Role of phosphatidylinositol 3,4,5-trisphosphate in regulating the activity and localization of 3-phosphoinositide-dependent protein kinase-1
Role of phosphatidylinositol 3,4,5-trisphosphate in regulating the activity and localization of 3-phosphoinositide-dependent protein kinase-1
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DOI：
10.1042/0264-6021:3370575
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发表时间：
1999-02-01
影响因子：
4.1
通讯作者：
Lucocq, J
中科院分区：
文献类型：
--
作者：
Currie, RA;Walker, KS;Lucocq, J
3-磷酸肌醇依赖性蛋白激酶-1（PDK 1）与PtdIns（3，4，5）P-3（K-D 1.6 nM）和PtdIns（3，4）P-2（K-D 5.2 nM）的D-对映体立体选择性相互作用，但与PtdIns 3 P或PtdIns（4，5）P-2的结合亲和力较低。通过在PDK 1的pleckstrin同源（PH）结构域中进行特异性突变或删除该结构域，PtdIns（3，4，5）P-3与PDK 1的结合大大降低，相同的突变也大大降低了PDK 1激活蛋白激酶B α的速率在含有PtdIns（3，4，5）P-3的脂质囊泡存在下，但不影响在PtdIns（3，4，5）P-3不存在下PDK 1激活缺乏PH结构域的PKB α突变体的速率。当在293或PAE细胞中过表达时，PDK 1位于质膜和胞质溶胶中，但被排除在细胞核之外。破坏PtdIns（3，4，5）P-3或PtdIns（4，5）P-2与PDK 1相互作用的突变可消除PDK 1与质膜的结合。生长因子刺激促进了转染的PKB α易位到质膜上，但对固定细胞的免疫电镜观察显示，对PDK 1的亚细胞分布没有影响。这一结论也得到了活细胞中绿色荧光蛋白-PDK 1的共聚焦显微镜的支持。这些结果与以前的观察结果一起表明，PtdIns（3，4，5）P-3在PDK 1诱导的PKB α激活中起几种作用。首先，它与PKB的PH结构域结合，改变其构象，使其可以被PDK 1激活。其次，与PtdIns（3，4，5）P-3的相互作用将PKB募集到质膜，由于其与PtdIns（3，4，5）P-3或PtdIns（4，5）P-2的强得多的相互作用，PDK 1与质膜组成性地定位。第三，PDK 1与PtdIns（3，4，5）P-3的相互作用促进其激活PKB的速率。
3-Phosphoinositide-dependent protein kinase-1 (PDK1) interacts stereoselectively with the D-enantiomer of PtdIns(3,4,5)P-3 (K-D 1.6 nM) and PtdIns(3,4)P-2 (K-D 5.2 nM), but binds with lower affinity to PtdIns3P or PtdIns(4,5)P-2. The binding of PtdIns(3,4,5)P-3 to PDK1 was greatly decreased by making specific mutations in the pleckstrin homology (PH) domain of PDK1 or by deleting it. The same mutations also greatly decreased the rate at which PDK1 activated protein kinase B alpha (PKB alpha) in vitro in the presence of lipid vesicles containing PtdIns(3,4,5)P-3, but did not affect the rate at which PDK1 activated a PKB alpha mutant lacking the PH domain in the absence of PtdIns(3,4,5)P-3. When overexpressed in 293 or PAE cells, PDK1 was located at the plasma membrane and in the cytosol, but was excluded from the nucleus. Mutations that disrupted the interaction of PtdIns(3,4,5)P-3 or PtdIns(4,5)P-2 with PDK1 abolished the association of PDK1 with the plasma membrane. Growth-factor stimulation promoted the translocation of transfected PKB alpha to the plasma membrane, but had no effect on the subcellular distribution of PDK1 as judged by immunoelectron microscopy of fixed cells. This conclusion was also supported by confocal microscopy of green fluorescent protein-PDK1 in live cells. These results, together with previous observations, indicate that PtdIns(3,4,5)P-3 plays several roles in the PDK1-induced activation of PKB alpha. First, it binds to the PH domain of PKB, altering its conformation so that it can be activated by PDK1. Secondly, interaction with PtdIns(3,4,5)P-3 recruits PKB to the plasma membrane with which PDK1 is localized constitutively by virtue of its much stronger interaction with PtdIns(3,4,5)P-3 or PtdIns(4,5)P-2. Thirdly, the interaction of PDK1 with PtdIns(3,4,5)P-3 facilitates the rate at which it can activate PKB.