Regulation of RelA Subcellular Localization by a Putative Nuclear Export Signal and p50

Abstract

Nuclear factor κB (NF-κB) represents a family of dimeric DNA binding proteins, the pleotropic form of which is a heterodimer composed of RelA and p50 subunits. The biological activity of NF-κB is controlled through its subcellular localization. Inactive NF-κB is sequestered in the cytoplasm by physical interaction with an inhibitor, IκBα. Signal-mediated IκBα degradation triggers the release and subsequent nuclear translocation of NF-κB. It remains unknown whether the NF-κB shuttling between the cytoplasm and nucleus is subjected to additional steps of regulation. In this study, we demonstrated that the RelA subunit of NF-κB exhibits strong cytoplasmic localization activity even in the absence of IκBα inhibition. The cytoplasmic distribution of RelA is largely mediated by a leucine-rich sequence homologous to the recently characterized nuclear export signal (NES). This putative NES is both required and sufficient to mediate cytoplasmic localization of RelA as well as that of heterologous proteins. Furthermore, the cytoplasmic distribution of RelA is sensitive to a nuclear export inhibitor, leptomycin B, suggesting that RelA undergoes continuous nuclear export. Interestingly, expression of p50 prevents the cytoplasmic expression of RelA, leading to the nuclear accumulation of both RelA and p50. Together, these results suggest that the nuclear and cytoplasmic shuttling of RelA is regulated by both an intrinsic NES-like sequence and the p50 subunit of NF-κB.

ACKNOWLEDGMENTS

We greatly acknowledge M. Yoshida for providing the LMB, W. C. Greene for the RelA expression vectors, and Dave Antonetti and the Penn State Retina Research Group for the use of their microscope.

E.W.H. is supported by NIH predoctoral training grant 5 T32 CA 6039-5. This study was supported by Public Health Service grant 1 R01 CA68471 to S.-C.S.