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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2003 Mar 7;100(6):3257–3262. doi: 10.1073/pnas.0637114100

Small ubiquitin-like modifier conjugation regulates nuclear export of TEL, a putative tumor suppressor

Lauren D Wood *, Brenda J Irvin *, Giuseppina Nucifora †, K Scott Luce *, Scott W Hiebert *,‡,§
PMCID: PMC152279  PMID: 12626745

Abstract

Posttranslational modification by small ubiquitin-like modifier (SUMO) conjugation regulates the subnuclear localization of several proteins; however, SUMO modification has not been directly linked to nuclear export. The ETS (E-Twenty-Six) family member TEL (ETV6) is a transcriptional repressor that can inhibit Ras-dependent colony growth in soft agar and induce cellular aggregation of Ras-transformed cells. TEL is frequently disrupted by chromosomal translocations such as the t(12;21), which is associated with nearly one-fourth of pediatric B cell acute lymphoblastic leukemia. In the vast majority of t(12;21)-containing cases, the second allele of TEL is deleted, suggesting that inactivation of TEL contributes to the disease. Although TEL functions in the nucleus as a DNA-binding transcriptional repressor, it has also been detected in the cytoplasm. Here we demonstrate that TEL is actively exported from the nucleus in a leptomycin B-sensitive manner. TEL is posttranslationally modified by sumoylation at lysine 99 within a highly conserved domain (the “pointed” domain). Mutation of the sumo-acceptor lysine or mutations within the pointed domain that affect sumoylation impair nuclear export of TEL. Mutation of lysine 99 also results in an increase in TEL transcriptional repression, presumably because of decreased nuclear export. We propose that the ability of TEL to repress transcription and suppress growth is regulated by sumoylation and nuclear export.

The function of posttranslational modification by ligation of a small ubiquitin-like modifier (SUMO) to a target protein appears to be diverse and substrate specific. RanGAP1 was the first protein shown to be modified by addition of SUMO (1–3). Unmodified RanGAP1 is diffusely cytoplasmic, whereas sumoylation targets RanGAP1 to nuclear pore complexes. Sumoylation also directs targeted proteins such as promyelocytic leukemia and the homeodomain-interacting protein kinase 2 to distinct subnuclear domains (nuclear bodies or speckles; refs. 4 and 5). SUMO modification appears to activate the heat shock transcription factors 1 and 2 (6, 7), whereas sumoylation may negatively regulate c-JUN and c-MYB activity (8, 9). Sumoylation also affects protein stability if the modified lysine is also used for ubiquitination. Both inhibitor of NF-κBα and murine double minute 2 are targeted for degradation by ubiquitination, but competition for the target lysine by SUMO stabilized these factors (10–12). Like ubiquitin, SUMO conjugation and deconjugation is a very dynamic process. Sumoylation studies are limited because of the rapid removal of the modification by SUMO-specific isopeptidases in cellular extracts. Thus, for each of these examples, the functional consequences of sumoylation were derived from conservative substitution of the modified lysine of the target protein.

Like ubiquitination, sumoylation is a three-step process involving an E1-activating enzyme heterodimer Aos/Uba2, the E2-conjugating enzyme Ubc9 and substrate-specific E3 ligases (for recent reviews, see refs. 13 and 14). The nucleoporin RanBP2/Nup358 and the protein inhibitors of activated signal transducer and activator of transcription family represent two types of E3 ligases (15–19). Sumoylation forms an isopeptide bond between the C-terminal carboxyl group of SUMO and the ɛ-amino group of a lysine residue in the target protein. Three SUMO family members have been identified (SUMO1, SUMO2, and SUMO3; ref. 20). SUMO2 and SUMO3 are 95% identical and 40–50% homologous to SUMO1, suggesting that SUMO2 and SUMO3 have similar functions. Several mammalian SUMO isopeptidases have been identified, including SENP1, Smt3IP1, SUSP1, and Smt3IP2/SENP2 (21–24). SENP2 and RanBP2 localize to the nuclear pore complexes, suggesting that SUMO can be added and/or removed as proteins pass between the cytoplasm and nucleus (25, 26).

