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)

Among the many phenotypic characteristics of multidrug resistance (MDR), the presence of P-glycoprotein is nearly always observed, and it appears that the plasma membrane of the multidrug resistant cell is integrally involved in controlling drug resistance. Another membrane-associated protein kinase, protein kinase C (PKC), has been shown to regulate the flow of information to the cell interior and to control the efflux of a number of different compounds. We therefore initiated a study of PKC and MDR. We found that multidrug resistant sublines from both mouse sarcoma 180 and human KB lines exhibited 80-90% increases in basal PKC activity. The mechanism of the increase appears to be quite different in the two cell lines. The human KB cells overexpress the alpha isozyme of PKC, commensurate with the increase in alpha-PKC protein, whereas the mouse cells do not overexpress alpha-mRNA but increase alpha-PKC protein. Furthermore, it appears that PKC activity plays a functional role in drug resistance, since inhibition of endogenous PKC activity by staurosporine resulted in decreased resistance to Adriamycin. We also found that phosphorylation of MDR cell membrane vesicles by purified PKC, followed by immunoprecipitation of P-glycoprotein with monoclonal antibody C219, resulted in a level of phosphorylation of P-glycoprotein that was greater than the endogenous phosphorylation level. The data presented indicate that MDR cells of diverse species exhibited enhanced PKC activity but that the mechanisms were different. The increased kinase activity may have biological relevance to MDR since PKC appears to be coupled to P-glycoprotein function.
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PMID:Human multidrug resistant KB cells overexpress protein kinase C: involvement in drug resistance. 257 53

8-Cl-cAMP, a site-selective analogue of cAMP, decreased mdr-1 expression in multidrug-resistant human breast cancer cells. A sixfold reduction of mdr-1 mRNA expression by 8-Cl-cAMP began within 8 h of treatment and was associated with a decrease in the synthesis of P-glycoprotein and with an increase in vinblastine accumulation. A reduction in mdr-1 expression after 8-Cl-cAMP treatment was also observed in multidrug-resistant human ovarian cancer cell lines. 8-Cl-cAMP is known to change the ratio between the two regulatory subunits, RI and RII, of protein kinase A (PKA). We observed that RI alpha decreased within 24 h of 8-Cl-cAMP treatment, that RII beta increased after as few as 3 h of treatment, and that PKA catalytic activity remained unchanged during 48 h of 8-Cl-cAMP treatment. The results are consistent with the hypothesis that mdr-1 expression is regulated in part by changes in PKA isoenzyme levels. Although 8-Cl-cAMP has been used to differentiate cells in other model systems, the only differentiating effect that could be detected after 8-Cl-cAMP treatment in the MCF-7TH cells was an increase in cytokeratin expression. Evidence that the reduction of mdr-1 mRNA occurred at the level of gene transcription was obtained by measuring chloramphenicol acetyltransferase (CAT) mRNA in MCF-7TH cells transfected with an mdr-1 promoter-CAT construct prior to 8-Cl-cAMP treatment. Thus, 8-Cl-cAMP is able to downregulate mdr-1 expression and suggests a new approach to reversal of drug resistance in human breast cancer.
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PMID:Downregulation of mdr-1 expression by 8-Cl-cAMP in multidrug resistant MCF-7 human breast cancer cells. 754 90

Cells expressing elevated levels of the membrane phosphoprotein P-glycoprotein exhibit a multidrug resistance phenotype. Studies involving protein kinase activators and inhibitors have implied that covalent modification of P-glycoprotein by phosphorylation may modulate its biological activity as a multidrug transporter. Most of these reagents, however, have additional mechanisms of action and may alter drug accumulation within multidrug resistant cells independent of, or in addition to, their effects on the state of phosphorylation of P-glycoprotein. The protein kinase(s) responsible for P-glycoprotein phosphorylation has(ve) not been unambiguously identified, although several possible candidates have been suggested. Recent biochemical analyses demonstrate that the major sites of phosphorylation are clustered within the linker region that connects the two homologous halves of P-glycoprotein. Mutational analyses have been initiated to confirm this finding. Preliminary data obtained from phosphorylation- and dephosphorylation-defective mutants suggest that phosphorylation of P-glycoprotein is not essential to confer multidrug resistance.
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PMID:Effects of phosphorylation of P-glycoprotein on multidrug resistance. 762 52

The development of cross-resistance to many natural product anticancer drugs, termed multidrug resistance (MDR), is one of the major reasons why cancer chemotherapy ultimately fails. This type of MDR is often associated with over-expression of the MDR1 gene product, P-glycoprotein (Pgp), a multifunctional drug transporter. The expression of MDR in breast tumors is related to their origination from a tissue that constitutively expresses Pgp as well as to the development of resistance during successive courses of chemotherapy. Therefore, understanding the mechanisms that regulate the transcriptional activation of MDR1 may afford a means of reducing or eliminating MDR. We have found that MDR1 expression can be modulated by type I cAMP-dependent protein kinase (PKA), opening up the possibility of modulating MDR by selectively down-regulating the activity of PKA-dependent transcription factors which upregulate MDR1 expression. High levels of type I PKA occurs in primary breast carcinomas and patients exhibiting this phenotype show decreased survival. The selective type I cAMP-dependent protein kinase (PKA) inhibitors, 8-Cl-cAMP and Rp8-Cl-cAMP[S] may be particularly useful for downregulating PKA-dependent MDR-associated transcription factors, and we have found these compounds to downregulate transient expression of a reporter gene under the control of several MDR1 promoter elements. Thus, investigations of this nature should not only lead to a greater understanding of the mechanisms governing the expression of MDR, but also provide a focus for pharmacologic intervention by a new class of inhibitors.
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PMID:Transcriptional regulation of multidrug resistance in breast cancer. 788 Nov 4

