Purification, cDNA‐cloning and expression of human diacylglycerol kinase

FEBS Letters

van Damme, Jose³  van der Bend, Rob L.²  Gussow, Detlef¹  de Widt, John²  van Blitterswijk, Whim J.²  Schaap, Dick²  Vandekerckhove, Joel³  Ploegh, Hidde L.²  van der Wal, Jose² 

[1] Division of Cellular and Biochemistry, Behringwerke, Marburg, FRG;Division of Cellular Biochemistry, The Netherlands Cancer Institute, (Antoni van Leeuwenhoek Huis), 1066 CX, Amsterdam, The Netherlands;Division of Cellular and Biochemistry, University of Gent, Gent, Belgium

关键词: Diacylglycerol kinase;    Human white blood cell;    EF-hand;    Cysteine repeat;    DOC;    deoxycholate;    DG;    1;    2-diacyl-sn-glycerol;    diolein;    1;    2-dioleoyl-sn-glycerol;    PA;    3-sn-phosphatidic acid;    PC;    3-sn-phosphatidylcholine;    PI;    3-sn-phosphatidylinositol;    PS;    3-sn-phosphatidylserine;    PCR;    polymerase chain reaction;    PKC;    protein kinase C;    WBC;    white blood cells;   

DOI  :  10.1016/0014-5793(90)81461-V

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Diacylglycerol (DG) kinase attenuates the level of the second messenger DG in signal transduction, and therefore possibly modulates protein kinase C (PKC). DG kinase was purified to homogeneity from human white blood cells, showing an M ₁ of 86 kDa as determined by SDS-PAGE and gel filtration. Two amino acid sequences of tryptic peptides from DG kinase were determined and degenerate oligonucleotides were prepared and used in the polymerase chain reaction. An amplified DNA fragment was subsequently used to clone the full-length human DG kinase cDNA. This sequence is the human homolog of porcine DG kinase cDNA sequence reported recently [1]. The sequence contains a double EF- hand structure typical for Ca²⁺ binding proteins. DG kinase further contains a double cysteine repeat that is present in all PKC isoforms, where it constitutes the phorbol ester (and most likely diacylglycerol) binding site. Therefore we speculate that the double cysteine repeat in DG kinase is involved in DG binding. DG kinase is transcribed as a single mRNA of 3.2 kb, that is highly expressed in T-lymphocytes. The human DG kinase cDNA when transfected in mammalian cells (COS-7) results in a 6–7-fold increase of DG kinase activity.

【 授权许可】

Unknown