Genotoxic Stress and Cellular Stress Alter the Subcellular Distribution of Human T-Cell Leukemia Virus Type 1 Tax through a CRM1-Dependent M
http://www.100md.com
《病菌学杂志》
Interdepartmental Program in Cell and Molecular Biology, Department of Molecular Virology and Microbiology, Baylor College of Medicine, One Baylor Plaza MS-385, Houston, Texas 77030
ABSTRACT
Human T-cell leukemia virus type 1 Tax is a predominantly nuclear viral oncoprotein that colocalizes with cellular proteins in nuclear foci known as Tax speckled structures (TSS). Tax is also diffusely distributed throughout the cytoplasm, where it interacts with and affects the functions of cytoplasmic cellular proteins. Mechanisms that regulate the distribution of Tax between the cytoplasm and nucleus remain to be identified. Since Tax has been shown to promote genome instability by perturbing cell cycle progression and DNA repair mechanisms following DNA damage, we examined the effect of genotoxic stress on the subcellular distribution and interacting partners of Tax. Tax localization was altered in response to various forms of cellular stress, resulting in an increase in cytoplasmic Tax and a decrease in Tax speckled structures. Concomitantly, colocalization of Tax with sc35 (a TSS protein) decreased following stress. Tax translocation required the CRM1 nuclear export pathway, and a transient interaction between Tax and CRM1 was observed following stress. These results suggest that the subcellular distribution of Tax and the interactions between Tax and cellular proteins respond dynamically to cellular stress. Changes in Tax distribution and interacting partners are likely to affect cellular processes that regulate cellular transformation.
INTRODUCTION
Human T-cell leukemia virus type 1 (HTLV-1) has been etiologically linked to the development of a rare but rapidly progressing and fatal malignancy, adult T-cell leukemia (ATL), as well as to chronic conditions including tropical spastic paraparesis/HTLV-1-associated myelopathy. The HTLV-1 genome encodes several proteins that contribute to infection and oncogenesis; however, Tax is the major viral oncoprotein (17). Tax has been shown to transcriptionally activate the promoter, located within the 5' long terminal repeat, and to regulate the transcriptional activity of cellular promoters by serving as a transcriptional cofactor for the CREB, NF-B, and SRF pathways (61). In addition to interacting with and altering the activity of cellular transcription factors, Tax has been shown to increase the genomic instability (36) and mutation frequency (40-42) of the host cell, in part by repressing DNA repair mechanisms (28, 40, 49) and dysregulating cell cycle progression (17).
Pleiotropic functions of Tax, which are propagated through interactions with cellular proteins, have been shown to contribute to cellular transformation. Tax is predominantly a nuclear protein that localizes, along with a number of cellular proteins, to heterogeneous nuclear foci known as Tax speckled structures (TSS). These structures contain a variety of cellular proteins, including transcription factors, splicing cofactors, and DNA damage recognition and cell cycle regulatory proteins (4, 19, 51, 55). Tax has been reported to interact with more than 20 cellular proteins, including a number of cytoplasmic proteins, such as MEKK1, MAD1, CBP, RelA, and IB kinase subunits, as well as other nuclear proteins that are not found in TSS, including p16INK4a and p15INK4b (3, 26, 58, 60). Interactions of Tax with these proteins have profound effects on normal host cell processes and in many cases have been shown to be essential for or to enhance cellular transformation.
TSS composition and Tax protein interactions are dynamic, and changes in these interactions due to cellular or other environmental cues are likely to enhance the oncogenic properties of Tax. The ability of Tax to shuttle between the nucleus and cytoplasm may contribute to its oncogenic activity by facilitating changes in interacting partners under certain conditions (6). Nuclear localization and nuclear export sequences (NES) have been identified within the Tax protein, and these sequences are believed to modulate the nucleocytoplasmic shuttling of Tax (1, 52). However, neither the condition(s) that regulates the cycling of Tax between the nucleus and the cytoplasm nor the mechanism of Tax export from the nucleus has been identified. Here we report that Tax translocates to the cytoplasm in response to genotoxic and cellular stress. This translocation results in altered interactions between Tax and cellular proteins. We further demonstrate that Tax translocation following stress requires the CRM1 (chromosome region maintenance 1) nuclear export pathway and that functional mutations in the Tax nuclear export sequence inhibit cytoplasmic translocation following stress. This study provides the first evidence that the localization and interacting partners of Tax are regulated in response to stress and identifies a mechanism and domain within Tax that is required for this translocation. Changes in Tax localization and its interactions with cellular proteins in response to genotoxic and cellular stresses may contribute to or enhance the oncogenic activity of Tax.
MATERIALS AND METHODS
Cell lines, plasmids, and transfections. Clonal rat embryo fibroblasts that stably express Tax (CREF-Tax) (27) and 293 cells were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum. C81-66, an HTLV-1-infected human T-cell line (51), and HuT102, an HTLV-1-producing lymphocyte line (ATCC, Manassas, VA), were maintained in RPMI medium containing 10% fetal bovine serum. All cells were grown at 37°C in 5% CO2. pCMV-Tax, pCMV-M33, and pCMV-M47 (52) have been previously described and were transfected into 293 cells by use of Fugene 6 (Roche, Indianapolis, IN) as described by the manufacturer.
Antibodies. Anti-Tax antibodies (Tab170 and 586) were previously described (AIDS Research and Reference Reagent Program, Germantown, MD). Anti-CRM1 antibodies (H-300 and C-20) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-sc35 antibody was purchased from BD-Pharmacia (San Diego, CA). Alexa-Fluor 594-conjugated goat anti-rabbit and Alexa-Fluor 488-conjugated goat anti-mouse secondary antibodies were purchased from Molecular Probes (Eugene, OR). Anti--tubulin and horseradish peroxidase-conjugated secondary antibodies were purchased from Sigma-Aldrich (St. Louis, MO).
