James S R, Downes C P, Gigg R, Grove S J, Holmes A B, Alessi D R
Department of Biochemistry, University of Dundee, Scotland.
Biochem J. 1996 May 1;315 ( Pt 3)(Pt 3):709-13. doi: 10.1042/bj3150709.
Recent evidence has suggested that activation of phosphoinositide 3-kinase (PI 3-kinase) is required for the activation of Akt-1 by growth factors and insulin. Here we demonstrate by two independent methods that Akt-1 from L6 myotubes binds to PtdIns(3,4,5)P3, PtdIns(3,4)P2 and PtdIns(4,5)P2 when presented against a background of phosphatidylserine (PtdSer) or a 1:1 mixture of PtdSer and phosphatidylcholine (PtdCho). No binding was observed with the lipids PtdIns(3,5)P2, PtdIns4P and PtdIns3P or background lipids. Activated, hyperphosphorylated forms of Akt-1 from insulin-stimulated L6 myotubes bound to PtdIns(3,4,5)P3 in a similar manner as inactive Akt-1. Quantitative analysis using surface plasmon resonance showed that the equilibrium association constant for the binding of Akt-1 to PtdIns(3,4,5)P3 was submicromolar and that PtdIns(3,4)P2 and PtdIns(4,5)P2 bound to Akt-1 with 3- and 6-fold lower affinities respectively. Interaction of Akt-1 with PtdIns(3,4,5)P3 did not activate the protein kinase activity, either before or after incubation with MgATP. A model is presented in which PtdIns(3,4,5)P3 may prime Akt-1 for activation by another protein kinase, perhaps by recruiting it to the plasma membrane.
最近有证据表明，生长因子和胰岛素激活Akt-1需要磷酸肌醇3激酶（PI 3激酶）的激活。在此，我们通过两种独立的方法证明，当以磷脂酰丝氨酸（PtdSer）或PtdSer与磷脂酰胆碱（PtdCho）的1:1混合物为背景时，L6肌管中的Akt-1与PtdIns(3,4,5)P3、PtdIns(3,4)P2和PtdIns(4,5)P2结合。未观察到与脂质PtdIns(3,5)P2、PtdIns4P和PtdIns3P或背景脂质的结合。胰岛素刺激的L6肌管中活化的、高度磷酸化的Akt-1形式与PtdIns(3,4,5)P3的结合方式与无活性的Akt-1相似。使用表面等离子体共振的定量分析表明，Akt-1与PtdIns(3,4,5)P3结合的平衡缔合常数为亚微摩尔，并且PtdIns(3,4)P2和PtdIns(4,5)P2与Akt-1结合的亲和力分别低3倍和6倍。在与MgATP孵育之前或之后，Akt-1与PtdIns(3,4,5)P3的相互作用均未激活蛋白激酶活性。本文提出了一个模型，其中PtdIns(3,4,5)P3可能使Akt-1为被另一种蛋白激酶激活做好准备，也许是通过将其募集到质膜上。