TEL (also know as ETV6) is an E-Twenty-Six (ETS)-family transcriptional repressor that is modified by sumoylation (27, 28). TEL is a frequent target of chromosomal translocations in both myeloid and lymphoid leukemias, which fuse the N-terminal pointed domain [PNT, also called helix–loop–helix or sterile α motif (SAM)] to tyrosine kinases including platelet-derived growth factor-β, c-ABL, and Janus kinase 2 (29, 30). However, the most frequent chromosomal translocation involving TEL is the t(12;21), which fuses the N terminus of TEL to the acute myeloid leukemia (AML)1 transcription factor (also called RUNX1; for comprehensive reviews, see refs. 29 and 31). The t(12;21) is the most common genetic rearrangement found in childhood pre-B cell acute lymphoblastic leukemia. In the majority of t(12;21)-containing acute lymphoblastic leukemia, the second allele of TEL is deleted, suggesting that TEL is a tumor suppressor.

Whereas oncogenic ETS factors are mediators of Ras signals, TEL inhibits Ras-dependent transformation of NIH 3T3 cells (32–34). TEL may oppose the transactivation functions of oncogenic ETS factors by actively repressing ETS factor target genes through the recruitment of corepressors such as mSin3A, nuclear hormone corepressor, and histone deacetylase-3 (35–38). When expressed in Ras-transformed NIH 3T3 cells, TEL induced cellular aggregation, directly implicating TEL in cell adhesion (32, 34). Cellular aggregation correlated with repression of stromelysin-1, which is involved in extracellular-matrix remodeling (34). A histone deacetylase inhibitor not only impaired TEL-dependent transcriptional repression of the stromelysin-1 promoter, but biologically inactivated TEL in cellular aggregation assays (34, 36).

The PNT domain of TEL is a focal point for regulation of TEL because this domain mediates associations with mSin3A, homodimerization, and binding to other ETS factors (39–42). In addition, the SUMO-conjugating enzyme Ubc9 binds to the PNT domain and ligates SUMO to lysine 99 (27, 28). Sumoylation appears to target TEL to specific nuclear speckles in a subset of the cell population (28). TEL is a predominately nuclear phosphoprotein that represses transcription by directly binding to the promoters of target genes. However, when TEL was expressed in fibroblasts, it was both nuclear and cytoplasmic (32). Here we show that TEL is actively exported from the nucleus. TEL proteins with mutations that disrupt the PNT domain accumulate in the nucleus, suggesting that this domain mediates nuclear export. The highly conservative change of lysine 99 to arginine, within the PNT domain, blocks SUMO modification and caused a dramatic accumulation of TEL in the nucleus, implying that sumoylation of TEL regulates the nuclear export of this tumor suppressor.

Experimental Procedures

Cells and Biological Assays.

COS-7 and HeLa cells were obtained from the American Type Culture Collection. COS-7, 293T, and HeLa cells were grown in DMEM with 10% FBS. Ras-transformed and nontransformed NIH 3T3 cells were grown in DMEM with 10% calf serum. Retroviral infections of Ras-transformed 3T3 cells with pBabePuro retroviruses, soft agar colony-formation assays, and luciferase assays were performed as described (34).

Plasmids.

pBabePuro, pCMV-TEL, and pCMV-TEL/AML were as described (43). pBabePuro-TEL, pCMV-TELΔP (residues 40–104 deleted), Δ122–268, Δ266–335, ΔETS (residues 374–385 deleted), and ΔP+Δ266 were as described (note Δ266–335 has an additional BglII site at the deletion site and was originally incorrectly called Δ268–333; refs. 34 and 36). Hemagglutinin (HA)-tagged TEL constructs were made by amplifying full-length TEL or deletions by using PCR and KpnI-linked 5′ primers and the XbaI-linked 3′ primer (5′-atctctagatcagcattcatcttcttg-3′) and ligated into a modified pCMV5 vector that has the HA tag inserted after the EcoRI site. Point mutations were made by using the QuikChange site-directed mutagenesis kit (Stratagene), and the sequence was confirmed. The GFP-SUMO1 and GFP-SUMO2/3 were as described (20, 44).

Immunoprecipitation and Immunoblot Analysis.

Immunoprecipitation and immunoblot analysis were performed as described (34, 35) except for the addition of 20 μM N-ethylmaleimide. Antibodies used for immunoprecipitation were anti-mSin3A or anti-HA tag. Polyclonal antibodies made against mSin3A, HA-tag, and c-Raf were obtained from Santa Cruz Biotechnology, and monoclonal anti-HA and anti-c-myc were from Covance (Berkeley, CA). Antibodies against C-terminal and N-terminal TEL were as described (34, 35). Cell fractionation was performed as described (45).

