Lombard Chloe K, Davis Audrey L, Inukai Takayuki, Maly Dustin J
Department of Chemistry , University of Washington , Seattle , Washington 98117 , United States.
Medicinal Chemistry Research Laboratories , Ono Pharmaceutical Company, Ltd. , 3-1-1 Sakurai , Shimamoto, Mishima, Osaka 618-8585 , Japan.
Biochemistry. 2018 Oct 9;57(40):5897-5909. doi: 10.1021/acs.biochem.8b00776. Epub 2018 Sep 25.
The c-Jun N-terminal kinases (JNKs) play a wide variety of roles in cellular signaling processes, dictating important, and even divergent, cellular fates. These essential kinases possess docking surfaces distal to their active sites that interact with diverse binding partners, including upstream activators, downstream substrates, and protein scaffolds. Prior studies have suggested that the interactions of certain protein-binding partners with one such JNK docking surface, termed the D-recruitment site (DRS), can allosterically influence the conformational state of the ATP-binding pocket of JNKs. To further explore the allosteric relationship between the ATP-binding pockets and DRSs of JNKs, we investigated how the interactions of the scaffolding protein JIP1, as well as the upstream activators MKK4 and MKK7, are allosterically influenced by the ATP-binding site occupancy of the JNKs. We show that the affinity of the JNKs for JIP1 can be divergently modulated with ATP-competitive inhibitors, with a >50-fold difference in dissociation constant observed between the lowest- and highest-affinity JNK1-inhibitor complexes. Furthermore, we found that we could promote or attenuate phosphorylation of JNK1's activation loop by MKK4 and MKK7, by varying the ATP-binding site occupancy. Given that JIP1, MKK4, and MKK7 all interact with JNK DRSs, these results demonstrate that there is functional allostery between the ATP-binding sites and DRSs of these kinases. Furthermore, our studies suggest that ATP-competitive inhibitors can allosterically influence the intracellular binding partners of the JNKs.
c-Jun氨基末端激酶（JNKs）在细胞信号传导过程中发挥着多种作用，决定着重要甚至不同的细胞命运。这些重要的激酶在其活性位点远端具有对接表面，可与多种结合伴侣相互作用，包括上游激活剂、下游底物和蛋白质支架。先前的研究表明，某些蛋白质结合伴侣与一个这样的JNK对接表面（称为D募集位点，DRS）的相互作用可以变构影响JNKs的ATP结合口袋的构象状态。为了进一步探索JNKs的ATP结合口袋与DRS之间的变构关系，我们研究了支架蛋白JIP1以及上游激活剂MKK4和MKK7的相互作用如何受到JNKs的ATP结合位点占据情况的变构影响。我们发现，JNKs对JIP1的亲和力可以被ATP竞争性抑制剂以不同方式调节，在最低亲和力和最高亲和力的JNK１-抑制剂复合物之间观察到解离常数相差50倍以上。此外，我们发现通过改变ATP结合位点的占据情况，可以促进或减弱MKK4和MKK7对JNK1激活环的磷酸化作用。鉴于JIP1、MKK4和MKK7都与JNK DRS相互作用，这些结果表明这些激酶的ATP结合位点和DRS之间存在功能性变构作用。此外，我们的研究表明，ATP竞争性抑制剂可以变构影响JNKs的细胞内结合伴侣。
