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Department of Pediatrics, Northwestern University Feinberg School of Medicine, Children's Memorial Research Center, Chicago, Illinois 60614
Received 25 May 2007/ Returned for modification 19 July 2007/ Accepted 12 December 2007
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Guided by their protein structures, Tbx5 and related T-box family members are thought to function as transcription factors. Several Tbx genes are implicated to function upstream or downstream of fibroblast growth factor (FGF) signal transduction, suggesting that growth control by Tbx genes could be a more general conserved function in a variety of developing organs (43). Experimental evidence indicates that Tbx5 regulates the expression of Fgf10 and Wnt2b genes, thereby controlling cell proliferation during limb development (37). In addition, Tbx5 interacts with other proteins to form functional complexes. For instance, the DNA-binding domain of the Tbx5 protein can interact with those of the heart-specific transcription factors Nkx2.5 and Gata4, and the transcription factor complexes act in a cooperative manner to regulate target gene activities (10).
In contrast to the N-terminal DNA-binding domain, LMP4, a PDZ-LIM protein, has been identified by our laboratory as specifically interacting with the C-terminal transactivation domains of Tbx5 and Tbx4, suggesting a different functional role for these transcription factors (30). PDZ-LIM family proteins are proposed to mediate protein association with the cytoskeleton and with proteins involved in signal transduction cascades that regulate cell lineage specification and organ development (6, 16, 17, 29). In transfected cells, coexpression of the cytoplasmic LMP4 induces the relocalization of Tbx5 from the nucleus to the cytoplasm, where the Tbx5-LMP4 complex associates with the actin cytoskeleton (30). In the presence of LMP4, Tbx5 shuttles dynamically between the nucleus and the cytoplasm, thus allowing LMP4 to regulate the nuclear availability of Tbx5 and in turn modulate the transcriptional activity of the protein (13).
Work with chicken primary epicardial cells revealed that endogenous Tbx5 and LMP4 subcellular localization changes in response to differentiation stimuli (13). While the nature of the signal remains elusive, these in vitro differentiation studies provided the first indications that Tbx5 subcellular localization is not dependent solely on the presence of LMP4 within the cell but, rather, on a regulated event. In vivo studies of Tbx5 and LMP4 protein expression during chicken embryonic heart development confirmed this hypothesis (9). The two proteins were dynamically expressed both temporally and spatially in the developing heart. In coexpressing cells, Tbx5 localization was strictly nuclear, nuclear and cytoplasmic, or strictly cytoplasmic, depending on the developmental stage and individual cardiac cell lineage. Cytoplasmic localization of the Tbx5-LMP4 complex was also demonstrated for the developing chicken wing; however, the ratio of nuclear to cytoplasmic distribution of Tbx5 varied in different regions of the limb. These in vivo observations for multiple tissues would indicate a more general functional role for nuclear and cytoplasmic Tbx5 distribution. Furthermore, the dynamic localization of Tbx5 in these tissues during chicken development reveals a striking correlation with the tissues affected in humans diagnosed with HOS. Taken together, these results strongly emphasize the importance of appropriate subcellular localization of the protein during developmental processes.
Nuclear localization signals (NLSs) were initially identified for human TBX5. An analysis of selected fragments of the C-terminal region of TBX5 identified a NLS within the transactivation domain (NLS2) (51). Work with a series of N- and C-terminal deletion constructs and single-amino-acid substitutions revealed a second NLS in the DNA-binding domain (NLS1) (15). From these studies, it remained unclear, however, to what extent the critical residues within each given NLS are important for function and whether the two NLS motifs have a synergistic effect on nuclear localization.
The identification of NLS motifs within Tbx5 provided only a partial insight into how the protein shuttles across the nuclear membrane. In the present study, we describe the identification of a putative nuclear export signal (NES) in the DNA-binding domain of Tbx5, a core of hydrophobic amino acids that appear to be evolutionarily conserved in all T-box protein family members. Site-directed mutagenesis of this NES and pharmacological interference studies in combination with confocal imaging and coimmunoprecipitation demonstrated that Tbx5 uses this NES for binding to the export protein CRM1 in order to relocate from the nucleus to cytoplasmic sites. The current work provides a deeper understanding of the molecular machinery that enables dynamic nuclear/cytoplasmic shuttling of the Tbx5 transcription factor. Our findings suggest that Tbx5 is using a mechanism for protein relocalization that evolved early in the generation of the T-box protein family, thus setting a new paradigm for nuclear and cytoplasmic functions of potentially many T-box proteins during developmental processes.
