha protein immunoprecipitated with an anti-influenza hemagglutinin antibody was more resistant than the wild type to OA, their IC50 values being 0.65 nM and 0.15 nM, and their IC80 values being 4.0 nM and 0.45 nM, respectively. The cysteine at residue 269 present only in highly OA-sensitive protein serine/threonine phosphatase catalytic subunit isozymes, PP2A alpha, PP2A beta, and PPX, is suggested to be involved in the binding of OA. CHO/OAR6-6 and CHO/OAR2-3 cells also overexpressed the P-glycoprotein, and the efflux of OA was more rapid. It is suggested that the PP2A alpha mutation in cooperation with a high level of P-glycoprotein makes the CHO-K1 variants highly resistant to OA.
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PMID:Characterization of the PP2A alpha gene mutation in okadaic acid-resistant variants of CHO-K1 cells. 793 53

Phosphorylation of P-glycoprotein (Pgp) by protein kinase C occurs on apparently the same sites in vitro and in intact cells (in situ) and is implicated in modulation of Pgp function. The region of the molecule which contains the in vitro phosphorylation sites and two specific sites within this region are now determined by peptide sequencing. Membrane vesicles from multidrug-resistant human KB-V1 cells were incubated with purified protein kinase C and [gamma-32P]ATP, and Pgp (containing 1 mol of phosphate/mol of protein) was purified to apparent homogeneity. Phosphorylation occurred exclusively on serine residues. Phosphopeptides were generated by digestion with Lys-C endoproteinase or trypsin, partially purified by high performance liquid chromatography, and further purified with strategies developed for individual phosphopeptides. Sequence analysis by Edman degradation and comparison with the deduced amino acid sequence of human (mdr 1) Pgp identified serines 661 and 671, and one or more of serines 667, 675, and 683, as sites of phosphorylation. These sites are clustered in the linker region located between the two homologous halves of Pgp. Our results identify a previously undefined, phosphorylatable domain of Pgp, smaller in size but analogous in location to the R-domain of the cystic fibrosis transmembrane conductance regulator. These data provide a basis for a better understanding of the role of phosphorylation in the mechanism of action and regulation of this important multidrug pump protein.
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PMID:Identification of specific sites in human P-glycoprotein phosphorylated by protein kinase C. 809 61

The serine residue located at position 939 and 941 in the predicted transmembrane segment 11 of P-glycoprotein (P-gp) encoded by mouse mdr3 and mdr1, respectively, appears to be important for interaction of chemotherapeutic drugs and reversal agents with P-gp. To further understand the role of this residue in this process and to identify the structural requirements involved, we have replaced this serine residue by alanine, cysteine, threonine, tyrosine, tryptophan, and aspartic acid and tested the effect of these mutations on the overall activity and substrate specificity of mdr1 and mdr3. All mutant proteins could be expressed at high levels in the membrane fractions of LR73 Chinese hamster cells transfected with the corresponding mutant cDNAs. All introduced mutations had limited effect on the capacity of mdr1 and mdr3 to confer resistance to vinblastine. The modulatory effect of mutations on resistance to colchicine, adriamycin, and actinomycin D was more dramatic. The hydroxyl group of serine did not seem essential for interaction with these drugs since mutant mdr1 and mdr3 bearing alanine or cysteine at that position behaved essentially as wild type, while threonine-bearing mutants showed significantly reduced resistance to these drugs. The insertion at that site of residues with bulkier side chains had more complex effects on P-gp function. While introducing tyrosine, tryptophan, or aspartic acid caused an almost complete loss of colchicine and adriamycin resistance in both mdr1 and mdr3, the replacement to tyrosine or tryptophan had the opposite effect on mdr1 and mdr3 for actinomycin D resistance, causing either a 3-fold increase or a 4-8-fold decrease in resistance to this drug, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Modulatory effects on substrate specificity of independent mutations at the serine939/941 position in predicted transmembrane domain 11 of P-glycoproteins. 810 79

The mechanism by which P-glycoprotein (P-gp) interacts with a number of structurally unrelated substrates or inhibitors remains unknown. We have recently shown that a serine residue within the predicted transmembrane (TM) domain 11 of P-gps encoded by mouse mdr1 (Ser941) and mdr3 (Ser939) plays an important role in the substrate specificity of P-gp. We wished to determine if Ser939/941 is also important for efficient interaction of P-gp with structurally different modulating agents, a cyclic peptide (cyclosporin A, CsA), a diaminoquinazoline (CP100356), and a chiral, tricyclic structure (CP117227). For this, the capacity of these compounds to modulate the vinblastine (VBL) resistance phenotype of transfected cells expressing similar levels of P-gps bearing either the wild-type Ser or a mutant Phe at position 941 (mdr1) or 939 (mdr3) was initially tested. The Ser-->Phe substitution indeed affected the potency and P-gp isoform specificity of some of the modulators, in particular that of CP117227 (racemic mixture and enantiomers), which were active against wild-type but not mutant mdr3. The modulatory effect of the mutation on CP117227-mediated reversal of VBL resistance was parallelled by a comparable modulation of the steady-state levels of VBL accumulation in Ser939- and Phe939-expressing cells, but was not linked to differential cellular accumulation of the modulator, which was identical in both cell types. To further assess the role of this amino acid residue in P-gp interactions with modulators, the effect of additional mutations (Ala, Cys, Thr, Asp, Tyr, Trp) at that site on potencies of CsA, CP117227 enantiomers, and CP100356 was evaluated.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Structurally distinct MDR modulators show specific patterns of reversal against P-glycoproteins bearing unique mutations at serine939/941. 817 79

