University of Dundee, Scotland, UK.
Biochem J. 2011 Jan 15;433(2):357-69. doi: 10.1042/BJ20101732.
PDK1 (3-phosphoinositide-dependent protein kinase 1) activates a group of protein kinases belonging to the AGC [PKA (protein kinase A)/PKG (protein kinase G)/PKC (protein kinase C)]-kinase family that play important roles in mediating diverse biological processes. Many cancer-driving mutations induce activation of PDK1 targets including Akt, S6K (p70 ribosomal S6 kinase) and SGK (serum- and glucocorticoid-induced protein kinase). In the present paper, we describe the small molecule GSK2334470, which inhibits PDK1 with an IC₅₀ of ~10 nM, but does not suppress the activity of 93 other protein kinases including 13 AGC-kinases most related to PDK1 at 500-fold higher concentrations. Addition of GSK2334470 to HEK (human embryonic kidney)-293, U87 or MEF (mouse embryonic fibroblast) cells ablated T-loop residue phosphorylation and activation of SGK isoforms and S6K1 induced by serum or IGF1 (insulin-like growth factor 1). GSK2334470 also inhibited T-loop phosphorylation and activation of Akt, but was more efficient at inhibiting Akt in response to stimuli such as serum that activated the PI3K (phosphoinositide 3-kinase) pathway weakly. GSK2334470 inhibited activation of an Akt1 mutant lacking the PH domain (pleckstrin homology domain) more potently than full-length Akt1, suggesting that GSK2334470 is more effective at inhibiting PDK1 substrates that are activated in the cytosol rather than at the plasma membrane. Consistent with this, GSK2334470 inhibited Akt activation in knock-in embryonic stem cells expressing a mutant of PDK1 that is unable to interact with phosphoinositides more potently than in wild-type cells. GSK2334470 also suppressed T-loop phosphorylation and activation of RSK2 (p90 ribosomal S6 kinase 2), another PDK1 target activated by the ERK (extracellular-signal-regulated kinase) pathway. However, prolonged treatment of cells with inhibitor was required to observe inhibition of RSK2, indicating that PDK1 substrates possess distinct T-loop dephosphorylation kinetics. Our data define how PDK1 inhibitors affect AGC signalling pathways and suggest that GSK2334470 will be a useful tool for delineating the roles of PDK1 in biological processes.
PDK1（3-磷酸肌醇依赖性蛋白激酶 1）激活了一组属于 AGC [PKA（蛋白激酶 A）/PKG（蛋白激酶 G）/PKC（蛋白激酶 C）]激酶家族的蛋白激酶，这些激酶在介导多种生物过程中发挥着重要作用。许多致癌驱动突变诱导 PDK1 靶标的激活，包括 Akt、S6K（核糖体 S6 激酶 p70）和 SGK（血清和糖皮质激素诱导的蛋白激酶）。在本文中，我们描述了小分子 GSK2334470，它对 PDK1 的抑制作用的 IC₅₀约为 10 nM，但在 500 倍更高浓度下不会抑制包括与 PDK1 最相关的 13 种 AGC-激酶在内的 93 种其他蛋白激酶的活性。在 HEK（人胚肾）-293、U87 或 MEF（小鼠胚胎成纤维细胞）细胞中添加 GSK2334470，可消除血清或 IGF1（胰岛素样生长因子 1）诱导的 SGK 同工型和 S6K1 的 T 环残基磷酸化和激活。GSK2334470 还抑制 Akt 的 T 环磷酸化和激活，但在响应血清等较弱激活 PI3K（磷酸肌醇 3-激酶）途径的刺激物时，对 Akt 的抑制作用更有效。GSK2334470 对缺乏 PH 结构域（pleckstrin 同源结构域）的 Akt1 突变体的激活抑制作用强于全长 Akt1，这表明 GSK2334470 对在细胞质中而不是在质膜中激活的 PDK1 底物更有效。与此一致的是，在表达无法与磷酸肌醇相互作用的 PDK1 突变体的 knock-in 胚胎干细胞中，GSK2334470 比在野生型细胞中更有效地抑制 Akt1 的激活。GSK2334470 还抑制 RSK2（p90 核糖体 S6 激酶 2）的 T 环磷酸化和激活，RSK2 是另一种被 ERK（细胞外信号调节激酶）途径激活的 PDK1 靶标。然而，需要延长细胞用抑制剂处理时间才能观察到 RSK2 的抑制，这表明 PDK1 底物具有不同的 T 环去磷酸化动力学。我们的数据定义了 PDK1 抑制剂如何影响 AGC 信号通路，并表明 GSK2334470 将是描绘 PDK1 在生物过程中的作用的有用工具。
