Inositol Signaling Group, Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, NIH, DHHS, Research Triangle Park, NC 27709, USA.
Biochem J. 2011 Mar 15;434(3):415-26. doi: 10.1042/BJ20101437.
The inositol pyrophosphates are multifunctional signalling molecules. One of the families of enzymes that synthesize the inositol pyrophosphates are the Vip1/PPIP5Ks (PP-InsP5 kinases). The kinase domains in Vip1/PPIP5Ks have been mapped to their N-terminus. Each of these proteins also possess a phosphatase-like domain of unknown significance. In the present study, we show that this phosphatase-like domain is not catalytically active. Instead, by using SPR (surface plasmon resonance) to study protein binding to immobilized lipid vesicles, we show that this domain is specialized for binding PtdIns(3,4,5)P3 (PPIP5K1 K(d)=96 nM; PPIP5K2 K(d)=705 nM). Both PtdIns(3,4)P2 and PtdIns(4,5)P2 are significantly weaker ligands, and no significant binding of PtdIns(3,5)P2 was detected. We confirm the functional importance of this domain in inositol lipid binding by site-directed mutagenesis. We present evidence that the PtdIns(3,4,5)P3-binding domain is an unusual hybrid, in which a partial PH (pleckstrin homology) consensus sequence is spliced into the phosphatase-like domain. Agonist-dependent activation of the PtdIns 3-kinase pathway in NIH 3T3 cells drives translocation of PPIP5K1 from the cytosol to the plasma membrane. We have therefore demonstrated receptor-regulated compartmentalization of inositol pyrophosphate synthesis in mammalian cells.
肌醇六磷酸是多功能信号分子。合成肌醇六磷酸的酶家族之一是 Vip1/PPIP5Ks（PP-InsP5 激酶）。Vip1/PPIP5Ks 的激酶结构域被定位在其 N 端。这些蛋白质中的每一种都还具有一个未知功能的磷酸酶样结构域。在本研究中，我们表明这个磷酸酶样结构域没有催化活性。相反，我们使用 SPR（表面等离子体共振）来研究蛋白质与固定化脂质体的结合，表明这个结构域专门用于结合 PtdIns(3,4,5)P3（PPIP5K1 K(d)=96 nM；PPIP5K2 K(d)=705 nM）。PtdIns(3,4)P2 和 PtdIns(4,5)P2 都是明显较弱的配体，并且没有检测到 PtdIns(3,5)P2 的显著结合。我们通过定点突变证实了这个结构域在肌醇脂质结合中的功能重要性。我们提供的证据表明，PtdIns(3,4,5)P3 结合结构域是一个不寻常的杂合体，其中部分 PH（pleckstrin homology）保守序列被拼接在磷酸酶样结构域中。NIH 3T3 细胞中 PtdIns 3-激酶途径的激动剂依赖性激活导致 PPIP5K1 从细胞质向质膜易位。因此，我们已经证明了哺乳动物细胞中肌醇六磷酸合成的受体调节区室化。
