Abstract：
The PKC-related kinases (PRKs, also termed PKNs) are important in cell migration, cancer, hepatitis C infection, and nutrient sensing. They belong to a group of protein kinases called AGC kinases that share common features like a C-terminal extension to the catalytic domain comprising a hydrophobic motif. PRKs are regulated by N-terminal domains, a pseudosubstrate sequence, Rho-binding domains, and a C2 domain involved in inhibition and dimerization, while Rho and lipids are activators. We investigated the allosteric regulation of PRK2 and its interaction with its upstream kinase PDK1 using a chemical biology approach. We confirmed the phosphoinositide-dependent protein kinase 1 (PDK1)-interacting fragment (PIF)-mediated docking interaction of PRK2 with PDK1 and showed that this interaction can be modulated allosterically. We showed that the polypeptide PIFtide and a small compound binding to the PIF-pocket of PRK2 were allosteric activators, by displacing the pseudosubstrate PKL region from the active site. In addition, a small compound binding to the PIF-pocket allosterically inhibited the catalytic activity of PRK2. Together, we confirmed the docking interaction and allostery between PRK2 and PDK1 and described an allosteric communication between the PIF-pocket and the active site of PRK2, both modulating the conformation of the ATP-binding site and the pseudosubstrate PKL-binding site. Our study highlights the allosteric modulation of the activity and the conformation of PRK2 in addition to the existence of at least two different complexes between PRK2 and its upstream kinase PDK1. Finally, the study highlights the potential for developing allosteric drugs to modulate PRK2 kinase conformations and catalytic activity.
摘要：
蛋白激酶C相关激酶（PRKs，也称为PKN)在细胞迁移中很重要，癌症,丙型肝炎感染，和营养传感。它们属于一组称为AGC激酶的蛋白激酶,其具有共同的特征,如C-末端延伸至包含疏水基序的催化结构域。PRK受N端结构域调控，一个伪底物序列，Rho结合结构域和参与抑制和二聚化的C2结构域，而Rho和脂质是活化剂。我们使用化学生物学方法研究了PRK2的变构调节及其与其上游激酶PDK1的相互作用。我们证实了PIF介导的PRK2与PDK1的对接相互作用，并表明这种相互作用可以变构调节。我们表明，多肽PIFtide和与PRK2的PIF口袋结合的小化合物是变构激活剂，通过从活性位点置换假底物PKL区。此外,与PIF袋结合的小化合物变构地抑制了PRK2的催化活性。一起,我们证实了PRK2和PDK1之间的对接相互作用和变构,并描述了PIF口袋和PRK2活性位点之间的变构通信,两者都调节ATP结合位点和假底物PKL结合位点的构象.除了PRK2及其上游激酶PDK1之间存在至少两种不同的复合物外，我们的研究还强调了PRK2活性和构象的变构调节。最后,该研究强调了开发变构药物以调节PRK2激酶构象和催化活性的潜力.
