Purification, cDNA-cloning and expression of human diacylglycerol kinase.
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 Mr 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 a porcine DG kinase cDNA sequence reported recently. 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.
Schaap
D
D
Division of Cellular Biochemistry, The Netherlands Cancer Institute (Antoni van Leeuwenhoek Huis), Amsterdam.
de Widt
J
J
van der Wal
J
J
Vandekerckhove
J
J
van Damme
J
J
Gussow
D
D
Ploegh
H L
HL
van Blitterswijk
W J
WJ
van der Bend
R L
RL
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002135:Calcium-Binding Proteins / Q000235:genetics; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D019852:Diacylglycerol Kinase; D015870:Gene Expression; D006801:Humans; D007962:Leukocytes / Q000201:enzymology; D008969:Molecular Sequence Data; D010770:Phosphotransferases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid; D013379:Substrate Specificity; D013552:Swine
D002135:Calcium-Binding Proteins; D012333:RNA, Messenger; D000255:Adenosine Triphosphate; D004247:DNA; D010770:Phosphotransferases; D019852:Diacylglycerol Kinase