The class II phosphoinositide 3-kinase PI3K-C2α is concentrated in the trans-Golgi network and present in clathrin-coated vesicles
The class II phosphoinositide 3-kinase PI3K-C2α is concentrated in the trans-Golgi network and present in clathrin-coated vesicles
复制标题
DOI：
10.1074/jbc.275.16.11943
复制
发表时间：
2000-04-21
影响因子：
4.8
通讯作者：
Waterfield, MD
中科院分区：
文献类型：
--
作者：
Domin, J;Gaidarov, I;Waterfield, MD
近年来，一个大的磷脂酰肌醇3-激酶(PI3K)同工酶家族已被鉴定和克隆。其中几种PI3K酶具有重叠的组织分布，目前尚不清楚它们的3-磷酸肌醇产物是否以及如何引起不同的细胞内效应。一种可能性是，PI3K酶在细胞内的分布受到限制，在特定的亚细胞隔间产生3-磷脂产物。在本研究中，我们研究了新的第II类PI3K同工酶PI3K-C2α在几种哺乳动物细胞中的亚细胞分布。COS-1和U937细胞的差速离心和Western印迹分析表明，PI3K-C2α与磷脂膜呈结构性结合。大鼠脑匀浆的离心和Western blotting表明，与IA类PI3K酶不同，PI3K-C2α可以与大量的笼蛋白包裹的小泡(CCV)结合。此外，在CCV制剂中检测到一种对Wortmannin处理无效的PI3K活性，这与PI3K-C2α同工酶的存在一致。这些生化观察得到了免疫荧光分析的支持，免疫荧光分析显示PI3K-C2a在核周部位具有点状分布和丰富的免疫反应性，与其在内质网或高尔基体中的存在一致。双标记免疫荧光显示，在该区域，PI3K-C2a的分布与Gamma-Adaptin的分布密切相关，Gamma-Adaptin是AP-1接头的一种成分，存在于反式高尔基体和反式高尔基体网络(TGN)驻留蛋白TGN-46中。PI3K-C2a的磷脂结合和亚细胞定位都不依赖于其COOH末端的Px或C2结构域。缺乏这些结构域的突变体在以重组蛋白的形式表达时，表现出与野生型酶相似的分布。灯盏花素A处理细胞后，破坏了PI3K-C2α和AP-1复合体的核周染色模式，表明这两个分子在TGN的定位取决于ADP-核糖化因子GTP酶的活性。
In recent years, a large family of phosphoinositide 3-kinase (PI3K) isozymes has been characterized and cloned. Several of these PI3K enzymes have overlapping tissue distributions and it remains unclear if and how their 3-phosphoinositide products elicit differential, intracellular effects. One possibility is that the PI3K enzymes display a restricted distribution within the cell to produce their 3-phospholipid products in specific, subcellular compartments. In the present study we characterize the subcellular distribution of the novel class II PI3K isozyme PI3K-C2 alpha in several mammalian cell types. Differential centrifugation of COS-1 and U937 cells together with Western blot analysis demonstrated that PI3K-C2 alpha is constitutively associated with phospholipid membranes. Centrifugation of rat brain homogenates and Western blotting revealed that in contrast to the class IA PI3K enzymes, PI3K-C2 alpha could be copurified with a population of clathrin-coated vesicles (CCVs). Furthermore, a PI3K activity refractory to wortmannin treatment was detected in CCV preparations consistent with the presence of the PI3K-C2 alpha isozyme. These biochemical observations were supported by immunofluorescence analysis that revealed PI3K-C2a to have a punctate distribution and an enrichment of immunoreactivity within a perinuclear site consistent with its presence in the endoplasmic reticulum or Golgi apparatus. Dual label immunofluorescence demonstrated that in this region, the distribution of PI3K-C2a closely paralleled that of gamma-adaptin, a component of the AP-1 adaptor that is present in the trans-Golgi and the trans-Golgi network (TGN) resident protein TGN-46. Neither the phospholipid association nor the subcellular localization of PI3K-C2a was dependent upon either its COOH-terminal PX or C2 domains. Mutants lacking these domains demonstrated a similar distribution to the wild type enzyme when expressed as recombinant proteins. Treatment of cells with brefeldin A disrupted the perinuclear staining pattern of both PI3K-C2 alpha and the AP-1 complex demonstrating that the localization of both molecules at the TGN is dependent upon ADP-ribosylation factor GTPase activity.