High affinity binding of inositol phosphates and phosphoinositides to the Pleckstrin homology domain of RAC protein kinase B and their influence on kinase activity
High affinity binding of inositol phosphates and phosphoinositides to the Pleckstrin homology domain of RAC protein kinase B and their influence on kinase activity
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DOI：
10.1074/jbc.272.13.8474
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发表时间：
1997-03-28
影响因子：
4.8
通讯作者：
Hemmings, BA
中科院分区：
文献类型：
--
作者：
Frech, M;Andjelkovic, M;Hemmings, BA
使用纯化的野生型和突变型激酶制剂和重组普列克底物蛋白同源（PH）结构域研究了肌醇磷酸和磷酸肌醇对RAG-蛋白激酶B的α亚型（RAC/PK B）的影响。肌醇磷酸和磷酸肌醇与PH结构域的结合以固有色氨酸荧光的猝灭来测量，磷酸肌醇和D3-磷酸化磷酸肌醇的结合亲和力分别为1-10 μ M和0.5 μ M。在荧光测定中使用从杆状病毒感染的Sf 9细胞表达和纯化的RAC/PKB获得类似的值。本文研究了合成的磷脂酰肌醇3，4-二磷酸和磷脂酰肌醇3，4，5-三磷酸的二辛酰衍生物对从转染的COS-1细胞中纯化的RAC/PKB活性的影响。发现3，4，5-三磷酸磷脂酰肌醇抑制RAC/PKB激酶活性，在2.5 μ M时具有半数最大抑制。相比之下，磷脂酰肌醇3，4-二磷酸刺激激酶活性（在2.5 μ M的半最大刺激）。缺乏PH结构域的突变RAC/PKB蛋白不受D3-磷酸化磷酸肌醇的影响。这些结果表明RAC/PKB的PH结构域以高亲和力结合肌醇磷酸和磷酸肌醇，并表明磷脂酰肌醇3-激酶的产物可以作为RAC/PKB活性的膜锚和调节剂。这些数据也为磷脂酰肌醇3-激酶和RAC/PKB调节之间的联系提供了进一步的证据。
The influence of inositol phosphates and phosphoinositides on the alpha isoform of the RAG-protein kinase B (RAC/PKB) was studied using purified wild type and mutant kinase preparations and a recombinant pleckstrin homology (PH) domain, Binding of inositol phosphates and phosphoinositides to the PH domain was measured as the quenching of intrinsic tryptophan fluorescence, Inositol phosphates and D3-phosphorylated phosphoinositides bound with affinities of 1-10 mu M and 0.5 mu M, respectively. Similar values were obtained using RAC/PKB expressed and purified from baculovirus-infected Sf9 cells in the fluorescence assay. The influence of synthetic dioctanoyl derivatives of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate on the activity of RAC/PKB purified from transfected COS-1 cells was studied. Phosphatidylinositol 3,4,5-trisphosphate was found to inhibit the RAC/PKB kinase activity with half-maximal inhibition at 2.5 mu M. In contrast, phosphatidylinositol 3,4-bisphosphate stimulated kinase activity (half-maximal stimulation at 2.5 mu M). A mutant RAC/PKB protein lacking the PH domain was not affected by D3-phosphorylated phosphoinositides. These results demonstrate that the PH domain of RAC/PKB binds inositol phosphates and phosphoinositides with high affinity, and suggest that the products of the phosphatidylinositide 3-kinase can act as both a membrane anchor and modulator of RAC/PKB activity. The data also provide further evidence for a link between phosphatidylinositide 3-kinase and RAC/PKB regulation.