König Hans-Georg, Watters Orla, Kinsella Sinéad, Ameen Mohammed, Fenner Beau J, Prehn Jochen H M
Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, 123 Saint Stephen's Green, Dublin 2, Ireland; Centre for the Study of Neurological Disorders, Royal College of Surgeons in Ireland, 123 Saint Stephen's Green, Dublin 2, Ireland.
Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, 123 Saint Stephen's Green, Dublin 2, Ireland.
Brain Res. 2018 Jan 1;1678:356-366. doi: 10.1016/j.brainres.2017.10.020. Epub 2017 Oct 24.
Previous studies provided evidence for an accumulation of IκB-kinase (IKK) α/β at the axon initial segment (AIS), a neuronal compartment defined by ankyrin-G expression. Here we explored whether the presence of the IKK-complex at the AIS was associated with the activation of IKK signaling at this site.
Proximity-ligation assays (PLAs) using pan-IKKα/β, phospho-IKKα/β-specific as well as ankyrin-G specific antibodies validated their binding to proximal epitopes in the AIS, while antibodies to other phosphorylated signaling proteins showed no preference for the AIS. Small-hairpin mediated silencing of IKKβ significantly reduced anti-phospho-IKKα/β-immunoreactivities in the AIS. ank3 gene-deficient cerebellar Purkinje cells also exhibited no phosphorylated IKKα/β at the proximal region of their axons. Transient ankyrin-G overexpression in PC12 cells augmented NF-κB transactivation in an ankyrin-G death-domain dependent manner. Finally, small molecule inhibitors of IKK-activity, including Aspirin, inhibited the accumulation of activated IKK proteins in the AIS.
Our data suggest the existence of a constitutively-active IKK signaling complex in the AIS.
先前的研究为IκB激酶（IKK）α/β在轴突起始段（AIS）的积累提供了证据，轴突起始段是由锚蛋白G表达所定义的神经元区室。在此，我们探究了AIS处IKK复合物的存在是否与该位点的IKK信号激活相关。
使用泛IKKα/β、磷酸化IKKα/β特异性以及锚蛋白G特异性抗体的邻近连接分析（PLA）证实了它们与AIS中近端表位的结合，而针对其他磷酸化信号蛋白的抗体对AIS无偏好性。小发夹介导的IKKβ沉默显著降低了AIS中的抗磷酸化IKKα/β免疫反应性。ank3基因缺陷的小脑浦肯野细胞在其轴突近端区域也未表现出磷酸化的IKKα/β。PC12细胞中瞬时过表达锚蛋白G以锚蛋白G死亡结构域依赖的方式增强了NF-κB反式激活。最后，包括阿司匹林在内的IKK活性小分子抑制剂抑制了AIS中活化IKK蛋白的积累。
我们的数据表明在AIS中存在组成性激活的IKK信号复合物。
