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Purification, cDNA‐cloning and expression of human diacylglycerol kinase
Author(s) -
Schaap Dick,
de Widt John,
van der Wal Jose,
Vandekerckhove Joel,
van Damme Jose,
Gussow Detlef,
Ploegh Hidde L.,
van Blitterswijk Whim J.,
van der Bend Rob L.
Publication year - 1990
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(90)81461-v
Subject(s) - diacylglycerol kinase , protein kinase c , complementary dna , microbiology and biotechnology , map2k7 , biology , cyclin dependent kinase 9 , kinase , protein kinase a , biochemistry , mitogen activated protein kinase kinase , cyclin dependent kinase 2 , gene
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 Ca 2+ 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.