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 regul ated by posttranslational phosphorylation and ubiquitination of its am ino-terminal signal response domain that targets I kappa B alpha for r apid proteolysis by the 26S proteasome. In an attempt to identify regu lators of the I kappa B alpha inhibitory activity, we undertook a yeas t two-hybrid genetic screen, using the amino-terminal end of I kappa B alpha as bait, and identified 12 independent interacting clones, Sequ ence analysis identified some of these cDNA clones as Dlc-1, a sequenc e 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 ful l-length I kappa B alpha protein but not with N-terminal-deletion-cont aining versions of I kappa B alpha. I kappa B alpha interacted in vitr o with a glutathione S-transferase-Dlc-1 fusion protein, and RelA(p65) did not displace this association, demonstrating that p65 and Dlc-1 c ontact different protein motifs of I kappa B alpha. Importantly, in He La and 293 cells, endogenous and transfected I kappa B alpha coimmunop recipitated with Myc-tagged or endogenous Dlc-1, Indirect immunofluore scence analyzed by confocal microscopy indicated that Dlc-1 and I kapp a B alpha colocalized with both nuclear and cytoplasmic distribution, Furthermore, Dlc-1 and I kappa B alpha were found to associate with th e microtubule organizing center, a perinuclear region from which micro tubules radiate, Likewise, I kappa B alpha colocalized with alpha-tubu lin filaments. Taken together, these results highlight an intriguing i nteraction between the I kappa B alpha protein and the human homolog o f a member of the dynein family of motor proteins and provide a potent ial link between cytoskeleton dynamics and gene regulation.