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MATERIALS AND METHODS |
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Cell culture, transfection, and pharmacological inhibitors. COS-7 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Cells were transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. For CRM1 inhibition studies, the cells were incubated in leptomycin B (LC Laboratories) overnight at the indicated concentrations followed by processing for confocal microscopy as described below.
Confocal microscopy. Transfected COS-7 cells were fixed in 4% paraformaldehyde followed by a brief wash with phosphate-buffered saline (PBS). Confocal microscopy was performed using a 510 Meta system (Carl Zeiss MicroImaging, Inc.) equipped with a Plan Apochromat 63x objective/1.4 numerical aperture oil differential interference contrast lens. Images were processed by using Photoshop CS2 (Adobe).
Luciferase reporter assays. For reporter assays, COS-7 cells were grown in 12-well culture dishes and transfected with 300 ng of atrial natriuretic factor (ANF)-luciferase reporter plasmid (12, 13), 10 ng of Renilla luciferase plasmid, 25 ng of Tbx5 expression plasmids, and 100 ng of LMP4 expression plasmid. The total amount of DNA transfected was held at 1 µg. Transfected cells were cultured for 36 h before lysis. Luciferase activity was measured by using a Dual-Luciferase assay system (Promega), and samples were read with a Lumat LB 9501 instrument (Bethold). All reporter assays were performed in triplicate, and the collected data from independent experiments were normalized to the Renilla luciferase activity. In order to compare values from independent experiments, the activation for wild-type Tbx5 alone was set at 100% and was used to standardize the experimental values from other data sets.
Immunoprecipitation and immunoblotting. COS-7 cells were grown to 80 to 90% confluence in 60-mm culture dishes and transfected with the described plasmids. A modification of the coimmunoprecipitation protocol from Rastogi et al. (44) was used. Forty-eight hours posttransfection, the medium was aspirated and the cells were scraped using 1 ml of cold PBS, collected, and centrifuged at 3,000 rpm for 2 min. The cells were washed three more times with cold PBS, resuspended in 150 µl of M2 buffer (20 mM Tris [pH 7.6], 0.5% NP-40, 250 mM NaCl, 3 mM EGTA, 3 mM EDTA, and protease inhibitor cocktail [Sigma-Aldrich]) and incubated with rocking at 4°C for 30 min followed by centrifugation at 14,000 rpm for 15 min. The supernatants were transferred to new tubes, and after the addition of 5 µl of anti-CRM1 (H-300; Santa Cruz Biotechnology), the samples were incubated with rocking at 4°C for 1 h. To each tube, 100 µl of immunoprecipitation buffer (20 mM HEPES [pH 7.9], 40 mM KCl, 1 mM MgCl2, 0.1 mM EGTA, 0.1 mM EDTA, 0.1 mM dithiothreitol, 0.1 mM NaF, 0.1 mM Na3VO4, 0.5% NP-40, 3 mg/ml bovine serum albumin) and 15 µl of protein G Sepharose for Fast Flow (Amersham Bioscience) were added with further rocking at 4°C for 1 h. The reaction was transferred to a Zeba Spin column (Pierce) and centrifuged at 5,000 rpm for 2 min followed by five washes with the immunoprecipitation buffer. The protein was eluted from the Sepharose beads by adding 30 µl of sodium dodecyl sulfate (SDS) buffer, boiling for 5 min, and centrifuging at maximum speed for 2 min. The protein samples were resolved by SDS-polyacrylamide gel electrophoresis (PAGE), electrotransferred to nitrocellulose membranes, and immunoblotted with the indicated antibodies (anti-CRM1, H-300; Santa Cruz Biotechnology; anti-HA, HA-7; Sigma-Aldrich). Anti-mouse or anti-rabbit horseradish peroxidase-linked secondary antibodies were used with SuperSignal West Femto reagents (Pierce) to visualize the immunoreactive protein bands on film.
