We show firstly that the deletion of eleven amino acids (Δ4–14) encompassing the putative NLS sequence KVKRQR at a.a. 8–13 of CtBP2 results in shift in the localisation of detectable CtBP2 from exclusively nuclear, to nuclear and partly cytoplasmic. This effect was observed in a number of cell lines of diverse origin. These initial findings confirm a recently published study which identified a role for a.a. 1–21 in the nuclear localisation and retention of CtBP2 , as well as another study which was published whilst this manuscript was being submitted . Our data further localises the critical sequence elements to a.a. 4–14. One of the cell lines, Cos-7, was chosen because a previous study had shown that transfected CtBP1 primarily localises to the cytoplasm in these cells, making them a useful experimental model . Amino acids 4–14 also direct nuclear accumulation of CtBP2 in Cos-7 cells. Therefore, whatever the mechanism that underlies the cytoplasmic localisation of CtBP1 expressed in Cos-7 cells, it does not affect the ability of a.a. 4–14 to localise CtBP2 to the nucleus. As a primary function of CtBP proteins is as nuclear transcriptional co-repressors, this sequence in CtBP2 is likely to play a key role in maintaining nuclear CtBP activity in cells where CtBP2 is expressed.
Before considering the role of a.a. 4–14 further, it is important to note that we, as well as Zhao et al  who examined the effects of an a.a. 1–21 deletion, observed that CtBP2 with these N-terminal sequences deleted still retained a predominantly nuclear localisation, with only a partial redistribution to the cytoplasm. Interestingly, Verger et al  found than a CtBP2Δ1–25 mutant localised almost exclusively in the cytoplasm in Cos-1 cells. This different result could be due to the slightly larger deletion that they used, the cell type, or experimental differences. However, our experiments clearly show that domains other than a.a. 4–14 can be important in defining the nuclear-cytoplasmic distribution of CtBP2. These could potentially become important under conditions whereby the N-terminal sequences of CtBP2 might be masked, such as following binding of another protein or through post-translational modification. As the localisation of CtBP2(1–445)Δ4–14mh in all four cell lines closely resembled that of transfected CtBP1 in HEK 293, HeLa and MCF-7 cells it is possible this a.a. 4–14-independent nuclear localisation occurs through the same mechanisms that regulate CtBP1. In studies on Cos-7 cells, Criqui-Philipe et al  showed that CtBP1 could be recruited to the nucleus through an association with PxDLS containing transcription factors. Structural studies on rat CtBP1-S (BARS) have characterised the PxDLS-binding interface, and identified mutations (e.g. V55R) that disrupt CtBP-binding to the C-terminal domain of E1A. We therefore examined the effects of generating the CtBP2 equivalent of the CtBP1-SV55R mutation. Our finding that this V72R mutation of CtBP2(1–445)Δ4–14mh does not affect its subcellular localisation excludes the PxDLS-binding interface as the major determinant for the a.a. 4–14-independent localisation of CtBP2 to the nucleus in these experiments. The N-terminal 119 a.a of CtBP2 is able to engage in other protein-protein interactions through less well-defined interfaces, e.g. , and a role for these interactions in CtBP2 subcellular distribution cannot yet be excluded.
Our experiments showed that CtBP2(1–119)Δ4–14mh has a markedly more cytoplasmic distribution than CtBP2(1–445)Δ4–14mh. This identifies a.a. 120–445 as having a role in nuclear localisation. When compared to domains in this region of CtBP1 with a known role in subcellular localisation, CtBP2 lacks the PDZ binding motif present at the extreme C-terminus of CtBP1 , as well as the equivalent of the sumoylation site at K428 . Sequences that are conserved which are good candidates for a role in CtBP2 nuclear localisation are the Pak1 phosphorylation site at Ser 164, given that the phosphorylation status of the corresponding site in CtBP1 (Ser158) regulates its subcellular localisation , and possibly the dimerisation domain. In the intact CtBP2 protein, the N-, C- and core domains do not function independently . It is quite possible that whilst a.a. 120–445 are necessary for optimal nuclear localisation, a functional interaction between this region of the molecule and other sequences within a.a. 1–119 is required for a.a. 4–14-independent nuclear accumulation.
