Imai Shin-Ichi, Yasuda Satoshi, Kai Masahiro, Kanoh Hideo, Sakane Fumio
Department of Biochemistry, Sapporo Medical University School of Medicine, South-1, West-17, Chuo-ku, Sapporo, 060-8556, Japan.
Biochim Biophys Acta. 2009 Apr;1791(4):246-53. doi: 10.1016/j.bbalip.2009.01.024. Epub 2009 Feb 7.
The delta-isozyme (type II) of diacylglycerol kinase (DGK) is known to positively regulate growth factor receptor signaling. DGKdelta, which is distributed to clathrin-coated vesicles, interacts with DGKdelta itself, protein kinase C and AP2alpha. To search for additional DGKdelta-interacting proteins, we screened a yeast two-hybrid cDNA library from HepG2 cells using aa 896-1097 of DGKdelta as a bait. We identified aa 184-317 (WD40 repeats 5-7) of receptor for activated C kinase 1 (RACK1), which interacts with various important signaling molecules, as a novel binding partner of DGKdelta. Co-immunoprecipitation analysis, using COS-7 cells co-expressing RACK1 and DGKdelta, revealed that RACK1 selectively interacted with DGKdelta, but not with type I DGKs, in mammalian cells. The interaction was dynamically regulated by phorbol ester. Intriguingly, DGKdelta appeared to recruit RACK1 to clathrin-coated vesicles and co-localized with RACK1. These results suggest that DGKdelta serves as an adaptor protein to regulate the localization of the versatile scaffold protein, RACK1.
已知二酰基甘油激酶（DGK）的δ同工酶（II型）对生长因子受体信号传导起正向调节作用。分布于网格蛋白包被小泡的DGKδ，可与自身、蛋白激酶C和AP2α相互作用。为了寻找其他与DGKδ相互作用的蛋白，我们以DGKδ的第896 - 1097氨基酸残基为诱饵，筛选了来自HepG2细胞的酵母双杂交cDNA文库。我们鉴定出活化C激酶1（RACK1）受体的第184 - 317氨基酸残基（WD40重复序列5 - 7）是DGKδ的一种新结合伴侣，RACK1可与多种重要信号分子相互作用。利用共表达RACK1和DGKδ的COS - 7细胞进行的免疫共沉淀分析表明，在哺乳动物细胞中，RACK1选择性地与DGKδ相互作用，而不与I型DGK相互作用。这种相互作用受佛波酯动态调节。有趣的是，DGKδ似乎将RACK1招募至网格蛋白包被小泡，并与RACK1共定位。这些结果表明，DGKδ作为一种衔接蛋白，可调节多功能支架蛋白RACK1的定位。
