Analysis of 3-phosphoinositide-dependent kinase-1 signaling and function in ES cells.
Analysis of 3-phosphoinositide-dependent kinase-1 signaling and function in ES cells.
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DOI：
10.1016/j.yexcr.2008.04.006
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发表时间：
2008-07
影响因子：
3.7
通讯作者：
Tanja M Tamgüney;Chao Zhang;D. Fiedler;K. Shokat;D. Stokoe
中科院分区：
文献类型：
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作者：
Tanja M Tamgüney;Chao Zhang;D. Fiedler;K. Shokat;D. Stokoe
3-磷酸肌醇依赖性激酶 1 (PDK1) 磷酸化并激活 cAMP 依赖性、cGMP 依赖性和蛋白激酶 C (AGC) 家族中的多种激酶。许多假定的 PDK1 底物已被鉴定，但尚未在 PDK1 活性瞬时和特异性抑制后进行分析。在这里，我们证明了先前表征的 PDK1 抑制剂 BX-795 显示出与 PDK1 抑制不一致的生物效应。因此，我们描述了 PDK1 突变体 L159G 的创建和表征，该突变体可以结合含有大基团的抑制剂类似物，这些基团阻碍野生型 (WT) 激酶的 ATP 结合口袋。当在 PDK1−/−ES 细胞中表达时，PDK1 L159G 恢复了已知在这些细胞中低磷酸化的 PDK1 靶点的磷酸化。多种抑制剂类似物的筛选表明，1-NM-PP1 和 3,4-DMB-PP1 可最佳地抑制表达 PDK1 L159G 但不表达 WT PDK1 的 PDK1−/−ES 细胞中 PDK1 靶标的磷酸化。这些化合物证实了之前假定的 PDK1 底物，但揭示了不同的去磷酸化动力学。虽然 PDK1 抑制对细胞生长影响不大，但它使细胞对凋亡刺激敏感。此外，PDK1 缺失会抑制同种异体移植肿瘤的生长。总之，我们描述了一个模型系统，该系统允许对细胞中的 PDK1 进行急性和可逆抑制，以探测生化和生物学后果。
3-Phosphoinositide-dependent kinase-1 (PDK1) phosphorylates and activates several kinases in the cAMP-dependent, cGMP-dependent and protein kinase C (AGC) family. Many putative PDK1 substrates have been identified, but have not been analyzed following transient and specific inhibition of PDK1 activity. Here, we demonstrate that a previously characterized PDK1 inhibitor, BX-795, shows biological effects that are not consistent with PDK1 inhibition. Therefore, we describe the creation and characterization of a PDK1 mutant, L159G, which can bind inhibitor analogues containing bulky groups that hinder access to the ATP binding pocket of wild type (WT) kinases. When expressed in PDK1−/−ES cells, PDK1 L159G restored phosphorylation of PDK1 targets known to be hypophosphorylated in these cells. Screening of multiple inhibitor analogues showed that 1-NM-PP1 and 3,4-DMB-PP1 optimally inhibited the phosphorylation of PDK1 targets in PDK1−/−ES cells expressing PDK1 L159G but not WT PDK1. These compounds confirmed previously assumed PDK1 substrates, but revealed distinct dephosphorylation kinetics. While PDK1 inhibition had little effect on cell growth, it sensitized cells to apoptotic stimuli. Furthermore, PDK1 loss abolished growth of allograft tumors. Taken together we describe a model system that allows for acute and reversible inhibition of PDK1 in cells, to probe biochemical and biological consequences.