Immunofluorescence.

Cells were transfected by using Superfect (Qiagen, Chatsworth, CA) according to the manufacturer's instructions. Leptomycin B (LMB), N-ethylmaleimide, normal goat serum, and Hoechst no. 33258 were obtained from Sigma. The cells were fixed in 3.7% formaldehyde/PBS, permeabilized in 0.1% Nonidet P-40/PBS, and blocked in 10% normal goat serum/PBS for 30 min. Cells were incubated with primary antibodies (diluted in 10% normal goat serum/PBS) for 60 min. Anti-TEL and anti-HA were used at a 1:1,000 dilution or a polyclonal antibody against the HA tag at a 1:250 dilution. Cells were then incubated for 60 min with the appropriate Alexa488-conjugated secondary antibodies. Anti-mouse and anti-rabbit Alexa488 or Alexa546 were obtained from Molecular Probes. Cells were incubated with 5 ng/ml Hoechst dye for 5 min to visualize DNA. Cells were analyzed on a Zeiss Axiophot microscope by using Zeiss 40 × 1.4 oil immersion objectives. For protein localization scoring, at least 200 cells for each condition were scored, with results presented as percentage of counted cells.

Results

Endogenous TEL Is Located in Both the Cytoplasm and Nucleus.

TEL functions in the nucleus as a transcriptional repressor (35, 37, 43, 46), yet, when TEL was expressed in NIH 3T3 cells, it was detected in both the cytoplasm and nucleus (32). Therefore, we examined the localization of endogenous TEL by fractionation of NIH 3T3 cells (Fig. 1A). We found a significant proportion of the endogenous TEL was cytoplasmic. In this experiment, the level of cytoplasmic TEL was >50%, although the amount varied from 30% to 70% between experiments (Fig. 1A). In addition, the cytoplasmic form of TEL appeared to include a hyperphosphorylated form (see arrowheads, Fig. 1A). A third form that is derived from internal translational initiation from methionine 43 (TEL-43; ref. 47) was consistently more nuclear in its localization (Fig. 1A). Detection of either nuclear mSin3A or cytoplasmic c-Raf was used as a control. SUMO-modified TEL represents only a small percentage of the total TEL protein. Whether this situation exists because only a small percentage of TEL is targeted for SUMO conjugation or whether SUMO is rapidly removed by SUMO-specific protease activity is unclear. To detect sumoylated TEL, we expressed epitope-tagged TEL (HA-TEL) in COS cells. As expected, fractionation of the HA-TEL-transfected COS cells revealed that the SUMO modified form was nuclear (data not shown; ref. 28).

Figure 1.

Figure 1

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Subcellular localization of endogenous TEL. (A) Cytoplasmic (Cyto) and nuclear (Nuc) fractions from NIH 3T3 cells were separated by SDS/PAGE and analyzed by immunoblotting with antibodies against mSin3A, c-Raf, and TEL. TEL and internally initiated TEL (TEL-43) forms are indicated; the arrowhead indicates hyperphosphorylated TEL. Expt. 1 and Expt. 2 refer to two separate cell fractionation experiments. (B) Endogenous TEL location in NIH 3T3 cells by immunofluorescence with an antibody against the C terminus of TEL. Arrows indicate cells with predominantly cytoplasmic TEL.

Cell fractionation determines the relative distribution of TEL in a population of cells. To examine the distribution of TEL in single cells, we performed immunofluorescence with a C-terminal TEL antibody. Endogenous TEL was detected in both the cytoplasm and the nucleus of most cells. However, in a limited number of cells, TEL appeared to be either excluded from the nucleus (Fig. 1B, see arrow in Center) or primarily nuclear (Fig. 1B Left). Although not representative of the population, Fig. 1B Right shows a cluster of cells in which TEL is mostly cytoplasmic. Similar staining of endogenous TEL was seen with an N-terminal TEL antibody that recognizes full-length TEL but not TEL-43 (data not shown).

LMB Inhibits TEL Export.