Three-dimensional cellular reconstruction. COS-7 cells transfected with EGFP-Tbx5 and HcRed-LMP4 were fixed and imaged using confocal microscopy as described above, and optical sections were collected for the entire thickness of the cell. The three-dimensional rendering of the cell and QuickTime movie making was done using Velocity 4.0.1 software (Improvision; Image Processing & Vision Company, Ltd.).
Protein structural modeling. Tbx5 three-dimensional protein structural modeling was done using NCBI's Molecular Modeling Database by utilizing the human TBX3 crystal structure (Protein Data Bank ID 1H6F) as a template. The resulting structure was developed using Cn3D, NCBI's three-dimensional structure viewing software, and the resulting image was processed with Photoshop CS2 (Adobe).
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RESULTS |
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-(X)2-3--(X)2-3--X-, where represents hydrophobic residues (L, I, F, V, M, P) and X represents any amino acid (22, 34). This conserved NES has been reported to be the target for binding by the nuclear exporting protein CRM1 (19, 42). We analyzed the Tbx5 protein for such a sequence motif and identified a putative NES (152LVSFQKLKL160) in the N-terminal DNA-binding domain between NLS1 and NLS2 (Fig. 2A). Having discovered the presence of an NES in Tbx5, we wondered whether this sequence motif was conserved among all members of the T-box protein family. An identical amino acid sequence was indeed present in Tbx4 (Fig. 2B), and in this context we note that Tbx4, similar to Tbx5, can also interact with LMP4 and colocalize in the cytoplasm at the actin cytoskeleton (30). Evolutionarily, the next closest pair of T-box proteins are Tbx2 and Tbx3, which together with the previous proteins define the Tbx2/3/4/5 subfamily. Tbx2 and Tbx3 share three of the four critical amino acids within the conserved NES motif. They diverge, however, in the initial position, and instead of a leucine, they have a proline and a valine, respectively (Fig. 2B). Going beyond the Tbx2/3/4/5 subfamily and including all T-box family members currently described for the mouse, we noticed the same theme: while the C-terminal three critical residues are well conserved, the N-terminal initial position is occupied by a range of amino acids, including L, P, V, I, M, E, and F. Thus, it appears that T-box proteins in general, in addition to NLS motifs, also contain a conserved NES motif.
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Protein-protein interaction between Tbx5 and CRM1. Having demonstrated that several members of the T-box family can bind to CRM1, we used Tbx5 as a model to investigate this interaction in more detail. To determine the core amino acids required for a physical interaction between Tbx5 and CRM1, COS-7 cells were transfected with expression plasmids containing HA-tagged wild-type and mutated forms to determine if they would coimmunoprecipitate with CRM1 from cell lysates. Equal protein levels before the immunoprecipitations were verified by SDS-PAGE and Western blots of the input cell lysate with antibodies against either CRM1 or the HA tag (see Fig. 4A). The remainder of the lysate was then subjected to immunoprecipitation with the CRM1 antibody. In order to detect coimmunoprecipitated Tbx5, a Western blot was performed using HA-tagged antibodies. The wild-type form of Tbx5 revealed a coimmunoprecipitate with CRM1, whereas the Tbx5 mutNES protein with all four critical NES residues exchanged with alanines did not bind to CRM1 (see Fig. 4A). Replacing the C-terminal NES residues with alanines (Fig. 3) similarly prevented the mutagenized protein from interacting with CRM1 (Fig. 4A). Surprisingly, single-amino-acid NES mutants in either the terminal or the initial position (Fig. 3) retained their ability to complex with CRM1 (Fig. 4A). In addition, an exchange of the initial leucine in the NES with either alanine or residues found in Tbx2, Tbx3, or Brachyury has no negative effect on binding the exporter protein. Thus, our work with wild-type and mutant forms of Tbx5 identifies a combination of three hydrophobic amino acids within the four-position NES to be essential for interaction with CRM1. Furthermore, the data suggest that apparently all T-box proteins contain a CRM1-interacting NES motif.