P-glycoprotein is phosphorylated in cells, and it has been suggested that phosphorylation may regulate the drug transport activity of P-glycoprotein. Domain mapping, utilizing a combination of cyanogen bromide digestion and immunoblot analysis, was used to reveal the major phosphorylation sites in murine mdr1b P-glycoprotein. After labeling of J7.V1-1 cells with [32P]Pi, or labeling membranes with [gamma-32P]ATP and either protein kinase A or protein kinase C, it was found that the majority of the label was contained within a single cyanogen bromide fragment (amino acid 627-682) that encompassed the majority of the linker region. The in vitro protein kinase C phosphorylation sites within this fragment were analyzed by a combination of fast atom bombardment mass spectrometry (FABMS) and two-dimensional phosphopeptide mapping. FABMS analysis of a protein kinase C-phosphorylated synthetic peptide, corresponding to a segment of the linker region of P-glycoprotein, identified serine 669 as the single site of phosphorylation. Comparison of two-dimensional tryptic phosphopeptide maps prepared from synthetic peptide and P-glycoprotein, both of which were phosphorylated in vitro with protein kinase C, revealed that serine 669 was also the major phosphorylation site in the intact glycoprotein. The in vitro protein kinase A phosphorylation site was identified as serine 681 by site-directed mutagenesis. Inspection of the gene organization and the deduced amino acid sequence of mdr1b P-glycoprotein revealed that the linker region, although shorter than the R domain (55 versus 241 amino acids), fits the operational definition of the R domain of cystic fibrosis conductance regulator. Like the R domain, the linker region is encoded by a single exon, is highly charged with alternating acidic and basic side chains, and contains several protein kinase A/protein kinase C consensus phosphorylation sites. Since the R domain is believed to be involved in the regulation of cystic fibrosis conductance regulator function by phosphorylation, it is possible that the linker region plays a similar regulatory role in P-glycoprotein function.
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PMID:Identification of the major phosphorylation domain of murine mdr1b P-glycoprotein. Analysis of the protein kinase A and protein kinase C phosphorylation sites. 790 Dec 20

A P-glycoprotein homologue has been previously identified in Plasmodium falciparum and was termed PGH 1. This paper describes studies analyzing the phosphorylation of the PGH 1 molecule. It was found, by metabolic labeling with [32P]orthophosphate, that PGH 1 was phosphorylated throughout the entire asexual erythrocytic life cycle of the parasite, with the maximum level of 32P incorporation during the trophozoite and schizont stages. Incubation of trophozoites with modulators of mammalian protein kinases suggests that a Ca(2+)-dependent protein kinase is involved in phosphorylation of PGH 1. PGH 1 could also be phosphorylated in the presence of gamma-32P ATP on purified digestive vacuoles where this protein has previously been localized. Two-dimensional phospho-amino acid analysis revealed that PGH 1 was phosphorylated on serine and threonine residues and the pattern of amino acid phosphorylation was similar for PGH 1 phosphorylated in infected red blood cells and on purified digestive vacuoles. PGH 1 phosphorylation in the presence of some antimalarial drugs was analyzed and it was found that neither chloroquine nor compounds that modulate chloroquine resistance had any effect on PGH 1 phosphorylation.
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PMID:Phosphorylation of a P-glycoprotein homologue in Plasmodium falciparum. 790 21

Specific sites in the linker region of human P-glycoprotein phosphorylated by protein kinase C (PKC) were identified by means of a synthetic peptide substrate, PG-2, corresponding to residues 656-689 from this region of the molecule. As PG-2 has several sequences of the type recognized by the cyclic AMP-dependent protein kinase (PKA), PG-2 was also tested as a substrate for PKA. PG-2 was phosphorylated by purified PKC in a Ca2+/phospholipid-dependent manner, with a Km of 1.3 microM, and to a maximum stoichiometry of 2.9 +/- 0.1 mol of phosphate/mol of peptide. Sequence analysis of tryptic fragments of PG-2 phosphorylated by PKC identified Ser-661, Ser-667 and Ser-671 as the three sites of phosphorylation. PG-2 was also found to be phosphorylated by purified PKA in a cyclic AMP-dependent manner, with a Km of 21 microM, and to a maximum stoichiometry of 2.6 +/- 0.2 mol of phosphate/mol of peptide. Ser-667, Ser-671 and Ser-683 were phosphorylated by PKA. Truncated peptides of PG-2 were utilized to confirm that Ser-661 was PKC-specific and Ser-683 was PKA-specific. Further studies showed that PG-2 acted as a competitive substrate for the P-glycoprotein kinase present in membranes from multidrug-resistant human KB cells. The membrane kinase phosphorylated PG-2 mainly on Ser-661, Ser-667 and Ser-671. These results show that human P-glycoprotein can be phosphorylated by at least two protein kinases, stimulated by different second-messenger systems, which exhibit both overlapping and unique specificities for phosphorylation of multiple sites in the linker region of the molecule.
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PMID:Phosphorylation by protein kinase C and cyclic AMP-dependent protein kinase of synthetic peptides derived from the linker region of human P-glycoprotein. 790 31

