Do you check for phospho-RelA or total RelA?  p50/RelA/IkB complexes continuously shuttle between the cytoplasm and the nucleus.  Only upon stimulation does the p50/RelA complex remain in the nucleus and bind the DNA.  You have more chances to see a difference if you look at p-RelA (activated) instead of total RelA.  Maximal stimulation of NF-kB DNA binding activity occurs later upon TCR/CD28 stimulation than TNF stimulation, with a maxima at 5 to 7h after stimulation compared to 30 min to 1h with TNF. See: Biochemistry 2003, 42, 8260-8271 Figs 5, 6 and 7.

Also:

Excerpt from the book NF-κB/Rel Transcription Factor Family

Editors:  Hsiou-Chi Liou Ph.D.

ISBN: 978-0-387-33572-8 (Print) 978-0-387-33573-5 (Online)

CHAPTER 4

Cellular Dynamics of NF-KB Associated Proteins

Daliya Banerjee and Ranjan Sen*

An important role for nuclear iKBa was firmly established by the identification of two nuclear export sequences (NES) in this protein. Both sequences (Fig. 1) are leucine/ isoleucine-rich motifs that interact with the exportin receptor CRMl in the presence of GTP-bound Ran protein. Proteins that contain such motifs bind to CRMl in the GTP-rich nuclear environment and the complex is brought to the nuclear pore for translocation to the cytoplasm. Upon hydrolysis of the Ran-bound GTP to GDP the complex falls apart and the cargo protein is delivered to the cytoplasm. This vectorial transport process critically depends upon the Ran (GTP)/Ran(GDP) gradient between the nucleus and the cytoplasm. The CRMl exportin is conserved from yeast to metazoa, though the metazoan species is selectively inhibited by the drug leptomycin B (LMB), which is widely used to study CRMl-dependent nuclear export.

The iKBa NESs determine sub-cellular location of iKBa itself, as well as iKBa-associated Rel proteins, in yeast and mammalian cells. A green fluorescent protein (GFP) iKBa fusion protein is located mainly in the cytoplasm of transfeaed mammalian cells or transformed yeast strains. However, inhibiting mammalian CRMl with LMB, or attenuating yeast CRMlp genetically, leads to exclusively nuclear localization in both situations. These observations suggest that that GFP- iKBa is in a state of dynamic flux between the nucleus and the cytoplasm. The appearance of most of the protein in the cytosol, either by direct visualization or in cellular extracts, thus reflects a snapshot of the state at which these measurements are made.

Continuous movement in and out of the nucleus requires a nuclear localization sequence, to direct the protein to the nucleus, in addition to the NES that directs it out of the nucleus. A classical lysine-rich NLS has not been identified in iKBa. Rather, nuclear localization function is attributed to the second ankyrin domain of iKBa. Consistent with the suggestion of a nonclassical NLS, nuclear import in vitro has been shown to be independent of importin alpha the major classical NLS-recognizing protein. Of the two NESs present in iKBa, mutation of the N-terminal NES, but not the C-terminal NES, is sufficient to eribrce nuclear localization. These observations suggest that the N-terminal NES is the stronger determinant of sub-cellular localization of iKBa.

Coexpression of p65/RelA and iKBa results in cytosolic localization of both proteins. However, as in the case of iKBa alone, this is also a momentary representation of a fluxional state. LMB-mediated inhibition of CRMl export in mammalian cells, or genetic mutation of the endogenous crml gene in yeast, leads to a complete shift of the complex to the nucleus indicative of ongoing shutding Two possible NLSs may mediate nuclear entry of the complex. First, the classical NLS of p65, though attenuated by interaction with iKBa, may still be available to interact with importin a. Accessibility of the p65 NLS in complex with iKBa is indicated in biochemical as well as X-ray crystallographic studies. Alternatively, the second ankyrin domain of iKBa may still be available for import duty. There is no direct experimental support for either model at present. Presumably, the two pathways can be distinguished by determining whether nuclear import is dependent, or independent, of importin a. Nuclear export of the complex is mediated by the iKBa NESs as well as an NES in the C-terminal domain of p65/ReLA (see below). In coexpression studies, mutation of either element does not significantly alter cytoplasmic localization, however, the p65/ iKBa complex is predominantly nuclear when both iKBa and p65/RelA NESs are simultaneously mutated. Thus, homodimers of p65 are not sequestered in the cytoplasm but shuttle continuously. While identification and characterization of sequences that regidate subcellular distribution of Rel complexes were carried out in transfection studies, the principles apply for endogenous Rel/iKBa complexes as well. For example, iKBa relocates to a primarily nuclear location in LMB-treated mammalian cells, and p65/RelA levels in the nucleus are also elevated under these conditions.

 By EMSA, it appears to occur around 1-6 hours after a strong (high concentration of) TCR/CD28 stimulation. For a positive control for Imagestream, I would recommend incubating your T cells with the nuclear export inhibitor, Leptomycin B, for 90 minutes. That will give you a robust nuclear p65 signal and ensure your staining/assay is working.

Article A nuclear export signal in the N-terminal regulatory domain ...