Regulation by phorbol ester and protein kinase C inhibitors, and by a protein phosphatase inhibitor (okadaic acid), of P-glycoprotein phosphorylation and relationship to drug accumulation in multidrug-resistant human KB cells. 1992

T C Chambers, and B Zheng, and J F Kuo
Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia 30322.

Covalent modification by phosphorylation is a characteristic of the P-glycoproteins expressed in multidrug-resistant cells. This report describes analysis of P-glycoprotein phosphorylation in multidrug-resistant human KB-V1 cells and a study of the relationship of phosphorylation and drug accumulation. In isolated membranes, phosphorylation of P-glycoprotein by purified protein kinase C (PKC) was rapid, and time-dependent dephosphorylation was inhibited by okadaic acid, an inhibitor of type 1 and type 2A protein phosphatases. In 32P-labeled intact KB-V1 cells, P-glycoprotein phosphorylation was stimulated by both 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of PKC, and okadaic acid. Two-dimensional thin layer tryptic phosphopeptide maps indicated that the sites of phosphorylation were similar in control, TPA-treated, and okadaic acid-treated cells and that they corresponded to those phosphorylated by PKC in vitro. The protein kinase inhibitor staurosporine, and the PKC-selective inhibitors calphostin C and the alkyl-lysophospholipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine, inhibited P-glycoprotein phosphorylation in vitro and in intact cells. Drug accumulation assays demonstrated that in KB-V1 cells TPA caused a decrease, whereas staurosporine and calphostin C caused an increase, in accumulation of [3H]vinblastine. These compounds did not significantly alter [3H]vinblastine levels in drug-sensitive KB-3 cells. These results suggest that PKC is chiefly responsible for P-glycoprotein phosphorylation in KB-V1 cells, that membrane-associated protein phosphatases 1 and 2A are active in dephosphorylation of P-glycoprotein, and that phosphorylation of P-glycoprotein may be an important mechanism for modulation of drug-pumping activity.

UI MeSH Term Description Entries
D007624 KB Cells This line KB is now known to be a subline of the ubiquitous KERATIN-forming tumor cell line HeLa. It was originally thought to be derived from an epidermal carcinoma of the mouth, but was subsequently found, based on isoenzyme analysis, HeLa marker chromosomes, and DNA fingerprinting, to have been established via contamination by HELA CELLS. The cells are positive for keratin by immunoperoxidase staining. KB cells have been reported to contain human papillomavirus18 (HPV-18) sequences. HeLa-KB Cells,Cell, HeLa-KB,Cell, KB,Cells, HeLa-KB,Cells, KB,HeLa KB Cells,HeLa-KB Cell,KB Cell
D008562 Membrane Glycoproteins Glycoproteins found on the membrane or surface of cells. Cell Surface Glycoproteins,Surface Glycoproteins,Cell Surface Glycoprotein,Membrane Glycoprotein,Surface Glycoprotein,Glycoprotein, Cell Surface,Glycoprotein, Membrane,Glycoprotein, Surface,Glycoproteins, Cell Surface,Glycoproteins, Membrane,Glycoproteins, Surface,Surface Glycoprotein, Cell,Surface Glycoproteins, Cell
D009281 Naphthalenes Two-ring crystalline hydrocarbons isolated from coal tar. They are used as intermediates in chemical synthesis, as insect repellents, fungicides, lubricants, preservatives, and, formerly, as topical antiseptics.
D010449 Peptide Mapping Analysis of PEPTIDES that are generated from the digestion or fragmentation of a protein or mixture of PROTEINS, by ELECTROPHORESIS; CHROMATOGRAPHY; or MASS SPECTROMETRY. The resulting peptide fingerprints are analyzed for a variety of purposes including the identification of the proteins in a sample, GENETIC POLYMORPHISMS, patterns of gene expression, and patterns diagnostic for diseases. Fingerprints, Peptide,Peptide Fingerprinting,Protein Fingerprinting,Fingerprints, Protein,Fingerprint, Peptide,Fingerprint, Protein,Fingerprinting, Peptide,Fingerprinting, Protein,Mapping, Peptide,Peptide Fingerprint,Peptide Fingerprints,Protein Fingerprint,Protein Fingerprints
D010641 Phenotype The outward appearance of the individual. It is the product of interactions between genes, and between the GENOTYPE and the environment. Phenotypes
D010742 Phospholipid Ethers Phospholipids which have an alcohol moiety in ethereal linkage with a saturated or unsaturated aliphatic alcohol. They are usually derivatives of phosphoglycerols or phosphatidates. The other two alcohol groups of the glycerol backbone are usually in ester linkage. These compounds are widely distributed in animal tissues. Ether Phosphatidates,Ether Phospholipids,Glycerol Phosphate Ethers,Glycerophosphate Ethers,1-Alkyl-2-Acylphosphatidates,1 Alkyl 2 Acylphosphatidates,Ethers, Glycerol Phosphate,Ethers, Glycerophosphate,Ethers, Phospholipid,Phosphate Ethers, Glycerol,Phosphatidates, Ether,Phospholipids, Ether
D010748 Phosphopeptides PEPTIDES that incorporate a phosphate group via PHOSPHORYLATION. Phosphopeptide
D010749 Phosphoprotein Phosphatases A group of enzymes removing the SERINE- or THREONINE-bound phosphate groups from a wide range of phosphoproteins, including a number of enzymes which have been phosphorylated under the action of a kinase. (Enzyme Nomenclature, 1992) Phosphoprotein Phosphatase,Phosphoprotein Phosphohydrolase,Protein Phosphatase,Protein Phosphatases,Casein Phosphatase,Ecto-Phosphoprotein Phosphatase,Nuclear Protein Phosphatase,Phosphohistone Phosphatase,Phosphoprotein Phosphatase-2C,Phosphoseryl-Protein Phosphatase,Protein Phosphatase C,Protein Phosphatase C-I,Protein Phosphatase C-II,Protein Phosphatase H-II,Protein-Serine-Threonine Phosphatase,Protein-Threonine Phosphatase,Serine-Threonine Phosphatase,Threonine Phosphatase,Ecto Phosphoprotein Phosphatase,Phosphatase C, Protein,Phosphatase C-I, Protein,Phosphatase C-II, Protein,Phosphatase H-II, Protein,Phosphatase, Casein,Phosphatase, Ecto-Phosphoprotein,Phosphatase, Nuclear Protein,Phosphatase, Phosphohistone,Phosphatase, Phosphoprotein,Phosphatase, Phosphoseryl-Protein,Phosphatase, Protein,Phosphatase, Protein-Serine-Threonine,Phosphatase, Protein-Threonine,Phosphatase, Serine-Threonine,Phosphatase, Threonine,Phosphatase-2C, Phosphoprotein,Phosphatases, Phosphoprotein,Phosphatases, Protein,Phosphohydrolase, Phosphoprotein,Phosphoprotein Phosphatase 2C,Phosphoseryl Protein Phosphatase,Protein Phosphatase C I,Protein Phosphatase C II,Protein Phosphatase H II,Protein Phosphatase, Nuclear,Protein Serine Threonine Phosphatase,Protein Threonine Phosphatase,Serine Threonine Phosphatase
D010766 Phosphorylation The introduction of a phosphoryl group into a compound through the formation of an ester bond between the compound and a phosphorus moiety. Phosphorylations
D011083 Polycyclic Compounds Compounds which contain two or more rings in their structure. Compounds, Polycyclic

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