Cellular stress. For UV irradiation, medium was removed from plates and cells were washed once with phosphate-buffered saline. A Stratalinker instrument (Stratagene, La Jolla, CA) was used to deliver 30 J/m2 of UV-C irradiation. The original medium was then replaced, and the cells were allowed to recover for a specified length of time. For ionizing radiation, cells were grown on 60-mm2 plates, exposed to 10 grays of radiation, and allowed to recover for a specified length of time. Sorbitol, anisomycin, sodium arsenite, hydrogen peroxide, SB203580, SP600125, and PD098059 were added directly to either DMEM or RPMI medium at the concentrations shown in Table 1. Cells were incubated in the presence of these agents for the specified lengths of time. For heat shock, cells were incubated at 42°C for 90 min.
Cell synchronization. Cells were synchronized in G0 phase as previously described (35). In short, CREF cells were seeded into 100-mm dishes at a density of 1 x 106 cells/ml. The cells were allowed to reach confluence and maintained at 100% confluence for 48 h. Cells were released from arrest by splitting them 1:10 into new 100-mm dishes with fresh medium.
Propidium iodide staining. Cells (1 x 106) were resuspended in 2 ml of 0.9% NaCl and then fixed with 5 ml of 95% ethanol. Fixed cells were incubated at room temperature for at least 30 min and stored at 4°C. For staining, the cell pellet was resuspended in 0.5 ml of 50-μg/ml propidium iodide (Sigma-Aldrich, St. Louis, MO) and 0.1 ml of 1-mg/ml RNase A (Sigma-Aldrich, St. Louis, MO). A flow cytometer (Epic Profile; Coulter, Colorado) was used to analyze cell cycle distribution. The percentage of cells in each phase of the cell cycle was determined using ModFit (Verity, Topsham, ME).
Cellular extracts. Whole-cell extracts were prepared as previously described (9, 45) with a few modifications. Briefly, 1 x 107 cells were resuspended in 20 ml of ice-cold buffer C (20 mM HEPES [pH 7.9], 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride [PMSF], 0.5 mM dithiothreitol [DTT]) plus 0.1% NP-40, incubated on ice for 10 min, and centrifuged for 10 min at 10,000 rpm at 4°C. The supernatant was diluted with 80 μl of ice-cold buffer D (20 mM HEPES [pH 7.9], 20% glycerol, 50 mM KCl, 0.2 mM EDTA, 0.5 mM PMSF, 0.5 mM DTT) per 1 x 107 cells.
Immunofluorescent staining. CREF-Tax cells were seeded on ethanol-washed coverslips and grown to approximately 50% confluence before exposure to genotoxic conditions. C81-66 and HuT102 cells were grown in suspension, exposed to genotoxic conditions, collected by centrifugation (1,000 rpm for 10 min), washed and resuspended in phosphate-buffered saline, and then cytospun (Shandon Cytospin3) onto ethanol-washed coverslips (1,000 rpm for 1 min). The cells were washed once with PEM buffer {80 mM potassium PIPES [piperazine-N,N'-bis(2-ethanesulfonic acid)] [pH 6.8], 5 mM EGTA [pH 7.0], 2 mM MgCl2} and fixed by incubation in 5% formaldehyde diluted in PEM buffer for 30 min at 4°C. To remove excess formaldehyde, cells were washed three times in PEM buffer and permeabilized by incubation in PEM buffer containing 0.5% Triton X-100 for 30 min at room temperature. Immunofluorescent staining was performed by incubation with primary antibody diluted in 2.5 to 5% bovine serum albumin containing TBS plus 0.1% Tween 20 (TBS-T) for a minimum of 3 h at room temperature. Excess antibody was removed by washing cells three times in TBS-T. Cells were incubated in the dark with a fluorophore-conjugated secondary antibody diluted in TBS-T for 40 min at room temperature. Excess antibody was removed by washing the coverslips three times with TBS-T. The cells were counterstained with DAPI (4',6'-diamidino-2-phenylindole) (Sigma-Aldrich, St. Louis, MO) to visualize the nucleus and mounted on slides using Slow-Fade antifade mounting medium (Molecular Probes, Eugene, OR). Cells were visualized with a Zeiss AxioPlan2 microscope by use of a CoolSnap HQ charge-coupled-device camera and analyzed using MetaView MetaMorph software. For deconvolved images (see Fig. 4), an Applied Precision microscope with SoftWoRx image restoration software was utilized.
Immunoprecipitation. Whole-cell extracts were prepared from 293 cells (1.5 x 107) that had been transfected with either pCMV-Tax or pCMV-M33. The extracts were incubated with either anti-Tax (568) or anti-CRM1 (H-300) antibodies diluted in incubation buffer (20 mM HEPES [pH 7.9], 75 mM KCl, 2.5 mM MgCl2, 1 mM DTT, 0.1% NP-40, 0.5 mM PMSF, 1 μg/ml aprotinin, 1 μg/ml pepstatin, 2 μg/ml leupeptin, 1 mM sodium orthovanadate) overnight at 4°C on a rotator. Thirty microliters of a 50% protein G-bead slurry (Upstate, Lake Placid, NY) was added to the mixture, and the mixture was incubated for 90 min at 4°C. Beads were collected by centrifugation, washed five times with 500 μl of incubation buffer, and resuspended in 100 μl sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample buffer. Samples were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10%), transferred to a nitrocellulose membrane, and incubated with either anti-Tax (Tab170) or anti-Crm1 (C-20) antibodies. The membrane was then incubated with the appropriate horseradish peroxidase-conjugated secondary antibody.
Leptomycin B treatment. Leptomycin B (LMB) (LC Laboratories, Woburn, MA) was resuspended in absolute ethanol and added at the described concentrations directly to the medium (DMEM) containing CREF-Tax cells. The cells were incubated at 37°C for 1 h and then exposed to the specified genotoxic conditions.
