Domin J, Gaidarov I, Smith M E, Keen J H, Waterfield M D
Ludwig Institute for Cancer Research, University College, London W1P 8BT, United Kingdom.
J Biol Chem. 2000 Apr 21;275(16):11943-50. doi: 10.1074/jbc.275.16.11943.
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-C2alpha in several mammalian cell types. Differential centrifugation of COS-1 and U937 cells together with Western blot analysis demonstrated that PI3K-C2alpha 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-C2alpha could be co-purified 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-C2alpha isozyme. These biochemical observations were supported by immunofluorescence analysis that revealed PI3K-C2alpha 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-C2alpha 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-C2alpha 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-C2alpha and the AP-1 complex demonstrating that the localization of both molecules at the TGN is dependent upon ADP-ribosylation factor GTPase activity.
近年来，一大类磷酸肌醇3激酶（PI3K）同工酶已被鉴定和克隆。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发不同的细胞内效应。一种可能性是PI3K酶在细胞内显示出受限的分布，以便在特定的亚细胞区室中产生其3-磷脂产物。在本研究中，我们描述了新型II类PI3K同工酶PI3K-C2α在几种哺乳动物细胞类型中的亚细胞分布。通过对COS-1和U937细胞进行差速离心并结合蛋白质印迹分析表明，PI3K-C2α与磷脂膜组成性相关。对大鼠脑匀浆进行离心并进行蛋白质印迹分析发现，与IA类PI3K酶不同，PI3K-C2α可与一群网格蛋白包被小泡（CCV）共同纯化。此外，在CCV制剂中检测到对渥曼青霉素处理具有抗性的PI3K活性，这与PI3K-C2α同工酶的存在一致。这些生化观察结果得到免疫荧光分析的支持，该分析显示PI3K-C2α具有点状分布，并且在核周部位免疫反应性增强，这与其存在于内质网或高尔基体中一致。双重标记免疫荧光表明，在该区域，PI3K-C2α的分布与γ-衔接蛋白密切平行，γ-衔接蛋白是存在于反式高尔基体和反式高尔基体网络（TGN）中的AP-1衔接蛋白的一个组分，也是驻留蛋白TGN-46。PI3K-C2α的磷脂结合和亚细胞定位均不依赖于其COOH末端的PX或C2结构域。当作为重组蛋白表达时，缺乏这些结构域的突变体显示出与野生型酶相似的分布。用布雷菲德菌素A处理细胞会破坏PI3K-C2α和AP-1复合物的核周染色模式，表明这两种分子在TGN处的定位依赖于ADP-核糖基化因子GTP酶活性。