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We wondered whether LMP4 would be part of the transport complex, temporarily entering the nucleus. Previous biochemical cell fractionation and coimmunoprecipitation experiments did not reveal any nuclear localization of LMP4 (13). However, here we confirmed an exclusive cytoplasmic distribution for LMP4 at the single-cell level by using a three-dimensional reconstruction of cells transfected with EGFP-Tbx5 and HcRed-LMP4 (see Movie S1 in the supplemental material).
Role of NES motif in Tbx5 subcellular localization. Consistent with our previous work (9, 13, 30), wild-type Tbx5 revealed strictly nuclear localization when expressed alone, but in the presence of LMP4, it was also distributed in the cytoplasm as a complex with LMP4 along the actin cytoskeleton (Fig. 5A). In order to investigate if the identified putative NES in Tbx5 was functional for export, the four critical amino acid residues in the conserved motif were replaced by alanines (Fig. 3). As with the NLS mutants, all of the NES mutant constructs were designed as EGFP fusions, but this time were coexpressed in COS-7 cells with an HcRed-LMP4 expression construct. Substitution of all the critical amino acids in the NES (mutNES) with alanines completely blocked the localization of Tbx5 to cytoplasmic sites (Fig. 5A). In support of the CRM1 coimmunoprecipitation results (Fig. 4A), the F155A/L158A/L160A mutant that did not bind CRM1 also did not localize outside of the nucleus (Fig. 5A). Interestingly, the apparent block of cytoplasmic localization of Tbx5 in the presence of LMP4 was recapitulated in each of the single point mutants (Fig. 5A) (see Fig. S1 in the supplemental material), even though they are capable of interaction with CRM1 (Fig. 4A). In order to verify that these observations were statistically significant, cells (n = 85 to 95 per construct) from two independent experiments were counted and scored for the number of cells expressing cytoplasmic Tbx5 in the presence of LMP4. The percentage of cotransfected cells with cytoplasmic Tbx5 dropped from 100% for wild-type Tbx5 to less than 10% for each of the NES mutants (Fig. 5B). In summary, these data suggest that Tbx5 contains two functional NLS motifs and, in addition, a novel NES motif that is responsible for the shuttling of the protein out of the nucleus.
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The data presented here provide evidence supporting the notion that Tbx5 cytoplasmic relocalization in the presence of LMP4 utilizes the CRM1 nuclear export pathway and that this process is dependent on the identified NES sequence present in the DNA-binding domain of the transcription factor. In addition to Tbx5, the data show that all T-box proteins contain an NES motif, are capable of interaction with CRM1, and potentially could shuttle between nuclear and cytoplasmic sites. In summary, new information presented in this study allowed an integration of previous and current findings into a new working model for Tbx5 nuclear-cytoplasmic shuttling as described in Fig. 7.
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DISCUSSION |
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In this study, we sought to gain a better understanding of the mechanisms facilitating Tbx5 transport across the nuclear envelope. Although two NLS motifs in Tbx5 had been recently identified, nuclear import was investigated with single-amino-acid point mutants and protein truncation constructs in one or the other NLS (15, 51). We confirmed and extended these studies demonstrating that NLS1 and NLS2 have a synergistic relationship and that both NLS motifs are essential for efficient nuclear localization. Surprisingly, but in agreement with the other studies, we could not achieve an exclusive cytoplasmic localization, even when both NLS1 and NLS2 were mutagenized. This result may suggest that additional, yet-unknown sites or mechanisms are involved in nuclear localization.
