Down syndrome (DS) is the genetic disease most frequently afflicting humans and is detected at a ratio of one out of every seven hundred births 1. Although most of those affected have an extra copy of chromosome 21, a few more rare cases involving partial trisomy define a region common to all carriers of the syndrome, called Down Syndrome Critical Region (DSCR), which is purportedly involved with the carriers' anomalous phenotype 23.

A gene located in this region, approximately 2 Mb above D21S55, has been dubbed DSCR1 – Down Syndrome Candidate Region 1 4. The genomic sequence of DSCR1 totals 45 kb, including 7 exons and 6 introns, and analyses of different cDNAs have found that the first four exons are alternative and code for two isoforms of 197 amino acids and one isoform of 171 amino acids, which differ in the N-terminal, the last 168 residues being common 5. Recent studies have indicated the presence of an alternative promoter region with approximately 900 bp, between exons 3 and 4, suggesting that the fourth isoform may derive from an alternative promoter 6. Analyses by Northern blot have detected high levels of transcripts in brain, striated skeletal and cardiac muscles, placenta and kidney 45789.

Through the analysis of amino acid sequences, a high identity has been found between DSCR1 and the human protein ZAKI-4 10, including a preserved region that is supposedly a phosphatase substrate (a serine-proline rich motif), another one assumed to be a calcineurin docking site, and an SH3 ligand domain, involved in protein dimerization 48. ZAKI-4 is a protein whose expression is positively activated by the thyroidal hormone triiodothyronine (T3); however, induction with T3 failed to produce any significant increase in the expression of DSCR1 6811. Because the human proteins DSCR1 and ZAKI-4 are capable of binding and inhibiting calcineurin signaling in myocytes, they were grouped as members of a new family of proteins called Myocyte-Enriched Calcineurin Interacting Protein 8. A new protein dubbed DSCR1L2, whose gene is located in chromosome 1 and which also presents a high identity with DSCR1 and ZAKI-4, has also been included in this family 12. Calcineurin, also known as protein phosphatase 2B (PP2B), mediates many cellular responses signalized by calcium, including the activation of lymphocytes, neurons and muscular development, which could be prevented by the addition of immunosuppressive drugs or overexpression of endogenous inhibitor proteins, as DSCR1 131415. A novel calcineurin-binding protein, called CBP1, from the pathogenic fungus Cryptococcus neoformans was recently identified. This protein presents a serine-proline rich motif identical to DSCR1 and to other calcineurin-inhibiting proteins and is detected in fungi to humans. Electrophoretic mobility experiments and mutations involving the serines have shown that CBP1 can be a phosphoprotein, a substrate of calcineurin and/or of calcium-calmodulin complex-dependent kinases 16.

Calcineurin, which is activated by calcium-calmodulin signaling, regulates the nuclear import of NF-AT (Nuclear Factor Activated T Cells) by dephosphorylation of specific serines, promoting translocation of NF-AT to the nucleus where, in a complex with AP1, it activates the transcription of specific genes such as IL2 17. It was recently discovered that the last 82 amino acid residues in the carboxi-terminal of DSCR1 inhibit the calcineurin signal, preventing the translocation of NF-AT to the nucleus, even when calcineurin activity is induced by ionophore and phorbol ester 7. Moreover, the DSCR1 promoter region presents a binding sequence for the NF-AT transcription factor, indicating that DSCR1 participates in a negative feedback-type regulation process mediated by calcineurin itself 618.

In this study, we investigated the intracellular location of DSCR1 in different cell lines and, by means of specific mutations, we detected some possible signaling regions within the DSCR1. Thus, we identified domains and amino acids that are prone to phosphorylation/dephosphorylation events that influence the protein's siting and that may be associated with the function of the protein, which, in turn, could be a candidate for involvement in DS pathogenesis.

Previous studies on the subcellular location of the DSCR1 protein have been carried out in C2C12 mouse myoblast cell line, in which the protein was detected in the nucleus 8; thus, our analyses corroborate that finding. Our experiments using fusions of DSCR1 with the green fluorescent protein detected the fusion protein preferentially in the nucleus of the analyzed cells. The detection was not altered by the insertion of DSCR1 in the GFP's N- or C-terminal, thus precluding any kind of artifact. The location was also independent of the cell line used and no differences were found for any of the isoforms.

Having analyzed the location of the DSCR1, our next step was to determine the regions responsible for the protein's location. To this end, we built truncated forms of the DSCR1, removing regions of the C- and the N-terminal. Using the pEGFP-DSCR1^Δ138–171 construction, we found that the absence of the last 33 amino acids in the carboxi-terminal altered the DSCR1's intracellular distribution, which was now detected preferentially in the cytoplasm of the analyzed cells. This finding was complemented by an analysis of cells transfected with the pEGFP-DSCR1^Δ1–137 plasmid, whose GFP is fused only with the 33 amino acids. An accumulation of the fusion protein in the nucleus was detected in the cells transfected by this construction. Hence, this region containing the 33 amino acids in the C-terminal is required for nuclear location, suggesting that it may act or contribute with a nuclear location signal (NLS). However, it is important to clarify that we used the third isoform for deletion studies because it was the first form described and we are of the opinion that it does not differ from the other forms insofar as subcellular location is concerned.