CHX treatment. Cycloheximide (CHX) (Sigma-Aldrich, St. Louis, MO) was resuspended in absolute ethanol and added at the described concentrations directly to the medium (DMEM) containing CREF-Tax cells. The cells were incubated for the specified lengths of time and then exposed to the described genotoxic conditions.
RESULTS
Genotoxic stress and cellular stress alter the subcellular localization of Tax. Tax is known to shuttle between the nucleus and cytoplasm; however, the mechanisms and cellular conditions that regulate Tax distribution remain unknown. Since Tax has been shown to promote genome instability in response to DNA damage (20, 28), we examined the effects of genotoxic stress on the subcellular distribution and cellular interacting partners of Tax. Immunofluorescent microscopy was used to examine the effects of UV irradiation on the subcellular distribution of Tax in asynchronously growing CREF-Tax, C81-66, and HuT102 cells. Consistent with previous reports (4, 6, 51), Tax was located predominantly in nuclear TSS in the absence of genotoxic stress in each of these cell lines (Fig. 1A, C, and E). However, UV irradiation (30 J/m2) followed by a 30-minute recovery period resulted in increased cytoplasmic Tax and decreased TSS (Fig. 1B, D, and F). Ten minutes after UV irradiation, most CREF-Tax cells (95.7% ± 0.8%) demonstrated Tax localization predominantly in TSS (Fig. 1G). The percentage of cells expressing TSS decreased until approximately 30 min after UV irradiation, when most cells (94.3% ± 1.4%) demonstrated predominantly cytoplasmic Tax and a significant decrease (>1 standard deviation from the mean of 85 TSS/cell) in the number of TSS (Fig. 1G). Predominantly cytoplasmic Tax distribution was maintained until approximately 8 h after UV irradiation, after which TSS had re-formed in the majority (72.9% ± 6.9%) of cells (Fig. 1G). The temporal and spatial distributions of Tax were consistent in each cell line tested, and the rate of translocation was consistent with those of other proteins known to undergo nuclear export following UV irradiation (10). To confirm these results, immunoblot analysis of nuclear and cytoplasmic fractions of unirradiated and UV-irradiated CREF-Tax cells showed a 2.9-fold increase in cytoplasmic Tax and a 50% decrease in nuclear Tax following UV irradiation (data not shown).
Lymphocytes are susceptible to a variety of genotoxic and cellular stresses, including oxidative and osmotic stress; various forms of DNA damage, including single- and double-stranded DNA breaks; and induction of bulky DNA adducts (2, 8, 16, 25, 30, 48). To determine whether genotoxic and cellular stresses other than UV irradiation could induce nucleocytoplasmic shuttling of Tax, Tax-expressing cells (CREF-Tax, C81-66, and HuT102) were exposed to ionizing radiation, which induces double-stranded DNA breaks; hydrogen peroxide, which induces oxidative damage; or sodium arsenite, which induces clastogenic mutations and aneuploidy (Table 1). In a manner similar to that of UV irradiation, each of these genotoxic stressors increased the amount of cytoplasmic Tax and decreased the number and intensity of nuclear TSS (Table 1).
Since multiple DNA-damaging agents altered Tax localization in similar ways, the effects of other types of cellular stress on Tax distribution were examined (Table 1). The effects of sorbitol, which causes osmotic stress, of anisomycin, an inhibitor of protein translation and activator of mitogen-activated protein kinase (MAPK) signaling pathways, or of heat shock, a destabilizer of tertiary protein conformation on Tax localization, were similar to the effects observed following UV and ionizing irradiation (Table 1). Cytoplasmic localization of Tax was stress dependent, because agents that do not induce stress (diluted media, ethanol, and MAPK-specific inhibitors SB203580, SP600125, and PD098059) did not affect the subcellular distribution of Tax. These results demonstrate that Tax localization is affected by genotoxic and cellular stress and that altered Tax localization is a consistent outcome following exposure to each form of stress examined.
Translation inhibition does not affect stress-induced Tax redistribution. The decrease in nuclear Tax and accumulation of cytoplasmic Tax observed following stress could result from Tax export out of the nucleus or from localized Tax degradation in the nucleus with concomitant Tax synthesis in the cytoplasm. To determine if new translation contributed to the appearance of Tax in the cytoplasm, CREF-Tax cells were treated with the translational inhibitor CHX for 30 minutes prior to either mock or UV irradiation, and Tax localization was examined by immunofluorescent microscopy. CHX treatment alone did not affect Tax localization (compare Fig. 2A [94.8% ± 2.2% cells express TSS] and C [94.3% ± 1.2% cells express TSS]). Importantly, CHX treatment did not reduce the accumulation of Tax in the cytoplasm of UV-irradiated cells (compare Fig. 2B and D). Consistent with our previous results (Fig. 1), very few (6.9% ± 2.3% mock-treated [Fig. 2B] versus 7.1% ± 1.2% CHX-treated [Fig. 2D]) UV-irradiated cells expressed TSS (Fig. 2E). These results indicate that new protein synthesis is not required for Tax to accumulate in the cytoplasm following stress and suggest that cytoplasmic Tax results from Tax transport out of the nucleus after stress.
To confirm that CHX treatment did in fact inhibit translation, Tax expression in CREF-Tax cells was examined by immunoblot analysis in the presence and absence of CHX and UV irradiation. Total Tax protein expression remained constant following UV irradiation (Fig. 2F). Since the half-life of Tax has been reported to be 15 h (21), CHX treatment caused a decrease in Tax protein at 5 and 7 h, which confirmed the inhibition of translation by CHX in this experiment (Fig. 2G). When translation was inhibited, similar levels of Tax protein expression were observed in the presence and absence of UV irradiation (compare Fig. 2H and G). In summary, UV irradiation had no apparent effect on Tax degradation or translation, indicating that cytoplasmic accumulation of Tax following stress is due to increased Tax export rather than nuclear degradation of Tax coupled with enhanced cytoplasmic translation.
