The stimulation of cells by TNF led to the breakdown of cytosolic IB, allowing N

Question

The stimulation of cells by TNF led to the breakdown of cytosolic IB, allowing NF-B to translocate to the nucleus; NF-kB then induced additional cytosolic IB expression, which inducedNF-kB to move back to the cytosol. If you were paying close attention you might have noticed a problem here: if the newIkB is in the cytosol, but the NF-B has already moved to the nucleus, how does IB interact with the NF-B to get it back out of the nucleus? Propose a hypothesis for how this happens and an experiment you could use to test your idea.

Explanation / Answer

Answer:

The classical view of the regulation of NF-B is that it is kept inactive by cytoplasmic retention due to binding to IB proteins. IB is thought to mask the nuclear localization signal (NLS) of NF-B, thereby preventing the interaction of NF-B with the nuclear import machinery. Moreover, the nonconventional NLS of IB itself, located within the second ankyrin repeat, appears to be blocked when IB is bound to NF-B.

Thus, the NLS of NF-B is unmasked upon breakdown of cytosolic IB and the transcription factor can be imported into the nucleus, where it binds to specific promoter elements. After translation in the cytosol, IB is imported into the nucleus, where it is assumed to dissociate NF-B from promoter regions. The newly formed NF-B·IB complex is then transported back to the cytosol by the means of a specific nuclear export sequence (NES) of IB.

Experiment:

In order to prove the hypothesis,constructs can be prepared where NF-B with NLS can be fused to GFP and the IB (with and without NLS) fused to the red fluorescent protein, DsRed. These constructs (NF-B+IB with NLS OR NF-B+IB without NLS ) can then be transiently or stably transfected in He La cells for analysis.

Upon translation of IB, nuclear translocation of the DsRed construct mediated by the NLS should takes place, which can be monitored through confocal laser scanning microscopy using optical sectioning parameters that eliminate an out-of-focus fluorescence. There should not be any nuclear transport of the IB construct without the NLS.

The interaction of the IB and NF-B can be inferred from the colocalization of the two proteins in the nucleus. The experiment also suggests that the NLS in IB mediates its import into the nucleus and facilitate its interaction with NF-B.

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