Abstract
NF-kappaB transcription factors are involved in the cellular response to stress, and are regulated by inhibitor (IkappaB) proteins, which prevent NF-kappaB-mediated transcription by maintaining NF-kappaB in the cytoplasm. Proteins from other pathways are also known to regulate NF-kappaB negatively, notably the glucocorticoid receptor (GR) and IL-4-responsive STAT6. Both pathways were shown to inhibit NF-kappaB-mediated transcription, by expressing either STAT6 or GR and activating the respective pathways. Using fluorescent fusion proteins, we show that GR alters the timing of activated p65 NF-kappaB nuclear occupancy by increasing the export rate of p65 and is independent of whether GR is present as a dimer or monomer. Expression of STAT6 was also shown to alter p65 nuclear occupancy but appeared to affect the import rate and hence the overall maximal level of p65 translocation. Activating STAT6 with IL-4 prior to activating NF-kappaB significantly increased this inhibition. Investigation of IkappaBa showed that activated STAT6 inhibited TNFalpha-mediated IkappaBa phosphorylation and degradation, whereas GR activation did not alter IkappaBalphakinetics. This demonstrates a clear separation of two distinct mechanisms of inhibition by STAT6 and GR upon the NF-kappaB pathway.
Highlights
There is increasing evidence that cross-talk between signalling pathways is essential to modulate the cellular response to external stimuli
We have previously shown, using fluorescent fusion proteins, that the rate of IκBα degradation depends on p65 expression levels and that the kinetics of p65 translocation are altered by IκBα overexpression (Nelson et al, 2002a)
The dose response of TAT3-luc to dexamethasone was significantly shifted to give a greater response at lower concentrations of dexamethasone (EC50=0.77±0.24 nM; Fig. 1A) than cells transfected with a GFP-expressing control plasmid, pEGFPN1 (EC50=2.88±0.27 nM; Fig. 1A)
Summary
There is increasing evidence that cross-talk between signalling pathways is essential to modulate the cellular response to external stimuli. The p65 and IκBα proteins shuttle independently between the cytoplasm and nucleus in resting cells, with the nuclear export sequence (NES) on IκBα maintaining a predominantly cytoplasmic localization of the NF-κB/IκB complex (Schmid et al, 2000). The NF-κB/IκB complex is the preferred substrate for IκB phosphorylation/degradation, rather than IκB alone, thereby only degrading bound but not free IκB (Nelson et al, 2002a). This releases NF-κB with an unmasked nuclear localization sequence (NLS), allowing translocation to the nucleus and activation of transcription. Downregulation does not require cytoplasmic relocalization of NF-κB, because transcription can be switched off even when NF-κB is trapped in the nucleus after stimulation (Nelson et al, 2002a)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
