Stimulated nuclear translocation of NF-κB and shuttling differentially depend on dynein and the dynactin complex

Abstract

Translocation from the cytoplasm to the nucleus is required for the regulation of gene expression by transcription factors of the nuclear factor kappa B (NF-B) family. The p65:p50 NF-B heterodimer that predominates in many cell types can undergo stimulated movement, following degradation of the I B inhibitor, as well as shuttling in the absence of stimulation with I B bound. Disruption of the dynactin complex and knockdown of endogenous dynein were used to investigate the nuclear translocation requirements for stimulated and shuttling movement of NF-B. A differential dependence of these two modes of transport on the dynein molecular motor and dynactin was found. NF-B used active dynein-dependent transport following stimulation while translocation during shuttling was mediated by a dynein-independent pathway that could be potentiated by dynactin disruption, consistent with a process of facilitated diffusion. Nuclear translocation and activation of NF-B-dependent gene expression showed a dependence on endogenous dynein in a variety of cell types and in response to diverse activating stimuli, suggesting that dyneindependent transport of NF-B may be a conserved mechanism in the NF-B activation pathway and could represent a potential point of regulation.NF-kappaB ͉ nuclear transport ͉ transcription ͉ molecular motor ͉ active transport T he NF-B or Rel transcription factors are ubiquitouslyexpressed regulators of gene expression that are essential for physiological processes ranging from innate and adaptive immunity to learning and memory. NF-B functions as a dimer and is retained in a latent form in the cytoplasm, bound to the inhibitor of NF-B (I B). In the canonical pathway, incoming stimuli trigger activation of the I B-kinase complex (IKK) that phosphorylates I B␣ and leads to its degradation. The subsequent translocation of active I B-free NF-B from cytoplasm to nucleus is critical for NF-B-dependent gene expression. In the absence of I B degradation, NF-B can undergo stimulusindependent movement back and forth between the nucleus and cytoplasm in a process termed shuttling (1). Shuttling of p65:p50 NF-B dimers is believed to result from incomplete masking of the p50 nuclear localization sequence (NLS) by bound I B␣ and can be observed by blocking exportin1/crm1-mediated nuclear export under unstimulated conditions (2).While NF-B requires importin family members to cross the nuclear pore (3), the molecular mechanisms underlying NF-B cytoplasmic movement remain poorly understood. Studies examining the role of microtubules are conflicting, possibly because the pharmacological agents that disrupt microtubules result in a confounding activation of NF-B (4-10). Microtubule-dependent transport can also be probed by disrupting the multisubunit dynactin complex, and it was recently reported that dynactin could play a role in the nuclear accumulation of neuronal NF-B (10). Dynactin regulates the processivity of molecular motors using microtubules, including cytoplasmic dynein and kinesin (11,12). Dynein is a retrograde m...

Nuclear signaling by the APP intracellular domain occurs predominantly through the amyloidogenic processing pathway

“…NGF-and BDNF-induced rapid nuclear signaling by trkA and trkB receptors has been shown to rely on dynein-based retrograde transport of signaling endosomes (Ehlers et al, 1995;Watson et al, 1999) that can similarly be disrupted by p50 dynamitin (Bhattacharyya et al, 2002). Recently, nuclear translocation and subsequent gene regulation by the transcription factor NF-κB has likewise been shown to be dependent on active retrograde transport (Shrum et al, 2009).…”

NF- B in the Nervous System

“…This retrograde transport is dependent on the p65 nuclear localization signal (NLS) and involves a dynein -dynactin motor protein complex gliding on microtubules (Mikenberg et al 2007;Shrum et al 2009). p65 NLS is also important for the contact of p65 and the motor protein complexes.…”

NuMA Recruits Dynein Activity to Microtubule Minus-Ends at Mitosis

“…Scale bar, 5 mm. DOI: https://doi.org/10.7554/eLife.29328.005 pDsRed-N1, Clontech, gift from T. Schroer, Johns Hopkins University) (Quintyne and Schroer, 2002); mCherry-p50 (chicken p50 in mCherry-C1, Clontech, gift from M. Moffert and T. Schroer, Johns Hopkins University) (Shrum et al, 2009); GFP-NuMA (human NuMA in pEGFP-N1, Clontech, gift from D. Compton, Dartmouth Medical School) (Kisurina-Evgenieva et al, 2004); GFP-CAM-SAP1 (human CAMSAP1 in pEGFP-C1, Clontech, gift from A. Akhmanova, Utrecht University) .…”