Tax localization is not affected by cell cycle progression. Tax was recently shown to interfere with the G1/S-phase cell cycle checkpoint that is typically induced in response to UV irradiation (20). Tax can also interact with and affect the activity of nuclear and cytoplasmic proteins that regulate normal cell cycle progression (26, 29, 37, 38). Therefore, we wanted to determine whether normal cell cycle progression was associated with any changes in Tax localization. To address this question, CREF-Tax cells were synchronized in the quiescent (G0) stage of the cell cycle by contact inhibition. Cells were released into the cell cycle by splitting into new plates containing sterile coverslips. At specific time points following G0-phase release, the coverslips were stained for Tax. Cells remaining on the dish were collected for propidium iodide staining and cell cycle analysis by flow cytometry. No difference in subcellular Tax distributions was observed in cells enriched in the G0/G1, S, or G2/M phases of the cell cycle (Fig. 3A to C). In each phase of the cell cycle, nearly all cells (>99%) showed a predominantly nuclear distribution of Tax, demonstrating that Tax localization is not affected during normal cell cycle progression.
Cellular stress affects the colocalization of Tax and sc35. Since Tax localization is altered in response to various forms of stress, we wanted to determine whether proteins remain associated with TSS, dissociate, or translocate with Tax to the cytoplasm. sc35, a splicing cofactor that has been reported to interact and colocalize with Tax in TSS, is commonly used as a marker of these structures (51). As previously reported for other cell lines (4, 6, 51), sc35 and Tax colocalize in asynchronous cells under normal cellular conditions (Fig. 4A to C). Consistent with previous data demonstrating the heterogeneous nature of TSS (51), Tax foci and sc35 foci that do not colocalize were readily identified (Fig. 4C).
We next examined of the effect of genotoxic stress on the colocalization of Tax and sc35. As shown in Fig. 1, Tax rapidly translocated to the cytoplasm following UV irradiation (Fig. 4E), while sc35 localization remained nuclear (Fig. 4D). As a result, a marked decrease in the number of overlapping foci was observed in the merged image following UV irradiation (Fig. 4F), suggesting a reduced interaction between Tax and sc35 following stress. These results are consistent with the previous report that heat shock disrupts the interaction between Tax and sc35 (51). When TSS re-formed 8 hours after UV irradiation (Fig. 1G), the percentage of speckles containing both Tax and sc35 (48.8% ± 4.3%) was similar to that seen in unirradiated cells (51.3% ± 0.8%). These results were consistent for each type of stress examined in this study (data not shown), suggesting that genotoxic stress and cellular stress affect both the localization of Tax and the interactions of Tax with cellular proteins.
Stress-induced changes in Tax localization are LMB sensitive. The functions and activities of many cellular and viral proteins depend upon their subcellular distributions (23). Nuclear export of protein macromolecules is regulated by interactions with specific nuclear export proteins. These interactions are controlled by NES located in the cargo protein (18, 44). A leucine-rich NES characterized by the canonical sequence LX2-3(L/I/M/V/F)X2-3LX(L/I), where X is any amino acid, was recently identified at amino acids 188 to 202 of Tax (1, 12, 57). In most cases, nuclear export of leucine-rich NES-containing proteins is regulated through an interaction with the nuclear export factor CRM1 (11, 14, 15, 53). When conjugated to green fluorescent protein, export of the Tax NES is CRM1 dependent. However, full-length Tax protein does not utilize this pathway under normal conditions, leading to speculation that the Tax NES is conditionally masked and that a conformational change in the protein, either through posttranslational modification or through protein interactions, is required to expose the NES (1).
To test the possibility that stress-induced changes in Tax localization involved a CRM1-dependent pathway, cells were pretreated with a CRM1 inhibitor (LMB) that interacts with CRM1 and prevents its binding to the NES of the cargo protein (31, 32). Proteins that require the CRM1 nuclear export pathway remain sequestered in the nucleus of LMB-treated cells. To examine the effect of LMB on Tax localization, CREF-Tax cells were pretreated with LMB or a control for 1 hour before exposure to genotoxic agents. Pretreatment of cells with an ethanol control did not affect the typical distribution of Tax in the absence (Fig. 5A) or presence (Fig. 5B) of stress. Consistent with previous studies (1), LMB alone had no obvious effect on Tax localization (Fig. 5C), suggesting that the normal distribution of Tax between the nucleus and cytoplasm of undamaged cells does not involve the CRM1 pathway. However, pretreatment of cells with LMB blocked the expected increase in cytoplasmic Tax following genotoxic stress. Instead, Tax remained in nuclear speckles (Fig. 5D). Consistent with our previous data, after a 30-min recovery period, most (99.3% ± 0.5%) unstressed and only a small percentage (7.3% ± 2.3%) of UV-irradiated CREF-Tax cells expressed TSS. In contrast, similar percentages of cells expressing TSS were observed in unirradiated (99.2% ± 0.4%) and UV-irradiated (97.8% ± 1.4%) CREF-Tax cells pretreated with LMB (Fig. 5E). Similar results were observed for each type of stress examined (data not shown), demonstrating that Tax translocation following genotoxic and cellular stress is LMB sensitive and suggesting that the CRM1 nuclear export pathway is involved in this process.