Recently, many potent inhibitors of protein serine/threonine phosphatases (PPs) have been found. Some of them have proven to be tumor promoters in mouse skin two-step carcinogenesis and rat liver medium-term tests. Among these inhibitors, okadaic acid (OA) selectively inhibits PP2A, and its use has therefore been proposed to facilitate analysis of biological roles of this phosphatase. OA shows bimodal effects on in vitro transformation and, in addition to such epigenetic changes, also induces marked genetic changes. OA treatment for more than 1 week flattened NIH 3T3 transformants irreversibly, with loss of the transfected genes. It is also known to induce diphtheria toxin-resistant mutations in Chinese hamster lung cells and sister chromatid exchanges (SCEs) in Chinese hamster ovary cells and human lymphocytes. To analyze roles of protein phosphatases in gene stability, we isolated OA-resistant mutants. They were proven to have a mutation in the PP2A alpha catalytic subunit, in which cysteine 269 had been substituted for glycine; and it was demonstrated that this region interacts with OA. The recombinant mutant protein was 4 approximately 9-fold more resistant to OA than the wild type. Although the OA resistant mutants of CHO cells expressed high levels of P-glycoprotein, inhibition of PP2A itself was suggested to lead to SCE induction. However, the number of molecular species of PP which are known to be sensitive to OA continues to increase, and we have isolated cDNA for a novel type of OA sensitive PP. Our studies indicate that the fact that the roles of PP2A cannot be elucidated using only OA is of crucial importance.
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PMID:Protein serine/threonine phosphatases as binding proteins for okadaic acid. 853 25

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-glyco-protein-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.
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PMID:Characterization of phosphorylation-defective mutants of human P-glycoprotein expressed in mammalian cells. 857 73

Protein kinase C (PKC) comprises a family of related phospholipid-dependent serine/threonine protein kinases. PKC has been implicated in the induction and maintenance of the multidrug-resistance (MDR) phenotype but the role of different isozymes is not well understood. We compared the expression and subcellular distribution, and membrane association and down-regulation induced by phorbol esters, of individual PKC isozymes in drug-sensitive KB-3 and multidrug-resistant KB-V1 human carcinoma cell lines. Immunoblotting with isozyme-specific antibodies indicated the presence of PKC alpha (cytosol only). PKC beta (membrane only). PKC epsilon (mainly membrane associated) and PKC zeta (both fractions). PKC delta and PKC gamma were not detected. The expression levels of PKC beta. PKC epsilon and PKC zeta were unchanged in KB-V1 cells; PKC alpha was modestly increased ( approximately 65%) in the resistant cells as further determined by enzyme assay. The cytosolic nature and increased expression of PKC alpha were confirmed by immunofluorescent localization studies. Revertant cells, obtained by culturing KB-V1 cells in a drug-free medium, regained drug sensitivity with a loss of P-glycoprotein and a concomitant decrease in expression of PKC alpha, KB-V1 cells were found to differ markedly from KB-3 cells with respect to the translocation and down-regulation specifically of PKC alpha upon exposure to 12-O-tetradecanoyl-1-phorbol-13-acetate (TPA). Treatment with 30 nM TPA for 24 h completely depleted KB-3 cells of PKC alpha whereas 1 microM TPA was required to deplete KB-V1 cells of PKC alpha. Similar results were obtained when phorbol-12, 13-dibutyrate was used instead of TPA. Defective TPA-mediated down-regulation of PKC alpha was also observed in another PKC alpha-overexpressing MDR cell line. KB-A1. Importantly, cellular uptake of radiolabeled phorbol ester was similar for both drug-sensitive and MDR cells. Sensitive and resistant cells exhibited similar expression levels of RACK1, a PKC-binding protein important in activation-induced translocation. These findings further highlight the importance of PKC alpha in the MDR phenotype, and suggest that this isozyme may be expressed in a modified form or be subject to an altered regulation in MDR cells.
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PMID:Expression, subcellular distribution and response to phorbol esters of protein kinase C (PKC) isozymes in drug-sensitive and multidrug-resistant KB cells evidence for altered regulation of PKC-alpha. 877 28

