Hans-Georg König1, Orla Watters2, Sinéad Kinsella3, Mohammed Ameen4, Beau J Fenner5, Jochen H M Prehn6. 1. 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. Electronic address: hgkoenig@rcsi.ie. 2. 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. Electronic address: orlawatters@rcsi.ie. 3. 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. Electronic address: sineadkinsella@rcsi.ie. 4. Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, 123 Saint Stephen's Green, Dublin 2, Ireland. Electronic address: mohammedameen@rcsi.ie. 5. Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, 123 Saint Stephen's Green, Dublin 2, Ireland. Electronic address: beau.fenner@mohh.com.sg. 6. 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. Electronic address: prehn@rcsi.ie.
Abstract
BACKGROUND: 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. METHODS AND RESULTS: 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. CONCLUSION: Our data suggest the existence of a constitutively-active IKK signaling complex in the AIS.
BACKGROUND: 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. METHODS AND RESULTS: 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. CONCLUSION: Our data suggest the existence of a constitutively-active IKK signaling complex in the AIS.