The Tax NES is required for stress-induced nucleocytoplasmic translocation. We next wanted to map the region of Tax responsible for its redistribution following genotoxic or cellular stress. Leucine or isoleucine residues at specific amino acid positions within the NES are required for its function, since a mutation within the Tax NES (L200A) resulted in exclusive nuclear localization (1). The Tax M33 mutant (52) contains two mutations within the Tax NES (S199A and L200S), which we predicted would eliminate NES function. We used this mutant to determine whether the Tax NES was required for redistribution of Tax following UV irradiation. Since we do not have a cell line that stably expresses the Tax M33 mutant, these studies were performed using transient expression. Wild-type Tax was first transiently expressed in 293 cells to determine whether the effect of UV irradiation on Tax localization was similar to that previously observed in cell lines that stably expressed Tax. Consistent with our previous results (Fig. 1), transfection of a Tax expression vector into 293 cells resulted in predominant nuclear localization in TSS (85.3% ± 1.6% of Tax-expressing cells displayed TSS; Fig. 6A and G). Thirty minutes after UV irradiation, fewer cells displayed TSS (20.5% ± 1.8%), and a significant increase in cytoplasmic Tax was observed (Fig. 6B and G). In contrast, the percentages of 293 cells transfected with the M33 Tax mutant that expressed TSS were similar in the presence (81.4% ± 2.2%; Fig. 6D) and absence (81.5% ± 1.5%; Fig. 6C) of UV irradiation. The localization of another Tax mutant which does not contain mutations in the NES, M47 (L319R, L320S), was similar to that of wild-type Tax (Fig. 6E and F). That is, most (82.8% ± 2.1%) unstressed and fewer (27.0% ± 4.1%) UV-irradiated 293 cells expressing the M47 Tax mutant demonstrated TSS (Fig. 6G). These results suggest that the NES regulates Tax nuclear export in response to genotoxic stress.
Tax and CRM1 interact following stress. As previously discussed, the Tax NES may be conditionally masked. Revealing the NES may require changes in protein interactions or posttranslational modifications (1). The CRM1 nuclear export pathway relies on a direct interaction between CRM1 and the NES of the cargo protein (14, 15, 53), which, together with the transport cofactor RanGTP, form a trimeric complex that is exported through the nuclear pore in an energy-dependent manner (18, 44). To examine the potential interaction of Tax with CRM1 in response to cellular stress, whole-cell lysates were prepared from Tax-expressing 293 cells that were either undamaged, UV irradiated (30 J/m2, 20-min recovery), or treated with sorbitol (850 mM for 35 min) or sodium arsenite (1 mM for 35 min). These lysates were immunoprecipitated using anti-CRM1 (Fig. 7A) or anti-Tax (Fig. 7B) antibodies and subsequently immunoblotted for Tax and CRM1. No interaction between Tax and CRM1 was detected in undamaged cells (Fig. 7A and B, lanes 1); however, a complex containing Tax and CRM1 was detected following stress (Fig. 7A and B, lanes 2 to 4). Similar results were observed when CREF-Tax cell extracts were used (data not shown).
To determine whether the Tax NES itself was responsible for the interaction between Tax and CRM1, similar experiments were performed using extracts from 293 cells transfected with a Tax M33 expression vector. As predicted, no interaction between the NES-defective M33 mutant and CRM1 was detected in the absence (Fig. 7A and B, lanes 5) or presence (Fig. 7A and B, lanes 6 to 8) of stress. These results, combined with results from the LMB and Tax mutant localization experiments, demonstrate that the CRM1 nuclear export pathway is involved in Tax nucleocytoplasmic translocation following cellular stress and confirm that the Tax NES is required for this process. Since Tax and CRM1 interact after, but not before, stress, a change in protein conformation, interactions, or posttranslational modifications is likely to make the Tax NES accessible to CRM1.
Tax dissociates from sc35 before interacting with CRM1. We previously showed that Tax interacts with CRM1 following stress. To determine whether Tax dissociates from TSS before interacting with CRM1 or whether the interaction between Tax and CRM1 facilitates the separation of Tax from TSS, we examined the composition of Tax speckles in response to LMB treatment and UV irradiation. If Tax dissociates from TSS before interacting with CRM1, then colocalization of Tax and sc35 should not be seen following LMB treatment and UV irradiation. Alternatively, since LMB blocks the ability of CRM1 to interact with Tax, if an interaction between Tax and CRM1 is required to dissociate Tax from TSS, then Tax should remain associated with sc35 following UV irradiation.
CREF-Tax cells were pretreated with 10 nM LMB for 1 h, exposed to 30 J/m2 of UV irradiation or mock treated, and allowed to recover for 30 min. As expected, in the presence of LMB both sc35 (Fig. 8D) and Tax (Fig. 8E) were retained in nuclear speckles following UV irradiation. In contrast to unirradiated cells, which showed extensive colocalization of these two proteins (Fig. 8C), UV-irradiated cells showed significantly less colocalization of Tax and sc35 (Fig. 8F). UV irradiation induced a significant increase (P < 0.05) in the number of nuclear speckles containing only Tax (22.0% ± 1.4% unirradiated; 41.4% ± 1.7% irradiated) or only sc35 (18.3% ± 0.7% unirradiated; 51.1% ± 1.1% UV irradiated) and a significant decrease (P < 0.05) in the number of speckles containing both Tax and sc35 (59.7% ± 2.1% unirradiated; 7.5% ± 0.6% UV irradiated) (Fig. 8G). These results, together with the observation that M33 and sc35 do not colocalize following stress (Fig. 6D), suggest that stress causes Tax to be released from TSS, which allows it to interact with CRM1, and is required for nuclear export of Tax.
DISCUSSION
The pleiotropic HTLV-1 Tax protein is known to interact with a number of cellular proteins to affect a variety of cellular processes. Understanding how these interactions and functions are regulated within the cell remains a subject of intense interest. This study examined the effects of genotoxic and cellular stress on the subcellular distribution of Tax. The dramatic redistribution of Tax in response to various forms of stress is characterized by a transient increase in cytoplasmic Tax and a decrease in the number and intensity of TSS (Fig. 1). Mutation of the NES in the carboxy terminus of Tax (M33) demonstrated that this sequence is required for nuclear export. We further demonstrated that Tax utilizes the CRM1 nuclear export pathway to facilitate nucleocytoplasmic translocation in response to stress by demonstrating an interaction between Tax and CRM1 that is present only in response to stress (Fig. 7). Pretreatment of CREF-Tax cells with the CRM1-specific inhibitor LMB inhibited the cytoplasmic translocation of Tax in response to stress, confirming that changes in Tax localization following genotoxic and cellular stress occur through the CRM1 nuclear export pathway (Fig. 5).
