Krugmann S, Anderson K E, Ridley S H, Risso N, McGregor A, Coadwell J, Davidson K, Eguinoa A, Ellson C D, Lipp P, Manifava M, Ktistakis N, Painter G, Thuring J W, Cooper M A, Lim Z-Y, Holmes A B, Dove S K, Michell R H, Grewal A, Nazarian A, Erdjument-Bromage H, Tempst P, Stephens L R, Hawkins P T
Inositide Laboratory, The Babraham Institute, Cambridge, CB2 4AT, United Kingdom.
Mol Cell. 2002 Jan;9(1):95-108. doi: 10.1016/s1097-2765(02)00434-3.
We show that matrices carrying the tethered homologs of natural phosphoinositides can be used to capture and display multiple phosphoinositide binding proteins in cell and tissue extracts. We present the mass spectrometric identification of over 20 proteins isolated by this method, mostly from leukocyte extracts: they include known and novel proteins with established phosphoinositide binding domains and also known proteins with surprising and unusual phosphoinositide binding properties. One of the novel PtdIns(3,4,5)P3 binding proteins, ARAP3, has an unusual domain structure, including five predicted PH domains. We show that it is a specific PtdIns(3,4,5)P3/PtdIns(3,4)P2-stimulated Arf6 GAP both in vitro and in vivo, and both its Arf GAP and Rho GAP domains cooperate in mediating PI3K-dependent rearrangements in the cell cytoskeleton and cell shape.
我们表明，携带天然磷酸肌醇拴系同源物的基质可用于在细胞和组织提取物中捕获和展示多种磷酸肌醇结合蛋白。我们展示了通过这种方法分离出的20多种蛋白质的质谱鉴定结果，这些蛋白质大多来自白细胞提取物：它们包括具有既定磷酸肌醇结合结构域的已知和新型蛋白质，以及具有令人惊讶和不寻常磷酸肌醇结合特性的已知蛋白质。一种新型的磷脂酰肌醇-3,4,5-三磷酸（PtdIns(3,4,5)P3）结合蛋白ARAP3具有不寻常的结构域结构，包括五个预测的PH结构域。我们表明，它在体外和体内都是一种特异性的PtdIns(3,4,5)P3/PtdIns(3,4)P2刺激的Arf6 GAP，其Arf GAP和Rho GAP结构域在介导细胞骨架和细胞形状的PI3K依赖性重排中协同作用。
