IκBα Physically Interacts with a Cytoskeleton-Associated Protein through Its Signal Response Domain

The IκBα protein is a key molecular target involved in the control of NF-κB/Rel transcription factors during viral infection or inflammatory reactions. This NF-κB-inhibitory factor is regulated by posttranslational phosphorylation and ubiquitination of its amino-terminal signal response domain that targets IκBα for rapid proteolysis by the 26S proteasome. In an attempt to identify regulators of the IκBα inhibitory activity, we undertook a yeast two-hybrid genetic screen, using the amino-terminal end of IκBα as bait, and identified 12 independent interacting clones. Sequence analysis identified some of these cDNA clones as Dlc-1, a sequence encoding a small, 9-kDa human homolog of the outer-arm dynein light-chain protein. In the two-hybrid assay, Dlc-1 also interacted with full-length IκBα protein but not with N-terminal-deletion-containing versions of IκBα. IκBα interacted in vitro with a glutathione S-transferase-Dlc-1 fusion protein, and RelA(p65) did not displace this association, demonstrating that p65 and Dlc-1 contact different protein motifs of IκBα. Importantly, in HeLa and 293 cells, endogenous and transfected IκBα coimmunoprecipitated with Myc-tagged or endogenous Dlc-1. Indirect immunofluorescence analyzed by confocal microscopy indicated that Dlc-1 and IκBα colocalized with both nuclear and cytoplasmic distribution. Furthermore, Dlc-1 and IκBα were found to associate with the microtubule organizing center, a perinuclear region from which microtubules radiate. Likewise, IκBα colocalized with α-tubulin filaments. Taken together, these results highlight an intriguing interaction between the IκBα protein and the human homolog of a member of the dynein family of motor proteins and provide a potential link between cytoskeleton dynamics and gene regulation.