In order to explain Tbx5 nuclear export, we wondered if the CRM1-dependent nuclear export pathway utilized by other shuttling proteins is also employed by T-box proteins (38, 42). CRM1 is known to recognize and bind a conserved NES motif of four mostly large hydrophobic amino acids, thereby targeting the respective protein for nuclear export (38, 42). Our analysis of T-box amino acid sequences revealed that all known T-box protein family members contain the hydrophobic amino acid NES core motif. The CRM1 binding studies with Tbx5 and two additional protein family members experimentally confirmed that T-box proteins use the CRM1 pathway for nuclear export. The two closest family members, Tbx5 and Tbx4, share an identical NES motif within the DNA-binding domain. Interestingly, the evolutionarily more-divergent family members Tbx2 and Tbx3 (3) share the core NES but contain a proline and a valine, respectively, in place of the initial leucine. In this context, it was of interest to compare the data with those for the cephalochordate amphioxus, which has been used to track the gene structure changes of T-box genes in evolution (45). While in this species Tbx2/3 and Tbx4/5 gene clusters have formed from a primordial Tbx2/3/4/5 gene, this two-gene cluster had not been further extended to individual Tbx2, -3, -4, and -5 genes as seen for vertebrates. We identified an NES motif in amphioxus Tbx2/3 and Tbx4/5 with a threonine and a valine in the initial position, respectively, suggesting that all T-box proteins are shuttling proteins and that the particular NES sequence in vertebrate Tbx5 and Tbx4 was acquired later in evolution when new functional roles were adopted.
The other T-box protein family members use a diverse but limited range of hydrophobic residues in the initial NES position. Tbx1, -10, and -13 have an isoleucine, while Tbx2, -6, and Brachyury (T) have a proline in this position. Of note, the T-box protein Tbr-1 with a glutamic acid in the initial position in the cytoplasm of adult rodent brain cells has been described previously (24). In addition, recent work on Brachyury during mouse gastrulation described the protein in various embryonic and extraembryonic tissues as localizing predominantly to the cytoplasm (25), and based on experiments presented in this report, as also being capable of interacting with the CRM1 exporter. Thus, it is probable that T-box proteins with various NES motifs can utilize the CRM1 pathway for nuclear export; however, the mechanism of cytoplasmic retention for these proteins is still unknown.
We find in the T-box protein family a higher level of conserved amino acids with predominantly leucines or phenylalanines in the more C-terminal NES positions compared to the initial position that reveals a wider range of amino acid options. Using Tbx5 as a model, we investigated the functional role of the various NES core amino acids with regard to the extent to which they contribute to CRM1 binding. This was of interest since almost all previous studies mutagenized the four critical positions as a whole but did not evaluate individual point mutations. A set of systematic point mutations of the Tbx5 NES revealed the importance of particularly the three C-terminal positions for binding of CRM1. While we found the first position dispensable for interaction, the C-terminal positions of the NES appeared sufficient for binding to the export protein. This is in line with the observation that for NES activity in other shuttling proteins, the residues in the C-terminal position are more susceptible to mutation than those in the N-terminal position (33, 49). Replacing the initial leucine of Tbx5 with alanines or residues found in Tbx2, Tbx3, or Brachyury did not change the ability of the mutagenized protein to interact with CRM1. This result is consistent with our finding that full-length Tbx3 and Brachyury recombinant proteins can also bind to the CRM1 export protein. Interestingly, when we kept the N-terminal NES motif in Tbx5 as the wild type and mutagenized only the last position of the NES core, the ability to interact with CRM1 was retained. Thus, while four amino acids had been described to be critical for NES function, our experiments demonstrate that three of the hydrophobic core residues are sufficient for effective CRM1 binding. A robust protein interaction may be constrained by the three-dimensional surface features and therefore relies on a combinatorial and synergistic effect of the individual NES core residues with the target site on the exporter protein. Moreover, this interaction undoubtedly influences the formation of the export protein complex with the other proteins necessary for complete and efficient shuttling out of the nucleus. Besides sequence pattern, accessibility and flexibility appear to be additional important properties of NESs (35). In this context, it may be no coincidence that in the T-box transcription factors, the NES is integrated in the DNA-binding domain, a designated site for DNA and protein interactions.
