Synthesis and phorbol ester binding of the cysteine-rich domains of diacylglycerol kinase (DGK) isozymes. DGKgamma and DGKbeta are new targets of tumor-promoting phorbol esters.
Diacylglycerol kinase (DGK) and protein kinase C (PKC) are two distinct enzyme families associated with diacylglycerol. Both enzymes have cysteine-rich C1 domains (C1A, C1B, and C1C) in the regulatory region. Although most PKC C1 domains strongly bind phorbol esters, there has been no direct evidence that DGK C1 domains bind phorbol esters. We synthesized 11 cysteine-rich sequences of DGK C1 domains with good sequence homology to those of the PKC C1 domains. Among them, only DGKgamma-C1A and DGKbeta-C1A exhibited significant binding to phorbol 12,13-dibutyrate (PDBu). Scatchard analysis of rat-DGKgamma-C1A, human-DGKgamma-C1A, and human-DGKbeta-C1A gave K(d) values of 3.6, 2.8, and 14.6 nm, respectively, suggesting that DGKgamma and DGKbeta are new targets of phorbol esters. An A12T mutation of human-DGKbeta-C1A enhanced the affinity to bind PDBu, indicating that the beta-hydroxyl group of Thr-12 significantly contributes to the binding. The K(d) value for PDBu of FLAG-tagged whole rat-DGKgamma (4.4 nm) was nearly equal to that of rat-DGKgamma-C1A (3.6 nm). Moreover, 12-O-tetradecanoylphorbol 13-acetate induced the irreversible translocation of whole rat-DGKgamma and its C1B deletion mutant, not the C1A deletion mutant, from the cytoplasm to the plasma membrane of CHO-K1 cells. These results indicate that 12-O-tetradecanoylphorbol 13-acetate binds to C1A of DGKgamma to cause its translocation.
Shindo
Mayumi
M
Applied Biosystems Japan Ltd., 4-5-4 Hacchobori, Chuo-ku, Tokyo 104-0032, Japan.
Irie
Kazuhiro
K
Masuda
Akiko
A
Ohigashi
Hajime
H
Shirai
Yasuhito
Y
Miyasaka
Kei
K
Saito
Naoaki
N
D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D003545:Cysteine / Q000737:chemistry*; D019852:Diacylglycerol Kinase / Q000737:chemistry* / Q000378:metabolism; D017353:Gene Deletion; D049452:Green Fluorescent Proteins; D006801:Humans; D007700:Kinetics; D008164:Luminescent Proteins / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009369:Neoplasms / Q000139:chemically induced; D010455:Peptides / Q000737:chemistry; D015240:Phorbol 12,13-Dibutyrate / Q000737:chemistry; D010703:Phorbol Esters / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013912:Threonine / Q000737:chemistry; D013997:Time Factors
D008164:Luminescent Proteins; D010455:Peptides; D010703:Phorbol Esters; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D013912:Threonine; D015240:Phorbol 12,13-Dibutyrate; D019852:Diacylglycerol Kinase; D003545:Cysteine