The observation that TEL can be detected in the cytoplasm or nucleus suggested that either a portion of TEL is retained in the cytoplasm or that TEL is exported from the nucleus. Many nuclear-cytoplasmic shuttling proteins depend on the nuclear export receptor Crm-1 for nuclear exit (48), and Crm-1 is inactivated by LMB treatment (49, 50). Therefore, we transiently expressed epitope-tagged (HA-tagged) TEL in NIH 3T3 cells and determined the cellular localization of TEL in the absence or presence of LMB. In the absence of LMB, HA-TEL appeared to localize predominantly to the cytoplasm in ≈60% of the cells (Fig. 2). In the remaining cells, TEL was more nuclear or equally distributed between the two compartments. Similar cytoplasmic staining of HA-TEL was observed in telomerase-immortalized human foreskin fibroblasts, HeLa, and COS-7 cells (data not shown). LMB treatment of the transfected NIH 3T3 cells resulted in a decrease in the number of cells displaying cytoplasmic localization of TEL (≈30% cells) with an increase in the number of cells displaying predominantly nuclear TEL. These results imply that TEL is actively exported from the nucleus in a Crm-1-dependent manner.

Figure 2.

Figure 2

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Nuclear export of TEL is inhibited by LMB. (A) Localization of HA-TEL expressed in NIH 3T3 cells was determined by immunofluorescence with anti-HA (Upper). Cells were counterstained with Hoechst dye to show nuclei (Lower). Shown are representative pictures with either cytoplasmic or nuclear localization of HA-TEL. (B) NIH 3T3 cells were transfected with HA-TEL and then treated with LMB (10 ng/ml) for 6 h. Location of HA-TEL was determined by immunofluorescence with anti-HA. NIH 3T3 cells from three separate experiments were scored for protein localization and placed in the following categories: cytoplasmic > nuclear (stippled bar), nuclear > cytoplasmic (black bar), and nuclear = cytoplasmic (gray bar). The bar graph shown is the percentage of cells with the indicated HA-TEL localization from a representative experiment.

TEL Pointed Domain Is Required for Cytoplasmic Localization.

Mutations within the DNA-binding domain of TEL resulted in the loss of the nuclear localization of TEL, whereas deletion of residues 53–116 in the PNT domain resulted in both cytoplasmic and nuclear localization of TEL (32). The PNT domain is important for association with mSin3A, and it mediates homodimerization. The central domain of TEL that lies between the pointed domain and the C-terminal ETS DNA-binding domain interacts with both histone deacetylase-3 and nuclear hormone corepressor (36). Therefore, we tested mutants of TEL to define whether association with nuclear proteins or DNA affects the nuclear accumulation of TEL. NIH 3T3 cells were transiently transfected with various HA-tagged TEL deletion mutants (Fig. 3A), and the localization was determined for each mutant protein (Fig. 3B). We found that deletion of residues 40–104 (ΔP) increased nuclear accumulation. Deletion of the middle region of TEL (Δ122–268) or central domain, in which histone deacetylase-3/nuclear corepressor binds (Δ266–335), behaved similarly to wild-type TEL in that the majority of the cells displayed cytoplasmic localization of TEL. Disruption of the DNA-binding domain (ΔETS) showed almost no nuclear localization possibly because of the lack of binding to DNA. Therefore, the PNT domain, which is sufficient for mSin3A association and homodimerization, contributes to the nuclear export of TEL.

Figure 3.

Figure 3

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The pointed domain of TEL is required for cytoplasmic localization. (A) Schematic representation of TEL internal deletion mutants. Numbers indicate the initial 5′ amino acid deleted in TEL. The stippled box indicates the PNT domain, and the black box indicates the ETS DNA binding domain. (B) Bar graph showing the percentage of cells with the indicated subcellular localization as determined by immunofluorescence with an anti-HA antibody. Results are the average and SD from three experiments (data for TELΔPNT+Δ266 and TELΔ266 are the average of two experiments). (C) Bar graph showing the percentage of cells expressing TEL, TEL/AML1, or AML1 from a representative experiment with the indicated subcellular localization as determined by immunofluorescence with anti-HA.

To confirm that the N terminus of TEL, including the PNT domain, is sufficient to direct nuclear export, we took advantage of the t(12;21) fusion protein, which fuses the N-terminal 336 residues of TEL to nearly all of the AML1 transcription factor. AML1 is exclusively nuclear and associates with the nuclear matrix (51). HA epitope-tagged AML1 and TEL-AML1 were transfected into COS-7 cells and their localization was determined by immunofluorescence. As expected, AML1 was predominantly localized to the nucleus (Fig. 3C). By contrast, TEL-AML1 was both nuclear and cytoplasmic (Fig. 3C), indicating that TEL encodes sequences that are sufficient to relocalize AML1 to the cytoplasm.