It was previously suggested that the Tax NES is not accessible and that posttranslational modification of Tax, or changes in its protein partners, may be required to expose the NES (1). We demonstrated that Tax and sc35 fail to colocalize (Fig. 8) following UV irradiation. This result was confirmed using the NES-defective Tax mutant, M33, which also failed to colocalize with sc35 following UV irradiation (Fig. 6). These results suggest that in response to stress, Tax dissociates from TSS without requiring an interaction with CRM1. However, the release of Tax from TSS does allow Tax to interact with CRM1, which then facilitates the nuclear export of Tax following exposure to genotoxic or cellular stress.
Tax has been shown to be posttranslationally ubiquitinated and phosphorylated, and these modifications of Tax and/or other TSS proteins are likely to regulate Tax localization. Posttranslational phosphorylation or ubiquitination has been shown to regulate nuclear export of other cellular proteins through the CRM1 pathway (5, 7, 13, 24, 39, 47). Sumoylation and ubiquitination of Tax at specific residues have recently been shown to play an integral role in regulating the subcellular distribution and functions of Tax (33, 43). It is also possible that posttranslational modification of one or more Tax-interacting proteins in response to stress may cause them to dissociate from Tax and enable Tax to interact with CRM1. The specific role of these mechanisms in regulating the nuclear export of Tax remains to be determined.
Genotoxic and cellular stressors, including each of those utilized in this study (Table 1), have been shown to induce MAPK signaling pathways (10, 46, 50). Tax was not able to translocate to the cytoplasm in the presence of the MAPK inhibitors (Table 1). Since many other proteins known to undergo nucleocytoplasmic shuttling in response to stress require the activity of these pathways (10), the activation of MAPK signaling may be important for regulating Tax localization in response to stress. However, additional studies will be required to identify the pathway and mechanisms responsible for this effect.
Although a number of studies have demonstrated that Tax functions both in the nucleus and in the cytoplasm, its cytoplasmic functions in particular remain to be fully elucidated. Cytoplasmic Tax has been shown to modulate the activity of a number of cellular proteins and pathways. In particular, Tax has been shown to interact with and target the retinoblastoma protein for proteasomal degradation and to enhance activation of the NF-B pathway through a variety of mechanisms, including activation of upstream kinases, interaction with and targeting of NF-B inhibitors for proteasomal degradation, and direct interaction with the NF-B transcription factor p65/RelA (3, 22, 54, 56, 60). Tax mutants that are deficient for cytoplasmic functions display a reduced transformation capacity (59), and the Tax M33 mutant, which is defective for nuclear export, is 50% less effective at repressing DNA repair than wild-type Tax (34). Therefore, transient cytoplasmic localization of Tax may interfere with the host response to genotoxic or cellular stress and have important implications for cellular transformation.
Tax-expressing cells do not efficiently repair DNA damage by either the nucleotide excision or the base excision DNA repair pathways (28, 49). In addition, Tax expression is associated with defects in cell cycle checkpoints (20). Together, these effects are believed to enhance a cell's propensity to accumulate mutations. Since lymphocytes are exposed to genotoxic and cellular stress on a regular basis (2, 8, 16, 25, 30, 48), HTLV-1-infected lymphocytes are likely to be susceptible to the introduction of mutations due to unrepaired DNA lesions. Indeed, Tax-expressing cells demonstrate higher gene amplification levels and higher mutation frequency than normal cells (36, 42). HTLV-1-infected cells and cells isolated from ATL patients also contain a large number of chromosomal abnormalities, including deletions, duplications, and translocations (17). Collectively, these results suggest that unrepaired DNA damage in Tax-expressing cells may contribute to cellular transformation and that changes in Tax localization due to genotoxic and cellular stress may enhance this process.
In summary, this study demonstrates that exposure of Tax-expressing cells to genotoxic or other forms of stress alters the subcellular distribution of Tax. Altered Tax localization may contribute to the enhanced mutation frequency observed in Tax-expressing cells by affecting DNA repair mechanisms and/or cell cycle progression and may ultimately play an important role in generating chromosomal abnormalities characteristic of HTLV-1 Tax-transformed cells and lymphocytes from ATL patients. Future studies to specifically address the biological consequences of Tax localization will likely reveal important insights into cellular transformation by this viral oncoprotein.
ACKNOWLEDGMENTS
We thank Ian Cushman (Duke University) and Mary Moore (Baylor College of Medicine) for their insights into nuclear export as well as Catherine Gatza (Baylor College of Medicine) and the members of the Marriott laboratory for helpful suggestions and editorial comments. We also acknowledge the AIDS Research and Reference Reagent Program for providing anti-Tax antibodies.
This study was supported, in part, by U.S. Public Service grant CA-77371 from the National Cancer Institute, National Institutes of Health, awarded to S.J.M. M.L.G. is supported in part by National Institutes of Health Training Grant CA-09197 and a Sigma Xi Grant-in-Aid of Research award.
REFERENCES
Alefantis, T., K. Barmak, E. W. Harhaj, C. Grant, and B. Wigdahl. 2003. Characterization of a nuclear export signal within the human T cell leukemia virus type I transactivator protein Tax. J. Biol. Chem. 278:21814-21822.
Andreoli, C., P. Leopardi, S. Rossi, and R. Crebelli. 1999. Processing of DNA damage induced by hydrogen peroxide and methyl methanesulfonate in human lymphocytes: analysis by alkaline single cell gel electrophoresis and cytogenetic methods. Mutagenesis 14:497-504.
