Whereas transcription factors had originally been thought to spend their complete life in the cell nucleus, members of several transcription factor families have been shown in the meantime to be able to both enter and leave the nucleus. These include the STAT and Smad transcription factors, several bZIP proteins, nuclear receptors and other zinc finger proteins as well as some Sox proteins (19
). To our knowledge, Oct-6 is the first POU protein shown to possess nucleocytoplasmic shuttling activity.
Similar to nuclear import, which is an active process that requires the presence of at least one NLS in the protein to be transported into the nucleus, nuclear export depends on the presence of nuclear export signals. In many cases, these NES are hydrophobic, leucine-rich stretches. This is also the case for the NES identified in Oct-6 which contains one isoleucine and three leucines in characteristic spacing.
We have previously identified the major NLS of Oct-6 at the N-terminal end of the POU homeodomain (14
). It is intriguing that the newly identified NES is also part of the POU homeodomain. One single domain is thus involved in nuclear import, DNA-binding and nuclear export. The NES localization in helix 2 and the connecting loop to helix 3 of the POU homeodomain ensures that the NES is not masked by DNA-binding so that even DNA-bound Oct-6 is not necessarily precluded from interaction with the nuclear export machinery. Given the fact that most hydrophobic leucine-rich NES are recognized by CRM1/Exp1, it is not surprising that Oct-6 nuclear export is also CRM1/Exp1-dependent.
Although Oct-6 has the capacity to constantly move between nucleus and cytoplasm, it is usually detected in the nucleus. However, this static picture only reflects the fact that nuclear import rates exceed those for nuclear export. That this is not always the case, is indicated by the cytoplasmic occurrence of significant amounts of Oct-6 in select neuronal subpopulations of the adult mouse brain (15
). Cytoplasmic localization has also been detected for other POU proteins such as Brn-2 and Oct-1 (34
The balance between nuclear export and import is often subject to regulatory mechanisms. Phosphorylation events, in particular, have been shown to alter NLS or NES activity (23
). In case of Oct-6, the NLS rather than the NES is a likely target for regulatory post-translational modifications as it is flanked on both sides by several putative phosphorylation sites.
Nuclear export is an essential regulatory mechanism to control the activity of transcription factors in response to different cues including oxidative stress, cell-density or extracellular environment (22
). In principle, many ways can be envisaged by which nuclear export influences activity of a transcription factor. When combined with cytoplasmic retention, nuclear export may sequester transcription factors away from their target genes. This could be important for POU proteins as developmental regulators with often transient function either as a mechanism to accumulate potentially active protein in the cytoplasm before a rapid induction event or as a mechanism to inactivate still existing protein after the phase ended during which the protein was active. Accumulation of potentially active protein in the cytoplasm is observed for Oct-1 in early Xenopus
embryos, where maternal Oct-1 protein is retained in the cytoplasm until mid-blastula transition (35
). Rapid inactivation may be the reason for the differential localization of Brn-2 in the sub-ventricular and intermediate zones of the developing spinal cord. As precursor cells in the developing spinal cord mature and move from the sub-ventricular zone to the intermediate zone, Brn-2 becomes relocated from nucleus to cytoplasm probably as a means of rapid inactivation (34
). Future experiments will have to address whether these mechanisms are also employed for the regulation of Oct-6 function.
Our studies also revealed that Oct-6 lost its transcriptional activity upon NES inactivation. Although the reduced DNA-binding of NES mutants almost certainly contributes to the observed loss of transcriptional activity, we do not consider this the sole cause as even saturating amounts of these NES mutants failed to activate Oct-6-dependent transcription despite their residual DNA-binding activity. Additionally, pharmacological inhibition of nuclear export by treatment with leptomycin B reduced the transcriptional activity of wild-type Oct-6. These results point to the possibility that repeated passage through the cytoplasm is required for Oct-6 to acquire and retain full activity as a transcription factor. A similar model has also been proposed for R-Smads and Sox proteins of group E after studying their nuclear export in cell and explant cultures (25
). What acts on these transcription factors in the cytoplasm, for instance by introducing post-translational modifications, is unclear at the moment.
The NES that we have identified in this study in Oct-6 is conserved in the homeodomain of most, if not all POU proteins. Brn-5 could be the exception, as the residue corresponding to L376 in the Oct-6 NES is replaced by a tyrosine residue (). Even more striking, other homeodomain proteins also contain the NES, such as the maternal effect protein Bicoid from Drosophila melanogaster
which functions both within the nucleus and the cytoplasm. Other homeodomain proteins including many Hox proteins have hydrophobic leucine-rich sequences that differ only slightly from those of canonical NES and might as well be functional during nuclear export (). Other homeodomain proteins such as Engrailed proteins contain hydrophobic, leucine-rich sequences in helix 3 and the preceding loop in a position that strongly overlaps with the location of the Oct-6 NES (). Despite the fact that the hydrophobic residues do not exhibit the typical spacing of an NES, they have been shown to mediate nuclear export (40
). Thus, many homeodomains appear to be capable of nuclear export. In evolutionary terms, it certainly makes sense to include into a DNA-binding domain the necessary information for both nuclear import and export. Any new protein that acquires a homeodomain as its DNA-binding module during evolution would then automatically be able to reach its targets in the nucleus and leave it again. It might, however, also point to the fact that many homeodomain proteins have the capacity for nucleocytoplasmic shuttling and in fact might require this capacity to obtain their full activity or be correctly regulated during development.