Point Mutations in the Pointed Domain of TEL Inhibit Nuclear Export.

Because the pointed domain of TEL mediates dimerization, we hypothesized that dimerization contributes to the nuclear export of TEL. This could be a regulatory signal, because high levels of nuclear TEL would promote dimerization and nuclear export. Structural analysis of the TEL pointed domain (also known as the SAM domain) revealed a helical head-to-tail polymer with two binding surfaces: the mid-loop and end-helix surfaces (52, 53). A point mutation in the mid-loop surface (A93D) creates a SAM domain that fails to homodimerize but can heterodimerize with the wild-type TEL-SAM domain (sequences in Fig. 4A; ref. 53). By contrast, a substitution in the end-helix surface (V112E) only partially impairs dimerization in that it retains the ability to weakly self-associate and strongly heterodimerize with the wild-type TEL-SAM domain (52, 53). Combining the two mutations resulted in failure to either homo- or heterodimerize (52, 53). After placing the point mutations within the context of full-length TEL, we obtained similar results in that the single point mutants still heterodimerized with wild-type TEL, but the double mutant failed to homo- or heterodimerize (data not shown). When these TEL mutants were expressed and localized by indirect immunofluorescence, all three mutants were predominantly nuclear in contrast to wild-type TEL (Fig. 4B).

Figure 4.

Figure 4

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Single amino acid mutations in the TEL dimerization interfaces interfere with nuclear export and sumoylation. (A) Pointed domain mutations. The residues within the mid-loop surface (light gray) and the end-helix surface (dark gray) that form the interface during TEL dimerization are shown (52, 53). The point mutations in the association interfaces (A93D and V112E) and the lysine that is sumoylated (K99R) are shown below. (B) Bar graph showing the percentage of cells with the indicated subcellular localization as determined by immunofluorescence with anti-HA. Results are the average from three experiments. (C) TEL dimerization-defective point mutants cannot repress the stromelysin-1 promoter. Luciferase assays were performed by using NIH 3T3 cells transfected with the pGL2–754TR luciferase reporter and either 40 ng (stippled bars) or 100 ng (black bars) of the indicated HA-tagged TEL constructs. pCMV-SEAP was used as an internal control. The normalized light units are shown from a representative experiment performed in triplicate. (D) TEL dimerization-defective point mutants are not sumoylated. Whole-cell lysates from COS-7 cells that were cotransfected with the indicated HA-TEL construct and pCMV-GFP-SUMO2 were analyzed by immunoblotting with anti-TEL antibodies. Notice the shift of the sumoylated TEL form to the slower-migrating form, indicative of the addition of GFP (single arrowhead indicates sumoylated TEL; double arrowhead indicates GFP-linked SUMO conjugated to TEL).

The nuclear localization of the dimerization mutants may suggest that homodimerization is required for nuclear export. However, despite the nuclear accumulation of these mutants, they were impaired in repressing transcription in reporter assays (Fig. 4C). TEL(A93D) was inactive, whereas TEL(V112E), while still active at higher levels of input expression plasmid, was impaired in its ability to repress the stromelysin-1 promoter. Therefore, we tested whether these mutants were able to associate with mSin3A. As predicted from the transcriptional analysis, TEL(A93D) failed to associate with mSin3A, whereas TEL(V112E) was impaired for binding to mSin3A (data not shown). Thus, the A93D substitution disrupts both dimerization and corepressor binding, whereas V112E is a less severe mutation when placed in the context of full-length TEL. However, both mutations affect nuclear export.

While monitoring the expression of the dimerization mutants, we noted that these mutants were not modified by sumoylation. Coexpression of wild-type TEL or these mutants with a GFP-linked SUMO1 (data not shown) or GFP-SUMO2 in COS-7 cells confirmed that the point mutants were not modified by the addition of GFP-SUMO2 (Fig. 4D; single arrowhead denotes SUMO-TEL; double arrowheads denote GFP-SUMO-TEL; note that the addition of GFP to SUMO2 reduces the mobility of SUMO-TEL). These results suggested that either wild-type levels of dimerization are necessary for SUMO conjugation or the structure of the TEL mutants is altered so that SUMO conjugation could not occur.

Sumoylation Contributes to the Nuclear Export of TEL.

