Collins Barry J, Deak Maria, Arthur J Simon C, Armit Laura J, Alessi Dario R
MRC Protein Phosphorylation Unit, MSI/WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, UK.
EMBO J. 2003 Aug 15;22(16):4202-11. doi: 10.1093/emboj/cdg407.
PKB/Akt, S6K, SGK and RSK are mediators of responses triggered by insulin and growth factors and are activated following phosphorylation by 3-phosphoinositide-dependent protein kinase-1 (PDK1). To investigate the importance of a substrate-docking site in the kinase domain of PDK1 termed the 'PIF-pocket', we generated embryonic stem (ES) cells in which both copies of the PDK1 gene were altered by knock-in mutation to express a form of PDK1 retaining catalytic activity, in which the PIF-pocket site was disrupted. The knock-in ES cells were viable, mutant PDK1 was expressed at normal levels and insulin-like growth factor 1 induced normal activation of PKB and phosphorylation of the PKB substrates GSK3 and FKHR. In contrast, S6K, RSK and SGK were not activated, nor were physiological substrates of S6K and RSK phosphorylated. These experiments establish the importance of the PIF-pocket in governing the activation of S6K, RSK, SGK, but not PKB, in vivo. They also illustrate the power of knock-in technology to probe the physiological roles of docking interactions in regulating the specificity of signal transduction pathways.
蛋白激酶B/蛋白激酶Akt（PKB/Akt）、核糖体S6激酶（S6K）、血清和糖皮质激素诱导激酶（SGK）以及核糖体S6激酶（RSK）是由胰岛素和生长因子触发的反应的介质，在被3-磷酸肌醇依赖性蛋白激酶-1（PDK1）磷酸化后被激活。为了研究PDK1激酶结构域中一个称为“PIF口袋”的底物对接位点的重要性，我们构建了胚胎干细胞（ES细胞），其中PDK1基因的两个拷贝都通过敲入突变进行了改变，以表达一种保留催化活性但PIF口袋位点被破坏的PDK1形式。敲入ES细胞能够存活，突变型PDK1以正常水平表达，胰岛素样生长因子1诱导PKB正常激活以及PKB底物糖原合成酶激酶3（GSK3）和叉头框蛋白O1（FKHR）正常磷酸化。相比之下，S6K、RSK和SGK未被激活，S6K和RSK的生理底物也未被磷酸化。这些实验证实了PIF口袋在体内调控S6K、RSK、SGK（而非PKB）激活中的重要性。它们还说明了敲入技术在探究对接相互作用在调节信号转导途径特异性中的生理作用方面的强大功能。
