Otras medidas cautelares

Nucleo-cytoplasmic shuttling of transcription factors is an active process that relies on the recognition of their nuclear localization and/or export signals (NLS and NESs, respectively) by proteins of the nuclear import and export machinery (Hood and Silver 1999). Protein export from the nucleus is often mediated by a Leucine-rich Nuclear Export Signal (NES). Recently, a database for NESs has been created, which collects experimentally validated Leucine-rich NESs (la Cour et al. 2004). Submission of NC2 sequences to this database indicated that NC2 does not contain a known NES. However, a manual examination for the generally accepted

78 Discussion

NES consensus L-x(2,3)-[LIVFM]-x(2,3)-L-x-[LI] (Bogerd et al. 1996), revealed a similar sequence in NC2β. This sequence is localized between the amino acids

99-LKRRKASSRLENLGI-113 and matches the C-terminal part of the consensus sequence (LENLGI = [LIVFM]-x(2,3)-L-x-[LI]). Many of the identified Leucine-rich NESs deviate significantly from the generally accepted loose consensus, although there are some preferences. A mutational study of the NES of the PKI protein indicated that the leucines in the C-terminal end of the signal are more important for function than the N-terminal ones (Wen et al. 1995), as confirmed from the high conservation of the C-terminal hydrophobic residues within a set of experimentally characterized NESs (la Cour et al. 2003). Consistent with that, the C-terminal - LENLGI- motif of the hypotetical NC2β NES matches the consensus NES, and

these amino acids are conserved from C. elegans _to H. sapiens_.

In contrast, the first part of the potential NC2β NES diverges from the consensus

sequence [L-x(2,3)]: after the first lysine, NC2β contains the NLS plus two

serines (-LKRRKASSR-), instead of 2,3 random residues. Interestingly, sequence alignment of different NES shows that serines, lysines and arginines are tolerated mainly after the first lysine, which is exactly the position of the NLS in NC2β. The

possibility that NC2β contains its NLS inside the NES is quite intriguing. There is

currently no functional evidence for the existence of a NES in NC2β, although the

observation that NC2β localizes also in the cytoplasm, whereas NC2α accumulates

exclusively in the nucleus, could support this possibility. Interestingly, the NLS is followed by two serines, which are potential phosphorylation sites. Many important regulatory proteins, including cell cycle regulators and transcription factors, contain a phosphorylation site within or adjacent to a classical NLS (Jans 1995; Hood and Silver 1999; Jans et al. 2000). Usually dephosphorylation exposes one or more NLS and induces nuclear import (e.g. _{NFAT; Zhu et al. 1998), whereas phosphorylation}

leads to the exposure of an NES resulting in nuclear export mediated by the exportin protein Crm1 (e.g. _{NFAT; Zhu et al. 1998; Zhu and McKeon 1999; Macian}

et al. 2001. FKHRL1, a Forkhead family of transcription factors; Biggs et al. 1999; Brunet et al., 1999; Brunet et al., 2001a). It is possible that the hypothetical NC2β

NES is activated after phosphorylation of the serines, whose negative charge can neutralize the positive charges of the NLS and unmask the NES. Modification of one or two serines could define two forms: one competent for nuclear import (serine unphosphorylated) and one recognized by an export factor (serine phosphorylated). Moreover, the NLS coincides with a DNA binding region and is followed by the TBP interaction domain (aa 113-133). The two serines (105-106) reside between these two domains: phosphorylation of these residues could release NC2 from the TBP- DNA complex, due to the repulsion with the negative charges of the DNA, and at

the same time unmask the NES, leading to export of the NC2β subunit.

These serines are phosphorylayion sites for CKII, PKA and, more interestingly, PKC (see Fig. 2b). In contrast to CKII and PKA, which are constitutively active kinases, PKC is activated upon phorbol ester stimulation. It could be that one or two of these serines residues are phosphorylated in certain conditions, and promote dissociation and export of NC2.

To test the hypothesis that phosphorylation at these residues can promote export of NC2β, the two serines were mutated into alanine, a neutral amino acid, or into

aspartate, to mimic the negative charge acquired by phosphorylation, and the resulting mutants were tested in immunofluorescence. Mutants and wt protein had the same localization, with the protein accumulating in the nucleus in both cases, suggesting that an eventual phosphorylation at these residues did not affect protein localization. Recently, it has been shown that phosphorylation at a site adjacent to an NLS decreases the binding affinity of the NLS for the importin-α (Harreman et al.

2004). Interestingly, in this paper the authors substituted a serine at the N-terminal of an NLS with glutamate, whose negative charge is more exposed than aspartate, and they did not observe a clear cytoplasmic retention of the mutant, except when they used a yeast strain whose importin-a is defective in the import of NLS cargoes (Harreman et al. 2004)). This result would suggest that phosphorylation of serines 105-106 in NC2β could affect the functionality of the NLS or activate a potential NES,

although we did not see a clear effect in our immunofluorescence experiments. Perhaps one should mutate the serines into glutamate to see an effect or use also

in vitro _{assays measuring the strength of the interaction between the cargo and the}

importin-a or the exportin protein Crm1.