Because TEL(V112E) can still weakly self-associate (52) and retained partial transcriptional function but was not efficiently sumoylated, it appeared that the failure of this mutant to be exported from the nucleus was because of a lack of sumoylation. To directly test this hypothesis, we tested whether the highly conservative substitution of arginine for lysine at residue 99, which is the major site for SUMO conjugation (28), would affect the nuclear export of TEL. When the cellular localization of TEL(K99R) was determined by indirect immunofluorescence in both stably infected or transiently transfected NIH 3T3 cells, it was predominantly nuclear (Fig. 5 A and B).

Figure 5.

Figure 5

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Mutation of lysine 99, a target for sumoylation, impairs TEL cytoplasmic localization. (A) NIH 3T3 cells were stably infected with retroviruses expressing either HA-TEL or HA-TEL(K99R) and the subcellular localization of TEL determined by immunofluorescence by using anti-HA. (B) Bar graph showing the percentage of cells with the indicated location in NIH 3T3 cells transfected with HA-TEL or HA-TEL(K99R). (C) mSin3A interacts with TEL(K99R). Lysates from COS-7 cells transfected with either HA-TEL or HA-TEL(K99R) were immunoprecipitated with antibodies against mSin3A (Sin3) or normal rabbit serum (NS). HA-TEL and mSin3A were detected by immunoblot. TEL or sumoylated TEL (+SUMO) are indicated. (D) TEL(K99R) inhibits soft agar colony formation of Ras-transformed cells. Cells were grown for 11 days in medium containing 0.375% agarose, as overlays on 0.8% agarose beds. A total of 1,500 cells of the indicated cell lines were plated per dish. Values shown are the mean and range of duplicate samples. (E) TEL(K99R) is a repressor of the stromelysin-1 promoter. Luciferase assays were performed on lysates of NIH 3T3 cells transfected with the pGL2–754TR luciferase reporter and 100 ng of pCMV-TEL, TELΔETS, TELΔP, or TEL(K99R) (Left) or with the indicated amounts (ng) of either CMV-TEL or CMV-TEL(K99R) (Right). pCMV-SEAP was used as an internal control.

To ensure that the K99R mutation did not disrupt the structure of the pointed domain, we tested TEL(K99R) in assays that depend on a functional pointed domain. Lysine 99 lies between the two dimerization surfaces of the pointed domain, and, as expected from the structural predictions, TEL(K99R) retained the ability to homo- and heterodimerize (data not shown). This finding suggested that the K99R mutation did not alter the structure of the pointed domain, and we confirmed that TEL(K99R) was still able to bind to mSin3A (Fig. 5C). In addition, TEL(K99R) was as efficient or better than wild-type TEL at inhibiting colony formation in soft agar by Ras-transformed NIH 3T3 cells (Fig. 5D). In more quantitative assays, TEL(K99R) was a better transcriptional repressor than wild-type TEL at equal levels of input expression plasmid (Fig. 5E). Immunoblot analyses of the lysates confirmed that the same level of TEL proteins was expressed at each concentration for each transfected construct (data not shown). Thus, the lack of nuclear export of TEL(K99R) functionally correlates with its greater ability to repress transcription and suggests that SUMO modification contributes to the nuclear export of TEL.

Discussion

SUMO modification of nuclear proteins is now recognized as a prevalent posttranslational modification. However, the functional consequences of this modification are known for only a few proteins. For instance, mutational analysis of the promyelocytic leukemia tumor suppressor indicates that sumoylation directs promyelocytic leukemia into nuclear promyelocytic leukemia oncogenic domains (4, 54, 55). Our data suggest an additional function for SUMO conjugation: regulation of nuclear export. TEL is exported from the nucleus (Fig. 1), and a single, highly conservative mutation impairs both SUMO modification and nuclear export (Figs. 4 and 5). This mutant form of TEL is capable of carrying out all of the known functions of wild-type TEL; moreover, it is hyperactive, owing to its lack of nuclear export (Fig. 5). Whereas many proteins that are exported from the nucleus are degraded in the cytoplasm, TEL appears to be a very stable protein (L.D.W. and S.W.H., unpublished data). Once TEL is exported, it may be retained in the cytoplasm or quickly reimported to the nucleus as needed. Therefore, nuclear export of TEL may be an efficient mechanism to regulate this important growth suppressor.

