Regulation of clathrin-dependent endocytosis by diacylglycerol kinase δ:: importance of kinase activity and binding to AP2α
Regulation of clathrin-dependent endocytosis by diacylglycerol kinase δ:: importance of kinase activity and binding to AP2α
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DOI：
10.1042/bj20070755
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发表时间：
2008-01-15
影响因子：
4.1
通讯作者：
Saito, Naoaki
中科院分区：
文献类型：
--
作者：
Kawasaki, Takumi;Kobayashi, Takeshi;Saito, Naoaki
DGK δ（二酰基甘油激酶3）磷酸化DAG（二酰基甘油）并将其转化为PA（磷脂酸），在信号转导中具有重要作用。在本研究中，我们已经证明了DGK δ介导的网格蛋白依赖性内吞调节的分子机制，该内吞控制受体的内化、回收和降解。DGK 6参与网格蛋白依赖性内吞作用的调节先前在全基因组RNAi（RNA干扰）筛选后提出。网格蛋白包被的纹孔主要由网格蛋白和AP-2（adaptor protein 2）复合物形成。这些蛋白质在膜上组装成多面体晶格，并聚集几种内吞辅助蛋白。由于DGK δ 2的细胞内定位与网格蛋白包被的凹坑重叠，我们预测了DGK δ 2可能对网格蛋白依赖的内吞作用的调节及其与一些内吞调节蛋白的相互作用。DGK δ 2含有DXF型结合基序，DGK δ 2与AP-2复合物的亚基AP 2 α结合。DGK δ 2通过DGK δ 2催化结构域中的（FDTFRIL）-D-369和（DPF）-P-746序列与AP 2 α耳结构域中的平台亚结构域相互作用。为了进一步了解DGK δ 2在网格蛋白依赖性内吞作用中的作用，我们测量了内源性DGK δ敲低下表达野生型或突变型DGK δ 2的转铁蛋白和EGF（表皮生长因子）的摄取。缺乏与AP 2 α结合能力的突变体以及激酶阴性突变体不能补偿siRNA（小干扰RNA）处理抑制的转铁蛋白摄取，而野生型DGK δ 2的过表达完全恢复了转铁蛋白摄取。这些结果表明，DGK δ 2和AP 2 α之间的结合参与转铁蛋白内化，并且DGK活性对于内吞过程的调节也是必需的。
DGK delta (diacylglycerol kinase 3), which phosphorylates DAG (diacylglycerol) and converts it into PA (phosphatidic acid), has an important role in signal transduction. In the present study, we have demonstrated the molecular mechanism of DGK delta-mediated regulation of clathrin-dependent endocytosis that controls the internalization, recycling and degradation of receptors. Involvement of DGK6 in the regulation of clathrin-dependent endocytosis was previously proposed following genome-wide RNAi (RNA interference) screening. Clathrin-coated pits are mainly formed by clathrin and AP-2 (adaptor protein 2) complex. These proteins assemble a polyhedral lattice at the membrane and gather several endocytic accessory proteins. As the intracellular localization of DGK delta 2 overlapped with clathrin-coated pits, we predicted the possible regulation of clathrin-dependent endocytosis by DGK delta 2 and its interaction with some endocytosis-regulatory proteins. DGK delta 2 contained the DXF-type binding motifs, and DGK delta 2 bound to AP2 alpha, a subunit of the AP-2 complex. DGK delta 2 interacted with the platform subdomain in the AP2 alpha ear domain via (FDTFRIL)-D-369 and (DPF)-P-746 sequences in the catalytic domain of DGK delta 2. For further insight into the role for DGK delta 2 in clathrin-dependent endocytosis, we measured the transferrin and EGF (epidermal growth factor) uptakeexpressing wild-type or mutant DGK delta 2 under knockdown of endogenous DGK delta. Mutants lacking binding ability to AP2 alpha as well as kinase-negative mutants could not compensate for the uptake of transferrin inhibited by siRNA (small interfering RNA) treatment, whereas overexpression of wild-type DGK delta 2 completely recovered the transferrin uptake. These results demonstrate that binding between DGK delta 2 and AP2 alpha is involved in the transferrin internalization and that DGK activity is also necessary for the regulation of the endocytic process.