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Evidence for phosphorylation requirement for human bilirubin UDP-glucuronosyltransferase (UGT1A1) activity. 2003

Nikhil K Basu, and Labanyamoy Kole, and Ida S Owens
Heritable Disorders Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-1830, USA.

Our discovery of rapid down-regulation of human bilirubin UDP-glucuronosyltransferase (UGT) in colon cell lines that was transient and irreversible following curcumin- and calphostin-C-treatment, respectively, suggested phosphorylation event(s) were involved in activity. Likewise, bilirubin-UGT1A1 expressed in COS-1 cells was inhibited by curcumin and calphostin-C. Because calphostin-C is a highly specific protein kinase C (PKC) inhibitor, we examined and found 4 to 5 predicted PKC phosphorylation sites in 11 UGTs examined. UGT1A1 incorporated [33P]orthophosphate, which was inhibited by calphostin-C. Also triple mutant, T75A/T112A/S435G-UGT1A1, at predicted PKC sites failed to incorporate [33P]orthophosphate. Individual or double mutants exhibited dominant-negative, additive, or no effect, while the triple mutant retained 10-15% activity towards bilirubin and two xenobiotics. Compared to wild-type, S435G and T112A/S435G shifted pH-optimum for eugenol, but not for bilirubin or anthraflavic acid, toward alkaline and acid conditions, respectively. This represents the first evidence that a UGT isozyme requires phosphorylation for activity.

UI MeSH Term Description Entries
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
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.
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
D011493 Protein Kinase C An serine-threonine protein kinase that requires the presence of physiological concentrations of CALCIUM and membrane PHOSPHOLIPIDS. The additional presence of DIACYLGLYCEROLS markedly increases its sensitivity to both calcium and phospholipids. The sensitivity of the enzyme can also be increased by PHORBOL ESTERS and it is believed that protein kinase C is the receptor protein of tumor-promoting phorbol esters. Calcium Phospholipid-Dependent Protein Kinase,Calcium-Activated Phospholipid-Dependent Kinase,PKC Serine-Threonine Kinase,Phospholipid-Sensitive Calcium-Dependent Protein Kinase,Protein Kinase M,Calcium Activated Phospholipid Dependent Kinase,Calcium Phospholipid Dependent Protein Kinase,PKC Serine Threonine Kinase,Phospholipid Sensitive Calcium Dependent Protein Kinase,Phospholipid-Dependent Kinase, Calcium-Activated,Serine-Threonine Kinase, PKC
D011994 Recombinant Proteins Proteins prepared by recombinant DNA technology. Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
D002384 Catalysis The facilitation of a chemical reaction by material (catalyst) that is not consumed by the reaction. Catalyses
D003474 Curcumin A yellow-orange dye obtained from tumeric, the powdered root of CURCUMA longa. It is used in the preparation of curcuma paper and the detection of boron. Curcumin appears to possess a spectrum of pharmacological properties, due primarily to its inhibitory effects on metabolic enzymes. 1,6-Heptadiene-3,5-dione, 1,7-bis(4-hydroxy-3-methoxyphenyl)-, (E,E)-,Curcumin Phytosome,Diferuloylmethane,Mervia,Turmeric Yellow,Phytosome, Curcumin,Yellow, Turmeric
D004305 Dose-Response Relationship, Drug The relationship between the dose of an administered drug and the response of the organism to the drug. Dose Response Relationship, Drug,Dose-Response Relationships, Drug,Drug Dose-Response Relationship,Drug Dose-Response Relationships,Relationship, Drug Dose-Response,Relationships, Drug Dose-Response
D004791 Enzyme Inhibitors Compounds or agents that combine with an enzyme in such a manner as to prevent the normal substrate-enzyme combination and the catalytic reaction. Enzyme Inhibitor,Inhibitor, Enzyme,Inhibitors, Enzyme
D005966 Glucuronidase Endo-beta-D-Glucuronidase,Endoglucuronidase,Exo-beta-D-Glucuronidase,beta-Glucuronidase,Endo beta D Glucuronidase,Exo beta D Glucuronidase,beta Glucuronidase

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