Azran, I., K. T. Jeang, and M. Aboud. 2005. High levels of cytoplasmic HTLV-1 Tax mutant proteins retain a Tax-NF-kappaB-CBP ternary complex in the cytoplasm. Oncogene 24:4521-4530.
Bex, F., A. McDowall, A. Burny, and R. Gaynor. 1997. The human T-cell leukemia virus type 1 transactivator protein Tax colocalizes in unique nuclear structures with NF-B proteins. J. Virol. 71:3484-3497.
Bex, F., K. Murphy, R. Wattiez, A. Burny, and R. B. Gaynor. 1999. Phosphorylation of the human T-cell leukemia virus type 1 transactivator Tax on adjacent serine residues is critical for Tax activation. J. Virol. 73:738-745.
Burton, M., C. D. Upadhyaya, B. Maier, T. J. Hope, and O. J. Semmes. 2000. Human T-cell leukemia virus type 1 Tax shuttles between functionally discrete subcellular targets. J. Virol. 74:2351-2364.
Chiari, E., I. Lamsoul, J. Lodewick, C. Chopin, F. Bex, and C. Pique. 2004. Stable ubiquitination of human T-cell leukemia virus type 1 Tax is required for proteasome binding. J. Virol. 78:11823-11832.
Courtemanche, C., A. C. Huang, I. Elson-Schwab, N. Kerry, B. Y. Ng, and B. N. Ames. 2004. Folate deficiency and ionizing radiation cause DNA breaks in primary human lymphocytes: a comparison. FASEB J. 18:209-211.
Dignam, J. D., R. M. Lebowitz, and R. G. Roeder. 1983. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11:1475-1489.
Ducret, C., S. M. Maira, A. Dierich, and B. Wasylyk. 1999. The net repressor is regulated by nuclear export in response to anisomycin, UV, and heat shock. Mol. Cell. Biol. 19:7076-7087.
Elfgang, C., O. Rosorius, L. Hofer, H. Jaksche, J. Hauber, and D. Bevec. 1999. Evidence for specific nucleocytoplasmic transport pathways used by leucine-rich nuclear export signals. Proc. Natl. Acad. Sci. USA 96:6229-6234.
Fischer, U., J. Huber, W. C. Boelens, I. W. Mattaj, and R. Luhrmann. 1995. The HIV-1 Rev activation domain is a nuclear export signal that accesses an export pathway used by specific cellular RNAs. Cell 82:475-483.
Fontes, J. D., J. M. Stawhecker, N. D. Bills, R. E. Lewis, and S. H. Hinrichs. 1993. Phorbol esters modulate the phosphorylation of human T-cell leukemia virus type I Tax. J. Virol. 67:4436-4441.
Fornerod, M., M. Ohno, M. Yoshida, and I. W. Mattaj. 1997. CRM1 is an export receptor for leucine-rich nuclear export signals. Cell 90:1051-1060.
Fukuda, M., S. Asano, T. Nakamura, M. Adachi, M. Yoshida, M. Yanagida, and E. Nishida. 1997. CRM1 is responsible for intracellular transport mediated by the nuclear export signal. Nature 390:308-311.
Gabbianelli, R., C. Nasuti, G. Falcioni, and F. Cantalamessa. 2004. Lymphocyte DNA damage in rats exposed to pyrethroids: effect of supplementation with vitamins E and C. Toxicology 203:17-26.
Gatza, M. L., C. Chandhasin, R. I. Ducu, and S. J. Marriott. 2005. Impact of transforming viruses on cellular mutagenesis, genome stability, and cellular transformation. Environ. Mol. Mutagen. 45:304-325.
Gorlich, D. 1998. Transport into and out of the cell nucleus. EMBO J. 17:2721-2727.
Haoudi, A., R. C. Daniels, E. Wong, G. Kupfer, and O. J. Semmes. 2003. Human T-cell leukemia virus-I tax oncoprotein functionally targets a subnuclear complex involved in cellular DNA damage-response. J. Biol. Chem. 278:37736-37744.
Haoudi, A., and O. J. Semmes. 2003. The HTLV-1 tax oncoprotein attenuates DNA damage induced G1 arrest and enhances apoptosis in p53 null cells. Virology 305:229-239.
Hemelaar, J., F. Bex, B. Booth, V. Cerundolo, A. McMichael, and S. Daenke. 2001. Human T-cell leukemia virus type 1 Tax protein binds to assembled nuclear proteasomes and enhances their proteolytic activity. J. Virol. 75:11106-11115.
Hirai, H., T. Suzuki, J.-I. Fujisawa, J. Inoue, and M. Yoshida. 1994. Tax protein of human T-cell leukemia virus type I binds to the ankyrin motifs of inhibitory factor kappaB and induces nuclear translocation of transcription factor NF-kappaB proteins for transcriptional activation. Proc. Natl. Acad. Sci. USA 91:3584-3588.
Hood, J. K., and P. A. Silver. 1999. In or out Regulating nuclear transport. Curr. Opin. Cell Biol. 11:241-247.
Ishida, N., T. Hara, T. Kamura, M. Yoshida, K. Nakayama, and K. I. Nakayama. 2002. Phosphorylation of p27Kip1 on serine 10 is required for its binding to CRM1 and nuclear export. J. Biol. Chem. 277:14355-14358.
Jaloszynski, P., M. Kujawski, M. Wasowicz, R. Szulc, and K. Szyfter. 1999. Genotoxicity of inhalation anesthetics halothane and isoflurane in human lymphocytes studied in vitro using the comet assay. Mutat. Res. 439:199-206.
Jin, D. Y., F. Spencer, and K. T. Jeang. 1998. Human T cell leukemia virus type 1 oncoprotein Tax targets the human mitotic checkpoint protein MAD1. Cell 93:81-91.