The multidrug resistant (MDR) phenotype is a well-studied subject that has been recognized as a determinant underlying specific types of drug resistance in human cancer. Although it is clear that the P-glycoprotein plays a major role in MDR, it is not clear whether post-translational modifications such as phosphorylation have any major impact on its modulation. The laboratory of Dr. Bruce Chabner was one of the first to describe increased expression and activity of protein kinase C (PKC) associated with the MDR phenotype. Since that time, a similar correlation has been observed in many other MDR cell lines. Most of these studies have been performed with doxorubicin-selected cells that have acquired MDR and have shown increased PKC activity, mainly for PKC-alpha isoenzyme. Intrinsic MDR in human renal cell carcinoma lines has been shown to correlate directly with PKC activity, but further studies with intrinsic MDR cell lines are needed before any conclusions can be drawn. More recent evidence suggests that there is a complex biochemical process by which PKC isoenzymes differentially phosphorylate specific serine residues in the linker region of P-glycoprotein which may lead to alterations in P-glycoprotein ATPase and drug-binding functions. To further complicate matters, PKC plays an important role in anti-apoptotic pathways, which can confound the dissection and elucidation of drug-resistance mechanisms. However, these areas are still under active investigation and not fully answered. Further studies are needed to specifically answer the question of whether PKC directly modulates basal and/or drug-stimulated P-glycoprotein function. This manuscript reviews the majority of the literature on PKC and MDR, as well as offers caveats for interpretation of these studies to answer the above questions.
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PMID:P-glycoprotein, multidrug resistance and protein kinase C. 882 Sep 51

The role of protein kinase C and protein phosphatases was examined in the control of mutagenic metabolites of aromatic amines. Various metabolic activating systems derived from rat liver were treated with: 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C modulator; okadaic acid (OA), a potent inhibitor of serine/threonine protein phosphatases (PP1 and PP2A); and ortho-vanadate (OV), an inhibitor of tyrosine phosphatases. TPA used over a wide concentration range (10(-9)-10(-6) M) did not affect the bacterial mutagenicity of the aromatic amines and of the aromatic amide investigated, 2-aminoanthracene, 2-aminofluorene and 2-acetylaminofluorene (2AAF). At the molecular level, TPA did not affect the function of cytochrome P450s 1A1 or 1A2, which are known key factors for the activation and inactivation of aromatic amines/amides. By contrast the OA and OV treatment of rat hepatocytes, rat liver homogenate, fraction S9 and the nuclear fraction drastically reduced (by > 80%) the mutagenicity of the aromatic amines/amide investigated. This is by far the most pronounced change in genotoxicity observed to date via modulation of phosphorylation. Whilst the mutagenicity of the primary toxication product 2-N-OH-acetylaminofluorene (2-N-OH-AAF) in the presence of exogenous activating systems (hepatocytes, S9-fraction, nuclear fraction) was also reduced by OV, OA had no influence. Thus the tyrosine protein phosphatase inhibitor and the serine/threonine protein phosphatase inhibitor influence the genotoxicity of aromatic amines/amides on different levels. Moreover, this shows that the drastic reduction in mutagenicity by OA was due to its influence on a step prior to the presence of the primary toxication product 2-N-OH-AAF. This reduction could be due to changes in the activity of cytochrome P4501A1 and/or 1A2. However, no incorporation of 32P-labelled phosphate from intracellularly prelabelled [32P]-ATP into cytochromes P450 1A1 or 1A2 nor any change in their catalytic activities was observed in the presence of OA. Furthermore, a phosphorylation dependent change in the function of P-glycoprotein (known for its role in the transport of diverse xenobiotic substances and their metabolites) was shown not to contribute to the observed decrease in mutagenicity. Our results reveal an important role for protein phosphatase 1 and/or 2A and tyrosine phosphatase(s) in the control of the genotoxicity of aromatic amines and amides. However, the present study does not distinguish between effects mediated by individual proteins affected by these protein phosphatases.
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PMID:Control of the mutagenicity of aromatic amines by protein kinases and phosphatases. I. The protein phosphatase inhibitors okadaic acid and ortho-vanadate drastically reduce the mutagenicity of aromatic amines. 933 96

Multidrug resistance is one of the major obstacles in cancer chemotherapy. In tumor cells, overexpression of the transmembrane P-glycoprotein 170 (P-gp) is associated with the multidrug resistance phenotype and serves as a drug efflux pump. The activation of P-gp has been suggested to occur at the post-translational level. Protein kinase C mediated phosphorylation may be associated with the drug effux mechanism but the overall phosphorylation pathway has not been completely defined. we report the novel finding of an increase in phosphatase 1B (a tyrosine phosphatase) and a decrease in PP1 and PP2A (serine/threonine phosphatases) expression and activity in our series of early (R65) and late (R500) stage adriamycin resistant MCF-7 cells. In addition, we show a decrease in protein kinase A (PKA) activity and an increase in protein kinase C (PKC) in our drug resistant cells. Analyses of PKC isoforms alpha through epsilon revealed that PKCbeta was not expressed and that all other isoforms increased with increasing resistance, except PKCgamma which was detected only in R65 cells. Our findings suggest that in drug resistant cells, there is a pattern consistant with the maintenance of serine and threonine residues in a phosphorylated state.
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PMID:Differential expression and activity of phosphatases and protein kinases in adriamycin sensitive and resistant human breast cancer MCF-7 cells. 962 6


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