Phosphatidylinositol 3-phosphate-interacting domains in PIKfyve - Binding specificity and role in PIKfyve endomembrane localization
Phosphatidylinositol 3-phosphate-interacting domains in PIKfyve - Binding specificity and role in PIKfyve endomembrane localization
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DOI：
10.1074/jbc.m110194200
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发表时间：
2002-02-22
影响因子：
4.8
通讯作者：
Shisheva, A
中科院分区：
文献类型：
--
作者：
Sbrissa, D;Ikonomov, OC;Shisheva, A
PIKfyve 是一种磷脂酰肌醇 (PtdIns) 3-磷酸 (P) 代谢酶，除了位于 C 末端的催化结构域外，还具有多个进化保守结构域，包括 FYVE 指。 FYVE 指结构域被认为可将蛋白质定位至细胞内膜 PtdIns 3-P。最近对几种 FYVE 结构域蛋白的研究挑战了这一普遍概念。在这里，我们在体外和体内检查了 PIKfyve 的 FYVE 结构域与 PtdIns 3-P 的结合，以及这种结合机制对全长蛋白的细胞内定位的合理贡献。我们现在记录了重组产生的 PIKfyve FYVE 结构域肽片段与含有 Ptdlns 3-P 的脂质体的特异性和高亲和力相互作用，这需要 FYVE 结构域内存在碱性残基的保守核心。发现 PlKfyve 对晚期内吞途径膜的定位绝对依赖于完整 FYVE 指的存在。用PI 3-激酶抑制剂渥曼青霉素处理细胞可解离内体结合的PlKfyve，表明该蛋白靶向膜PtdIns 3-P。发现 PlKfyve 催化结构域的酶促无活性肽片段也与含有 Ptdlns 3-P 的脂质体特异性结合，其中残基 Lys-1999 在相互作用中至关重要。然而，这种结合的亲和力相对较低，并且在细胞环境中，发现无法有效地将分子引导至富含 Ptdlns 3-P 的内体。总的来说，这些结果表明 FYVE 结构域与 PtdIns 3-P 的相互作用对于 PIKfyve 靶向晚期内吞途径的膜是绝对必要的，并确定 PlKfyve 作为 PtdIns 3-P 的下游效应子。
PIKfyve is a phosphatidylinositol (PtdIns) 3-phosphate (P)-metabolizing enzyme, which, in addition to a C-terminally positioned catalytic domain, harbors several evolutionarily conserved domains, including a FYVE finger. The FYVE finger domains are thought to direct the protein localization to intracellular membrane PtdIns 3-P. Recent studies with several FYVE domain proteins challenge this general concept. Here we have examined the binding of PIKfyve's FYVE domain to PtdIns 3-P in vitro and in vivo and a plausible contribution of this binding mechanism for the intracellular localization of the full-length protein. We document now a specific and high affinity interaction of a recombinantly produced PIKfyve FYVE domain peptide fragment with Ptdlns 3-P-containing liposomes that requires the presence of the conservative core of basic residues within the FYVE domain. PlKfyve localization to membranes of the late endocytic pathway was found to be absolutely dependent on the presence of an intact FYVE finger. Cell treatment with PI 3-kinase inhibitor wortmannin dissociated endosome-bound PlKfyve, indicating that the protein targeted the membrane PtdIns 3-P. An enzymatically inactive peptide fragment of the PlKfyve catalytic domain was found to also specifically bind to Ptdlns 3-P-containing liposomes, with residue Lys-1999 being critical in the interaction. This binding, however, was of relatively low affinity and, in the cellular context, was found ineffective in directing the molecule to Ptdlns 3-P-enriched endosomes. Collectively, these results demonstrate that interaction of the FYVE domain with PtdIns 3-P is absolutely necessary for PIKfyve targeting to the membranes of the late endocytic pathway and determine PlKfyve as a downstream effector of PtdIns 3-P.