Synthesis and phorbol ester binding of the cysteine-rich domains of diacylglycerol kinase (DGK) isozymes -: DGKγ and DGKγ are new targets of tumor-promoting phorbol esters

被引:57
作者
Shindo, M
Irie, K
Masuda, A
Ohigashi, H
Shirai, Y
Miyasaka, K
Saito, N
机构
[1] Kyoto Univ, Grad Sch Agr, Div Food Sci & Biotechnol, Sakyo Ku, Kyoto 6068502, Japan
[2] Appl Biosyst Japan Ltd, Chuo Ku, Tokyo 1040032, Japan
[3] Kobe Univ, Biosignal Res Ctr, Kobe, Hyogo 6578501, Japan
关键词
D O I
10.1074/jbc.M300400200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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-GK beta-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.
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收藏
页码:18448 / 18454
页数:7
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