Dynamic Shuttling of Nuclear Factor κB between the Nucleus and Cytoplasm as a Consequence of Inhibitor Dissociation*

Activation of the nuclear factor κB (NFκB) transcription factor is intimately associated with its translocation from the cytoplasm to the nucleus. Using the nuclear export inhibitor leptomycin B, we demonstrate shuttling of the RELA subunit of NFκB and the inhibitory subunit IκBα between these two compartments in unstimulated cells. Determination of the kinetics of nuclear entry shows marked differences for the two components; the entry of IκBα occurs more rapidly than RELA. The shuttling is suggested to be a consequence of the cytoplasmic dissociation of the NFκB·IκB complex rather than its direct nuclear import or degradation and resynthesis of IκBα. Using previously published kinetic data, this proposition is born out by the deduction that 17% of NFκB is not complexed to IκBα in a resting cell. A numerical model is presented to validate the proposed regulation of NFκB subcellular localization consequent in part on the nuclear export function and in part on the cytoplasmic retention function of IκBα. We suggest that the non-saturated interaction of NFκB with the inhibitor may enhance the specificity of action of IκB proteins on different NFκB dimers and allow additional modes of regulation of IκB function.

[1]  E M Schwarz,et al.  Rel/NF-kappa B/I kappa B family: intimate tales of association and dissociation. , 1995, Genes & development.

[2]  A. Baldwin,et al.  Tumor necrosis factor and interleukin-1 lead to phosphorylation and loss of I kappa B alpha: a mechanism for NF-kappa B activation , 1993, Molecular and cellular biology.

[3]  W C Greene,et al.  NF-kappa B controls expression of inhibitor I kappa B alpha: evidence for an inducible autoregulatory pathway. , 1993, Science.

[4]  David Baltimore,et al.  Inducibility of κ immunoglobulin enhancer-binding protein NF-κB by a posttranslational mechanism , 1986, Cell.

[5]  T. McKinsey,et al.  Basal phosphorylation of the PEST domain in the I(kappa)B(beta) regulates its functional interaction with the c-rel proto-oncogene product , 1996, Molecular and cellular biology.

[6]  M. Lindsay,et al.  IκBα Degradation and Nuclear Factor-κB DNA Binding Are Insufficient for Interleukin-1β and Tumor Necrosis Factor-α-induced κB-dependent Transcription* , 1998, The Journal of Biological Chemistry.

[7]  S. Dower,et al.  Activation of Nuclear Transcription Factor NF-κB by Interleukin-1 Is Accompanied by Casein Kinase II-mediated Phosphorylation of the p65 Subunit* , 1997, The Journal of Biological Chemistry.

[8]  F. Carlotti,et al.  Activation of nuclear factor kappaB in single living cells. Dependence of nuclear translocation and anti-apoptotic function on EGFPRELA concentration. , 1999, The Journal of biological chemistry.

[9]  T. Hope,et al.  An N‐terminal nuclear export signal is required for the nucleocytoplasmic shuttling of IκBα , 1999 .

[10]  A. Baldwin,et al.  The I kappa B proteins: multifunctional regulators of Rel/NF-kappa B transcription factors. , 1993, Genes & development.

[11]  A. Baldwin,et al.  Activation of Nuclear Factor-κB-dependent Transcription by Tumor Necrosis Factor-α Is Mediated through Phosphorylation of RelA/p65 on Serine 529* , 1998, The Journal of Biological Chemistry.

[12]  S. Harrison,et al.  Structure of an IκBα/NF-κB Complex , 1998, Cell.

[13]  M. Yanagida,et al.  Molecular Cloning and Cell Cycle-dependent Expression of Mammalian CRM1, a Protein Involved in Nuclear Export of Proteins* , 1997, The Journal of Biological Chemistry.

[14]  V. Bours,et al.  The oncoprotein Bcl-3 directly transactivates through κB motifs via association with DNA-binding p50B homodimers , 1993, Cell.

[15]  D. Baltimore,et al.  I kappa B: a specific inhibitor of the NF-kappa B transcription factor. , 1988, Science.

[16]  J. McNally,et al.  The glucocorticoid receptor: rapid exchange with regulatory sites in living cells. , 2000, Science.

[17]  G. Nolan,et al.  The candidate proto-oncogene bcl-3 encodes a transcriptional coactivator that activates through NF-kappa B p50 homodimers. , 1993, Genes & development.

[18]  M J May,et al.  NF-kappa B and Rel proteins: evolutionarily conserved mediators of immune responses. , 1998, Annual review of immunology.

[19]  Minoru Yoshida,et al.  A nuclear export signal in the N-terminal regulatory domain of IκBα controls cytoplasmic localization of inactive NF-κB/IκBα complexes , 2000 .

[20]  G. Ghosh,et al.  IκBα Functions through Direct Contacts with the Nuclear Localization Signals and the DNA Binding Sequences of NF-κB* , 1998, The Journal of Biological Chemistry.

[21]  H. Pahl,et al.  Phosphorylation of human I kappa B‐alpha on serines 32 and 36 controls I kappa B‐alpha proteolysis and NF‐kappa B activation in response to diverse stimuli. , 1995, The EMBO journal.

[22]  Y. Ben-Neriah,et al.  Rapid proteolysis of IκB-α is necessary for activation of transcription factor NF-κB , 1993, Nature.

[23]  I. Verma,et al.  Autoregulation of I kappa B alpha activity. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[24]  Jill K Thompson,et al.  Nuclear Retention of IκBα Protects It from Signal-induced Degradation and Inhibits Nuclear Factor κB Transcriptional Activation* , 1999, The Journal of Biological Chemistry.

[25]  C. Mueller-Dieckmann,et al.  Tyrosine Phosphorylation of IκB-α Activates NF-κB without Proteolytic Degradation of IκB-α , 1996, Cell.

[26]  H. Erdjument-Bromage,et al.  The Transcriptional Activity of NF-κB Is Regulated by the IκB-Associated PKAc Subunit through a Cyclic AMP–Independent Mechanism , 1997, Cell.

[27]  G. Nabel,et al.  Tumor necrosis factor alpha and interleukin 1 stimulate the human immunodeficiency virus enhancer by activation of the nuclear factor kappa B. , 1989, Proceedings of the National Academy of Sciences of the United States of America.