IPO3-mediated Nonclassical Nuclear Import of NF-κB Essential Modulator (NEMO) Drives DNA Damage-dependent NF-κB Activation

Activation of I kappa B kinase (IKK) and NF-kappa B by genotoxic stresses modulates apoptotic responses and production of inflammatory mediators, thereby contributing to therapy resistance and premature aging. We previously reported that genotoxic agents induce nuclear localization of NF-kappa B essential modulator (NEMO) via an undefined mechanism to arbitrate subsequent DNA damage-dependent IKK/NF-kappa B signaling. Here we show that a nonclassical nuclear import pathway via IPO3 (importin 3, transportin 2) mediates stress-induced NEMO nuclear translocation. We found putative nuclear localization signals in NEMO whose mutations disrupted stress-inducible nuclear translocation of NEMO and IKK/NF-kappa B activation in stably reconstituted NEMO-deficient cells. RNAi screening of both importin alpha and beta family members, as well as co-immunoprecipitation analyses, revealed that a nonclassical importin beta family member, IPO3, was the only importin that was able to associate with NEMO and whose reduced expression prevented genotoxic stress-induced NEMO nuclear translocation, IKK/NF-kappa B activation, and inflammatory cytokine transcription. Recombinant IPO3 interacted with recombinant NEMO but not the nuclear localization signal mutant version and induced nuclear import of NEMO in digitonin-permeabilized cells. We also provide evidence that NEMO is disengaged from IKK complex following genotoxic stress induction. Thus, the IPO3 nuclear import pathway is an early and crucial determinant of the IKK/NF-kappa B signaling arm of the mammalian DNA damage response.