Waugh Mark G, Minogue Shane, Blumenkrantz Deena, Anderson J Simon, Hsuan J Justin
Centre for Molecular Cell Biology, Department of Medicine, Royal Free and University College Medical School, University College London, Rowland Hill Street, London NW3 2PF, UK.
Biochem J. 2003 Dec 1;376(Pt 2):497-503. doi: 10.1042/BJ20031212.
The seven known polyphosphoinositides have been implicated in a wide range of regulated and constitutive cell functions, including cell-surface signalling, vesicle trafficking and cytoskeletal reorganization. In order to understand the spatial and temporal control of these diverse cell functions it is necessary to characterize the subcellular distribution of a wide variety of polyphosphoinositide synthesis and signalling events. The predominant phosphatidylinositol kinase activity in many mammalian cell types involves the synthesis of the signalling precursor, phosphatidylinositol 4-phosphate, in a reaction catalysed by the recently cloned PI4KIIalpha (type IIalpha phosphatidylinositol 4-kinase). However the regulation of this enzyme and the cellular distribution of its product in different organelles are very poorly understood. This report identifies the existence, in unstimulated cells, of two major subcellular membrane fractions, which contain PI4KIIalpha possessing different levels of intrinsic activity. Separation of these membranes from each other and from contaminating activities was achieved by density gradient ultracentrifugation at pH 11 in a specific detergent mixture in which both membrane fractions, but not other membranes, were insoluble. Kinetic comparison of the purified membrane fractions revealed a 4-fold difference in K (m) for phosphatidylinositol and a 3.5-fold difference in V (max), thereby indicating a different mechanism of regulation to that described previously for agonist-stimulated cells. These marked differences in basal activity and the occurrence of this isozyme in multiple organelles emphasize the need to investigate cell signalling via PI4KIIalpha at the level of individual organelles rather than whole-cell lysates.
已知的七种多磷酸肌醇参与了广泛的细胞调节和组成性功能，包括细胞表面信号传导、囊泡运输和细胞骨架重组。为了理解这些多样的细胞功能的时空控制，有必要对多种多磷酸肌醇合成和信号传导事件的亚细胞分布进行表征。在许多哺乳动物细胞类型中，主要的磷脂酰肌醇激酶活性涉及信号前体磷脂酰肌醇4-磷酸的合成，该反应由最近克隆的PI4KIIα（IIα型磷脂酰肌醇4-激酶）催化。然而，对这种酶的调节及其产物在不同细胞器中的细胞分布了解甚少。本报告确定了在未受刺激的细胞中存在两个主要的亚细胞膜组分，它们含有具有不同内在活性水平的PI4KIIα。通过在特定去污剂混合物中于pH 11下进行密度梯度超速离心，将这些膜彼此分离以及与污染活性分离，在该混合物中两个膜组分不溶，而其他膜可溶。对纯化的膜组分进行动力学比较发现，磷脂酰肌醇的米氏常数（K(m)）有4倍差异，最大反应速度（V(max)）有3.5倍差异，从而表明其调节机制与先前描述的激动剂刺激细胞的调节机制不同。这些基础活性的显著差异以及该同工酶在多个细胞器中的存在，强调了有必要在单个细胞器而非全细胞裂解物水平上研究通过PI4KIIα进行的细胞信号传导。
