I kappa B alpha physically interacts with a cytoskeleton-associated protein through its signal response domain

The I kappa B alpha protein is a key molecular target involved in the control of NF-kappa B/Rel transcription factors during viral infection or inflammatory reactions. This NF-kappa B inhibitory factor is regulated by posttranslational phosphorylation and ubiquitination of its amino-terminal signal response domain that targets I kappa B alpha for rapid proteolysis by the 26S proteasome. In an attempt to identify regulators of the I kappa B alpha inhibitory activity, we undertook a yeast two-hybrid genetic screen, using the amino-terminal end of I kappa B alpha 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 I kappa B alpha protein but not with N-terminal-deletion-containing versions of I kappa B alpha. I kappa B alpha 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 I kappa B alpha. Importantly, in HeLa and 293 cells, endogenous and transfected I kappa B alpha coimmunoprecipitated with Myc-tagged or endogenous Dlc-1, Indirect immunofluorescence analyzed by confocal microscopy indicated that Dlc-1 and I kappa B alpha colocalized with both nuclear and cytoplasmic distribution, Furthermore, Dlc-1 and I kappa B alpha were found to associate with the microtubule organizing center, a perinuclear region from which microtubules radiate, Likewise, I kappa B alpha colocalized with alpha-tubulin filaments. Taken together, these results highlight an intriguing interaction between the I kappa B alpha 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.