The experimental data obtained from our analysis of truncations and mutants of CtBP2 also provides additional insight into the mechanism of regulation of subcellular localisation by a.a. 4–14 containing the putative CtBP2 NLS. Zhao et al  demonstrated that this region does not, in fact, function as a classical NLS, but rather that it is necessary for lysines within it, primarily lysine 10, to be acetylated for it to direct localisation in the nucleus. Specifically they showed that lysines in this sequence are acetylated in vivo and that this is likely to be through the actions of the p300 acetyltransferase, a known CtBP binding protein. Their experiments using a non-acetylatable K10R mutant of CtBP2 showed that this acetylation is required for retention of CtBP2 in the nucleus, and that this mutant actually enhances CtBP2 nuclear export. Analogous to our experiments with the V72R mutant, they also showed that a different mutant in the PxDLS binding domain (A58E) does not affect acetylation by p300, and therefore p300 must bind to different sequences on CtBP2 than other PxDLS transcription factors. In contrast, Verger et al  concluded that this N-terminal lysine-arginine rich region functions as a classical nuclear localisation signal, with a role in nuclear import, rather than retention.
The experiments that we have performed do not distinguish between these two alternative mechanisms. Our finding that a.a 1–119 of CtBP2 is sufficient to drive efficient nuclear accumulation of EGFP provides an important advance in our understanding of the regions that regulate the subcellular distribution of CtBP2. However, the data are consistent with a role of a.a. 4–14 in either nuclear retention or import. Both previous studies demonstrated that the N terminal 25 amino acids cannot, alone, target a heterologous protein to the nucleus. This could be either due to an NLS or nuclear retention signal not being correctly presented to their target binding proteins in the context of these molecules, or the requirement for docking of acetyltransferases to a separate part of the molecule in order to achieve activation of the nuclear retention signal by acetylation. In CtBP2(1–119)-EGFP either the sequences may simply be sufficiently spaced from the EGFP for a.a. 4–14 to be correctly presented as an NLS, or this region may include the p300 binding site, allowing activation of the nuclear retention signal by acetylation. CtBP2(1–119)-EGFP is small enough to enter the nucleus by passive diffusion, and therefore the presence of a nuclear retention signal would be sufficient for nuclear accumulation. Expression of a.a. 1–119 in the context of a fusion with 2XEGFP would generate a larger protein that could only accumulate in the nucleus if it were actively imported through nuclear pores. However further experimentation would be required to determine conclusively whether this was due to a.a. 4–14 functioning as an NLS, or interaction of a.a. 1–119 with other actively imported proteins such as PxDLS containing transcription factors , HDM2 , or possibly p300 acetyltransferase .
Finally, we have identified heterodimerisation with CtBP2 as a novel mechanism that can promote the nuclear localisation of CtBP1. This interplay between the two proteins has also, very recently, been demonstrated by other investigators . It will be interesting to determine the extent to which this heterodimerisation contributes to CtBP1 subcellular distribution in different cell types, compared with the other mechanisms that have been described previously. It is important to note that CtBP2 expression is clearly not an absolute requirement for nuclear CtBP1 activity in many cell types . The contrasting subcellular localisations of over-expressed CtBP1 and CtBP2 in Cos-7 cells add weight to the growing argument that the two proteins are regulated differently. Indeed, studies on the role of CtBP1 and CtBP2 during murine development revealed a more severe and lethal phenotype in Ctbp2-/- mice compared to Ctbp1-/- mice . This has been attributed to temporal and spatial differences in the expression of Ctbp1 and Ctbp2 during development . Alternatively, it could be explained by the different modes of regulation of protein localisation and function between these two proteins, implying that perturbation of the constitutive nuclear function of CtBPs is responsible for the embryonic lethality of Ctbp2-/- animals. CtBP2 with an N-terminal motif that promotes its nuclear localisation is present in mice, man and fish. It is not yet known whether the smaller KRSR sequence in Drosophila CtBP is functional, and Xenopus CtBP does not contain any such motif in its N-terminus. Therefore, CtBP in Xenopus, and possibly Drosophila, will likely be dependent upon other protein-protein interactions for its recruitment to the nucleus. It is tempting to speculate, therefore, that this is an indicator of an increased importance of the nuclear activities of CtBP proteins in the regulation of the complex patterns of gene expression in higher organisms.