Our studies demonstrated that the identified NES is responsible for CRM1-mediated nuclear export of Tbx5. This export activity is abolished by treatment with leptomycin B or site-directed mutagenesis of critical amino acids within the NES motif. Moreover, LMP4-mediated regulation of Tbx5 transcriptional activity is no longer functional when nuclear export is blocked. Replacement of just the initial leucine of the NES motif sequence does not interfere with CRM1 binding; however, it is sufficient to prevent cytoplasmic retention of Tbx5 as a complex with LMP4 and actin. Thus, the initial residue of the NES may be involved in the specific recognition of the cytoplasmic interacting protein LMP4. In this context, we recall that Tbx4 shares the identical NES sequence with Tbx5, interacts also with LMP4, and is retained in the cytoplasm. Brachyury, on the other hand, has a quite different NES, including a proline in the initial position but, as we have demonstrated, it does bind the CRM1 export protein and, as reported by others, it also localizes to cytoplasmic sites (25). In contrast to Tbx5 and Tbx4, for Brachyury and other T-box proteins the respective cytoplasmic interacting proteins are not yet known. It would not be surprising if there is a defined group of cytoplasmic proteins targeted for binding by individual T-box family members with unique NES sequences. Their identification will facilitate a deeper and more general understanding of T-box protein nuclear export and cytoplasmic retention and function.
In addition, specific residues in T-box proteins may be involved in posttranslational modifications and therefore contribute to the regulation of the subcellular localization of the protein. For example, phosphorylation has been shown to play a role in the nuclear export of several proteins that contain a CRM1-dependent NES sequence motif (27, 28, 40). It is therefore conceivable that regulated phosphorylation of T-box proteins may be involved in fine-tuning the protein's localization and its overall activity. Nuclear import and export sequences are important regulators of the subcellular localization of proteins. Thus, they provide new opportunities to manipulate the subcellular distribution of T-box proteins in vivo in order to investigate the resulting functional consequences on development, work that is ongoing in our laboratory.
The human disorder HOS is caused by mutations in TBX5, leading to skeletal and cardiac malformations. The majority of mutations critical for disease manifestation are considered to result in early protein terminations and haploinsufficiency (8). However, chromosome 12q2 duplications, which lead to increased TBX5 dosage, have also been reported to cause HOS (21, 48). These results suggest that a balanced level of Tbx5 protein in the cell is critical for its function, as under- and overexpression result in similar phenotypes. Tbx5 contains functional NLS and NES motifs, and the protein dynamically shuttles between nuclear and cytoplasmic sites (13). Our modeling studies revealed that amino acids linked with HOS manifestation are in close proximity to the NES, facing the inner surface of the protein dimer. It is therefore possible that conformational changes induced by these point mutations may also affect the NES interaction with proteins important for nuclear export. Thus, disease manifestation in HOS is potentially caused by compromised regulation of the subcellular location of TBX5, in addition to altered transcriptional activity, and therefore affects protein function in both nuclear and cytoplasmic cell compartments. We note a highly related NES motif in Tbx1, making this family member also a candidate for changing its localization between the nucleus and cytoplasm. In humans, TBX1 is linked to DiGeorge syndrome, a disease with phenotypes ranging from cardiac malformations, facial dismorphogenesis, and cleft palate to hypoplasia of the thymus and parathyroid glands (4, 5, 46), all attributed to disruption of TBX1 transcriptional activity. Based on sequence homology in the putative NES, it is possible that TBX1 may have additional functions through other, yet-unidentified binding partners, facilitating shuttling and distribution of the protein in the cytoplasm. As suggested for TBX5, alternative explanations for the observed disease phenotypes may rely on functions different from transcriptional regulation. The NES appears to be an evolutionarily highly conserved motif in T-box proteins. Consequently, we expect to see protein family members with distribution patterns and functions inside and outside the nucleus, calling for a paradigm shift of how we interpret T-box protein function. Our efforts to understand how this dynamic protein shuttling is regulated at the cell and tissue levels may also provide new insights into the signaling pathways that control the T-box protein subcellular localization and function.
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ACKNOWLEDGMENTS |
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This work was supported by a grant from the National Institutes of Health (HL085834-01 to H.-G.S.).
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FOOTNOTES |
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Published ahead of print on 26 December 2007.
Supplemental material for this article may be found at http://mcb.asm.org/.
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