In yeast, temperature-sensitive mutants of the SUMO conjugation pathway block nuclear import of proteins containing a classic nuclear localization signal (56). However, the block in import was attributed to the failure of the importin-α subunit Srp1 to be recycled by export from the nucleus (56). Although the failure to export Srp1 is likely indirect, because a SUMO-modified form of Srp1 was not detected, our data indicate that the lack of sumoylation directly impairs the nuclear export of TEL. Coupled with the genetic study of general sumoylation in yeast, we hypothesize that sumoylation may be a more general mechanism for regulating nuclear export.

It is notable that several ETS family members such as NET and YAN are regulated by nuclear export, and both NET and YAN contain putative SUMO acceptor sites. YAN is the closest Drosophila homolog to TEL, and, like TEL, YAN-mediated repression is regulated by nuclear export (57). Sumoylation of YAN has yet to be addressed, but, phosphorylation on multiple residues appears to redirect YAN from the nucleus to the cytoplasm, thereby relieving repression (57, 58). In several examples, phosphorylation regulates SUMO modification (4, 8, 59, 60), and in our initial cell-fractionation experiments we noted that the hyperphosphorylated forms of full-length TEL were predominantly cytoplasmic (Fig. 1). In this regard, we note that ≈5–15% of the cells had cytoplasmic localization of TEL(K99R) (Fig. 5). Thus, it is possible that phosphorylation of TEL can also contribute to its nuclear export. Further analysis of other ETS factors will be required to determine whether sumoylation plays a more widespread role in nuclear export and to define whether phosphorylation regulates sumoylation and/or nuclear export of TEL.

Because of the labile nature of SUMO modification, it is difficult to directly assess how sumoylation of TEL might mediate nuclear export. K99 lies between two dimerization interfaces (see Fig. 4 and refs. 52 and 53), so SUMO conjugation may alter the conformation of TEL to expose a nuclear export signal or create a binding site for an export factor. TEL contains three weak nuclear export signals based on the LMB-sensitive export motif (LX (1–3)LX (1–3)LXL). Mutation of the leucines within the first two of these sequences yielded no effect on TEL nuclear export, but mutations in the third putative nuclear export signal (residues 113–121) did block cytoplasmic localization (L.D.W., unpublished data). However, this motif lies within the second dimerization interface of the pointed domain, and this TEL mutant failed to dimerize and was not sumoylated. We were therefore unable to unambiguously identify a nuclear export signal. Alternatively, TEL has been detected in subnuclear “speckles” in a subset of transfected cells, whereas the sumo-deficient mutant TEL(K99R) is excluded from these domains (28). These speckles were not readily apparent in the majority of our experiments, but were observed occasionally. It is possible that these speckles represent “loading docks” for nuclear export, or they may be an alternative functional domain unrelated to nuclear export. Regardless of the precise mechanism, when the N terminus of TEL is added to AML1 by the t(12;21), it caused cytoplasmic localization of the fusion protein, indicating that TEL residues 1–336 contain a region that mediates nuclear export.

Similarly to TEL, TEL/AML1 is also sumoylated (28), and mutation of the SUMO target lysine (Lys-99) in TEL/AML1 results in increased nuclear retention (L.D.W., unpublished data). The portion of TEL that is found fused to AML1 (RUNX1) is also fused to tyrosine kinases (TEL/platelet-derived growth factor receptor-β, TEL/Janus kinase 2, and TEL/ABL) in chronic myelogenous leukemia and AML (29, 30). Although most of these kinases are normally cytoplasmic, it will be of interest to determine whether mutation of K99 within these tyrosine kinase fusion proteins can cause inappropriate nuclear localization and impair the transformation ability of these fusion proteins.

Acknowledgments

We acknowledge the invaluable assistance provided by Sam Wells (at the Vanderbilt University Medical Center Cell Imaging Core) and the Vanderbilt–Ingram Cancer Center sequencing facility. We thank Yue Hou for expert technical assistance. This work was supported by National Institutes of Health Grants RO1-CA77274, RO1-CA64140, and RO1-CA87549; a Center grant from the National Cancer Institute (CA68485); the Vanderbilt–Ingram Cancer Center; an American Chemical Society postdoctoral fellowship to L.D.W., and a Leukemia and Lymphoma Society postdoctoral fellowship to B.J.I.

Abbreviations

SUMO

small ubiquitin-like modifier

PNT

N-terminal pointed domain

SAM

sterile α motif

AML

acute myeloid leukemia

HA

hemagglutinin

LMB

leptomycin B

ETS

E-Twenty-Six

Footnotes

This paper was submitted directly (Track II) to the PNAS office.

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