IKK alpha plays a major role in canonical NF-B-K signalling in colorectal cells

Inhibitor of kappa B (I Kappa B) kinase beta (IKK beta) has long been viewed as the dominant IKK in the canonical nuclear factor-Kappa B (NF-Kappa B) signalling pathway, with IKK alpha being more important in non-canonical NF-Kappa B activation. Here we have investigated the role of IKK alpha and IKK beta in canonical NF-Kappa B activation in colorectal cells using CRISPR-Cas9 knock-out cell lines, siRNA and selective IKK beta inhibitors. IKK alpha and IKK beta were redundant for I Kappa B alpha phosphorylation and turnover since loss of IKK alpha or IKK beta alone had little (SW620 cells) or no (HCT116 cells) effect. However, in HCT116 cells IKK alpha was the dominant IKK required for basal phosphorylation of p65 at S536, stimulated phosphorylation of p65 at S468, nuclear translocation of p65 and the NF-Kappa B-dependent transcriptional response to both TNF alpha and IL-1 alpha. In these cells, IKK beta was far less efficient at compensating for the loss of IKK alpha than IKK alpha was able to compensate for the loss of IKK beta. This was confirmed when siRNA was used to knock-down the non-targeted kinase in single KO cells. Critically, the selective IKK beta inhibitor BIX02514 confirmed these observations in WT cells and similar results were seen in SW620 cells. Notably, whilst IKK alpha loss strongly inhibited TNF alpha dependent p65 nuclear translocation, IKK alpha and IKK beta contributed equally to c-Rel nuclear translocation indicating that different NF-Kappa B subunits exhibit different dependencies on these IKKs. These results demonstrate a major role for IKK alpha in canonical NF-Kappa B signalling in colorectal cells and may be relevant to efforts to design IKK inhibitors, which have focused largely on IKK beta to date.