We have previously shown that GTP can replace ATP as an energy source to support vinblastine transport by the multidrug transporter P-glycoprotein (Pgp) in plasma membrane vesicles isolated from the multidrug resistant cell line KB-V1 [Lelong et al. (1992) FEBS Lett. 304, 256-260]. Like [gamma-32P]ATP, [gamma-32P]GTP was also able to phosphorylate Pgp in vitro. Unlabeled GTP enhanced the phosphorylation of the transporter by [gamma-32P]ATP, whereas unlabeled ATP inhibited incorporation of label. While phosphorylation by [gamma-32P]ATP was Mg(2+)-dependent, the enhanced phosphorylation of Pgp by GTP was supported by Mg2+ or Mn2+ and to a lesser extent, Ca2+. Specific inhibitors of cAMP-dependent protein kinase, protein kinase C and cGMP-dependent protein kinase, did not affect phosphorylation. The phosphoprotein phosphatase inhibitor okadaic acid slightly enhanced phosphorylation, and vanadate more dramatically increased phosphorylation of the transporter. Tryptic maps of Pgp phosphorylated peptides indicate that addition of GTP altered the relative labeling of phosphopeptides. These results suggest that the overall phosphorylation of Pgp in vitro is determined by several different protein kinases and phosphatases, at least one of which may be GTP-regulated.
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PMID:GTP-stimulated phosphorylation of P-glycoprotein in transporting vesicles from KB-V1 multidrug resistant cells. 791 30

The activity of several proteins involved in the development of antitumor drug resistance is regulated by protein phosphorylation. These proteins include the mdr-1-encoded P-glycoprotein (Pgp) and topoisomerase II (topo II). The corresponding evidence is reviewed and attempts to modulate multidrug resistance (MDR) by protein kinase C inhibitors are described. The expression of several proteins which are essential in drug resistance is regulated at the transcriptional level, involving protein phosphorylation by members of the protein kinase C (PKC) family, casein kinase II (CKII), and others. These proteins include mdr-1-encoded P-glycoprotein, metallothionein, glutathione S-transferase (GST), dTMP synthase, and the proteins Fos and Jun. The corresponding genes are under positive regulation of ras, which in turn requires the activation of a protein kinase cascade for its function. Protein kinases are therefore potentially useful targets in reducing the expression of proteins involved in the development of multifactorial drug resistance caused by the expression of transforming ras-genes. Attempts to inhibit the ras-induced fos expression by an inhibitor of protein kinase C (ilmofosine) are described. Protein kinase inhibitors are also able to synergistically enhance the cytotoxicity of cis-platinum, which is discussed as resulting from a reduction of PKC-dependent fos expression.
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PMID:Role of protein kinases in antitumor drug resistance. 806 Nov 7

Inhibition by staurosporine derivatives of cyclic AMP-dependent protein kinase (A-kinase) and protein kinase C (C-kinase), and drug resistance has been investigated. The substitution of an acetyl or an ethoxycarbonyl group for the amine N-ethoxycarbonyl-7-oxostaurosporine moiety on the tetrahydropyran ring of staurosporine decreased inhibition of both protein kinases, but increased selectivity for C-kinase by further modification of the lactam moiety to the imide (NA-382). The activities of SF-2370 on protein kinases were decreased by decarboxylation and hydroxyalkylation. These staurosporine derivatives enhanced accumulation of vinblastine in adriamycin-resistant P388 (P388/ADR) cells in a dose-dependent manner. The potency for the drug accumulation of these compounds was correlated with their inhibitory activity on the drug efflux, but was not correlated with their activity on protein kinases. Staurosporine and NA-382, with high potency for vinblastine accumulation, inhibited the photolabelling of [3H]azidopine on 140 kDa P-glycoprotein in the plasma membrane. The tetrahydrofuran compounds and NA-357, which had low potency for the drug accumulation, hardly interacted with azidopine on P-glycoprotein. Most of these compounds were highly cytotoxic by themselves, and only NA-382 was less cytotoxic among them and completely reversed the vinblastine-resistance of P388/ADR cells at a non-cytotoxic concentration. These results suggest that staurosporine derivatives can enhance drug accumulation and inhibit drug resistance through their direct action on the P-glycoprotein.
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PMID:Effect of staurosporine derivatives on protein kinase activity and vinblastine accumulation in mouse leukaemia P388/ADR cells. 809 45


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