Literature context: 000, H-470, scbt, Cat# sc-7607, RRID:AB_675667), a rabbit polyclonal anti-Ser1
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.
Literature context: RRID:AB_675667 (WB)), MYC
The ubiquitin ligase TRAF6 is a key regulator of canonical IκB kinase (IKK)/NF-κB signaling in response to interleukin-1 (IL-1) stimulation. Here, we identified the deubiquitinating enzyme YOD1 (OTUD2) as a novel interactor of TRAF6 in human cells. YOD1 binds to the C-terminal TRAF homology domain of TRAF6 that also serves as the interaction surface for the adaptor p62/Sequestosome-1, which is required for IL-1 signaling to NF-κB. We show that YOD1 competes with p62 for TRAF6 association and abolishes the sequestration of TRAF6 to cytosolic p62 aggregates by a non-catalytic mechanism. YOD1 associates with TRAF6 in unstimulated cells but is released upon IL-1β stimulation, thereby facilitating TRAF6 auto-ubiquitination as well as NEMO/IKKγ substrate ubiquitination. Further, IL-1 triggered IKK/NF-κB signaling and induction of target genes is decreased by YOD1 overexpression and augmented after YOD1 depletion. Hence, our data define that YOD1 antagonizes TRAF6/p62-dependent IL-1 signaling to NF-κB.
Literature context: sc-7607, RRID:AB_675667, 1:500), a
The axon initial segment (AIS) is a neuronal compartment defined by ankyrin-G expression. We here demonstrate that the IKK-complex co-localizes and interacts with the cytoskeletal anchor protein ankyrin-G in immunoprecipitation and proximity-ligation experiments in cortical neurons. Overexpression of the 270 kDa variant of ankyrin-G suppressed, while gene-silencing of ankyrin-G expression increased nuclear factor-κB (NF-κB) activity in primary neurons, suggesting that ankyrin-G sequesters the transcription factor in the AIS. We also found that p65 bound to the ank3 (ankyrin-G) promoter sequence in chromatin immunoprecipitation analyses thereby increasing ank3 expression and ankyrin-G levels at the AIS. Gene-silencing of p65 or ankyrin-G overexpression suppressed ank3 reporter activity. Collectively these data demonstrate that p65/NF-κB controls ankyrin-G levels via a negative feedback loop, thereby linking NF-κB signaling with neuronal polarity and axonal plasticity.
Literature context: technology, Inc., Cat# sc-7607, RIID: AB_675667), a rabbit polyclonal anti-PARP
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of motoneurons in the spinal cord, brainstem and motor cortex. Mutations in the superoxide dismutase 1 (SOD1) gene represent a frequent genetic determinant and recapitulate a disease phenotype similar to ALS when expressed in mice. Previous studies using SOD1(G93A) transgenic mice have suggested a paracrine mechanism of neuronal loss, in which cytokines and other toxic factors released from astroglia or microglia trigger motoneuron degeneration. Several pro-inflammatory cytokines activate death receptors and may downstream from this activate the Bcl-2 family protein, Bid. We here sought to investigate the role of Bid in astrocyte activation and non-cell autonomous motoneuron degeneration. We found that spinal cord Bid protein levels increased significantly during disease progression in SOD1(G93A) mice. Subsequent experiments in vitro indicated that Bid was expressed at relatively low levels in motoneurons, but was enriched in astrocytes and microglia. Bid was strongly induced in astrocytes in response to pro-inflammatory cytokines or exposure to lipopolysaccharide. Experiments in bid-deficient astrocytes or astrocytes treated with a small molecule Bid inhibitor demonstrated that Bid was required for the efficient activation of transcription factor nuclear factor-κB in response to these pro-inflammatory stimuli. Finally, we found that conditioned medium from wild-type astrocytes, but not from bid-deficient astrocytes, was toxic when applied to primary motoneuron cultures. Collectively, our data demonstrate a new role for the Bcl-2 family protein Bid as a mediator of astrocyte activation during neuroinflammation, and suggest that Bid activation may contribute to non-cell autonomous motoneuron degeneration in ALS.