Identification of a proline-rich Akt substrate as a 14-3-3 binding partner
Identification of a proline-rich Akt substrate as a 14-3-3 binding partner
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DOI：
10.1074/jbc.m210837200
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发表时间：
2003-03-21
影响因子：
4.8
通讯作者：
Roth, RA
中科院分区：
文献类型：
--
作者：
Kovacina, KS;Park, GY;Roth, RA
Akt（也称为蛋白激酶B）是磷脂酰肌醇3激酶途径的主要下游靶点之一。这种蛋白激酶与胰岛素信号传导、刺激细胞生长、抑制细胞凋亡和细胞转化有关。虽然许多细胞蛋白已被确定为该酶的假定靶标，但其他底物可能在该酶引发的各种反应中发挥作用。我们使用了14-3-3结合和抗体识别的方法来鉴定和分离Akt的一个主要底物，该底物也是14-3-3结合蛋白。这种40 kda的蛋白被命名为PRAS40，是一种富含脯氨酸的Akt底物。证明它是Akt的底物是通过证明1)PRAS40在体外被纯化的Akt磷酸化，其磷酸化位点与胰岛素处理细胞磷酸化的位点相同；2)诱导Akt单独激活足以刺激PRAS40的磷酸化；3)缺乏Akt1和Akt2的细胞磷酸化该蛋白的能力减弱。因此，PRAS40是Akt的新型底物，其磷酸化导致该蛋白与14-3-3结合。
Akt (also called protein kinase B) is one of the major downstream targets of the phosphatidylinositol 3-kinase pathway. This protein kinase has been implicated in insulin signaling, stimulation of cellular growth, and inhibition of apoptosis as well as transformation of cells. Although a number of cellular proteins have been identified as putative targets of the enzyme, additional substrates may play a role in the varied responses elicited by this enzyme. We have used a combination of 14-3-3 binding and recognition by an antibody to the phosphorylation consensus of the enzyme to identify and isolate one of the major substrates of Akt, which is also a 14-3-3 binding protein. This 40-kDa protein, designated PRAS40, is a proline-rich Akt substrate. Demonstration that it is a substrate of Akt was accomplished by showing that 1) PRAS40 was phosphorylated in vitro by purified Akt on the same site that was phosphorylated in insulin-treated cells; 2) activation of an inducible Akt was alone sufficient to stimulate the phosphorylation of PRAS40; and 3) cells lacking Akt1 and Akt2 exhibit a diminished ability to phosphorylate this protein. Thus, PRAS40 is a novel substrate of Akt, the phosphorylation of which leads to the binding of this protein to 14-3-3.