The DSCR1 possesses a putative NLS in the 33 C-terminal amino acids (RRPKPK). However, point mutations of arginines into alanines in the RRPKPK motif did not prevent the protein's migration to the nucleus (data not shown), suggesting that this domain either does not constitute a NLS or that the mutations into alanines were insufficient for its inactivation.

By means of site-directed mutagenesis, we identified amino acids that are supposedly a target of phosphorylation/dephosphorylation events and that are important for the perfect location of DSCR1. A simple substitution of a residue of threonine (DSCR1^A166) for alanine resulted in cytoplasmic accumulation of the DSCR1. This amino acid is contained in a probable SH2 ligand domain 4, identified in the last 33 amino acids of the protein. Based on these results, we hypothesize that the nuclear accumulation of DSCR1 can be controlled at two levels: phosphorylation within a probable NLS and/or formation of a heterodimer with a specific protein. SH2 domains participate in protein dimerization processes and can recognize phosphorylated residues close to the tyrosine of the SH2 ligand domain 2122. Hence, the threonine residue in the supposed SH2 ligand domain of the DSCR1, possibly phosphorylated, may be important for its recognition, which is assumed to be involved in controlling the protein's translocation to the nucleus.

It was recently discovered that DSCR1 interacts physically and inhibits calcineurin signaling 6789111213. Two specific regions within the DSCR1 interact with calcineurin: a serine-proline rich region and a region close to the carboxi-terminal, called "calcineurin docking site" 8. By point mutations in the probable calcineurin docking site (PKIIQT), another putative signaling amino acid (threonine) was identified, whose substitution by alanine provoked a cytoplasmic accumulation of DSCR1. In addition, mutants DSCR1^A160 and DSCR1^A166 showed an accumulation of the protein in the form of granules, suggesting its possible association with other proteins or specific structures, even of the cellular cytoskeleton.

The serine-proline rich motif of DSCR1 is highly conserved and found from fungi to humans, acting as a substrate of calcineurin 7812161823. Calcineurin acts in the dephosphorylation of the serines inside the serine-proline motif in the several proteins in which it is present, differing in the number of repetitions, with three found in NF-AT and two in DSCR1 82425. Acting in this region, as described for CBP1 16, kinases and phosphatases may be present, forming a complex. The nuclear location of the E2F transcription factor is dependent on the formation of a heterodimer 26, which is regulated by phosphorylation of the two serine residues that facilitate protein-protein interaction 27.

The mutant DSCR1^A108+A112, in which the serine residues of the serine-proline rich motif were substituted for alanines, showed preferential cytoplasmic location in the form of small points dispersed throughout the cytoplasm. This location pattern may even be related to the accumulation of DSCR1 in the cellular cytoskeleton, as described for protein 1B associated with microtubules 28, or to mitochondria since, in the hamster DSCR1 ortholog isoform adapt78, isolated from a Chinese hamster fibroblast culture induced by oxidative stress, a probable target signal region for mitochondria was described in the N-terminal 29. An analysis by co-visualization of this mutant form, together with a fluorescent red reporter protein carrying a target sequence for mitochondria, failed to reveal superimposition of the distribution patterns of the two proteins (data not shown). Be that as it may, it seems clear that the serines found in the SPPASPP motif, which may be targets of phosphorylation/dephosphorylation events, are important for the nuclear location and cellular activity of the DSCR1. Recently, it was confirmed that the DSCR1 can be phosphorylated by mitogen-activated protein kinase (MAPK) and glycogen synthase kinase-3 (GSK-3), forming a substrate to calcineurin. Moreover, a specific serine residue within the conserved SP repeat domain was phosphorylated by GSK-3 and dephosphorylated by calcineurin in vitro 30.

In addition, we carried out treatments with PMA/Ionomycin, which activate calcium signaling and, inversely, with Cyclosporin A, an immunosuppressor that blocks the interaction of calcineurin with other proteins. These treatments failed to change the location of the mutants described herein, confirming that the eliminated or substituted amino acids effectively participate in a specific signal and are independent of these treatments.

By means of mutational analyses, therefore, our studies identified amino acids that participate in the location of the DSCR1 and that are probably responsible for a specific cellular response. DSCR1 can represent a phosphoprotein in which the state of phosphorylation is essential for a given location pattern and consequent activity.

DSCR1 has been demonstrated previously to be a nuclear protein. In the work reported on herein, this finding has been confirmed in different cell lines. Furthermore, we have demonstrated the importance of the 33 C-terminal amino acids in this location. Besides, punctual mutations in amino acids that are prone to events of phosphorylation/dephosphorylation altered the DSCR1 location, suggesting that its subcellular location and activity may be regulated by kinases and/or phosphatases.