Kao, S. Y., F. J. Lemoine, and S. J. Marriott. 2001. p53-independent induction of apoptosis by the human T cell leukemia virus type I Tax protein following UV irradiation. Virology 291:292-298.
Kao, S. Y., and S. J. Marriott. 1999. Disruption of nucleotide excision repair by the human T-cell leukemia virus type 1 Tax protein. J. Virol. 73:4299-4304.
Kehn, K., C. L. Fuente, K. Strouss, R. Berro, H. Jiang, J. Brady, R. Mahieux, A. Pumfery, M. E. Bottazzi, and F. Kashanchi. 2005. The HTLV-I Tax oncoprotein targets the retinoblastoma protein for proteasomal degradation. Oncogene 24:525-540.
Kim, C. H., and N. D. Vaziri. 2005. Hypertension promotes integrin expression and reactive oxygen species generation by circulating leukocytes. Kidney Int. 67:1462-1470.
Kudo, N., N. Matsumori, H. Taoka, D. Fujiwara, E. P. Schreiner, B. Wolff, M. Yoshida, and S. Horinouchi. 1999. Leptomycin B inactivates CRM1/exportin 1 by covalent modification at a cysteine residue in the central conserved region. Proc. Natl. Acad. Sci. USA 96:9112-9117.
Kudo, N., B. Wolff, T. Sekimoto, E. P. Schreiner, Y. Yoneda, M. Yanagida, S. Horinouchi, and M. Yoshida. 1998. Leptomycin B inhibition of signal-mediated nuclear export by direct binding to CRM1. Exp. Cell Res. 242:540-547.
Lamsoul, I., J. Lodewick, S. Lebrun, R. Brasseur, A. Burny, R. Gaynor, and F. Bex. 2005. Exclusive ubiquitination and sumoylation on overlapping lysine residues mediate NF-B activation by the human T-cell leukemia virus Tax oncoprotein. Mol. Cell. Biol. 25:10391-10406.
Lemoine, F. J., S. Y. Kao, and S. J. Marriott. 2000. Suppression of DNA repair by HTLV-I Tax correlates with Tax transactivation of PCNA gene expression. AIDS Res. Hum. Retrovir. 16:1623-1627.
Lemoine, F. J., and S. J. Marriott. 2001. Accelerated G1 phase progression induced by the human T cell leukemia virus type I (HTLV-I) Tax oncoprotein. J. Biol. Chem. 276:31851-31857.
Lemoine, F. J., and S. J. Marriott. 2002. Genomic instability driven by the human T-cell leukemia virus type I (HTLV-I) oncoprotein, Tax. Oncogene 21:7230-7234.
Li, J., H. Li, and M. D. Tsai. 2003. Direct binding of the N-terminus of HTLV-1 Tax oncoprotein to cyclin-dependent kinase 4 is a dominant path to stimulate the kinase activity. Biochemistry 42:6921-6928.
Liang, M. H., T. Geisbert, Y. Yao, S. H. Hinrichs, and C. Z. Giam. 2002. Human T-lymphotropic virus type I oncoprotein Tax promotes S-phase entry but blocks mitosis. J. Virol. 76:4022-4033.
Lohrum, M. A., D. B. Woods, R. L. Ludwig, E. Balint, and K. H. Vousden. 2001. C-terminal ubiquitination of p53 contributes to nuclear export. Mol. Cell. Biol. 21:8521-8532.
Majone, F., and K. T. Jeang. 2000. Clastogenic effect of the human T-cell leukemia virus type I Tax oncoprotein correlates with unstabilized DNA breaks. J. Biol. Chem. 275:32906-32910.
Majone, F., O. J. Semmes, and K.-T. Jeang. 1993. Induction of micronuclei by HTLV-I Tax: a cellular assay for function. Virology 193:456-459.
Miyake, H., T. Suzuki, H. Hirai, and M. Yoshida. 1999. Trans-activator Tax of human T-cell leukemia virus type 1 enhances mutation frequency of the cellular genome. Virology 253:155-161.
Nasr, R., E. Chiari, M. El-Sabban, R. Mahieux, Y. Kfoury, V. Yazbeck, O. Hermine, H. De The, C. Pique, and A. Bazarbachi. 19 January 2006, posting date. Tax ubiquitylation and sumoylation control critical cytoplasmic and nuclear steps of NF-kB activation. Blood [Online.] doi:10.1182/blood-2005-09-3572.
Nigg, E. A. 1997. Nucleocytoplasmic transport: signals, mechanisms and regulation. Nature 386:779-787.
Osborn, L., S. Kunkel, and G. J. Nabel. 1989. Tumor necrosis factor alpha and interleukin 1 stimulate the human immunodeficiency virus enhancer by activation of the nuclear factor kappa B. Proc. Natl. Acad. Sci. USA 86:2336-2340.
Pearson, G., F. Robinson, T. Gibson, B. Xu, M. Karandikar, K. Berman, and M. Cobb. 2001. Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions. Endocrinol. Rev. 22:153-183.
Peloponese, J. M., Jr., H. Iha, V. R. Yedavalli, A. Miyazato, Y. Li, K. Haller, M. Benkirane, and K. T. Jeang. 2004. Ubiquitination of human T-cell leukemia virus type 1 Tax modulates its activity. J. Virol. 78:11686-11695.
Peluso, M., M. Neri, G. Margarino, C. Mereu, A. Munnia, M. Ceppi, M. Buratti, R. Felletti, F. Stea, R. Quaglia, R. Puntoni, E. Taioli, S. Garte, and S. Bonassi. 2004. Comparison of DNA adduct levels in nasal mucosa, lymphocytes and bronchial mucosa of cigarette smokers and interaction with metabolic gene polymorphisms. Carcinogenesis 25:2459-2465.
Philpott, S. M., and G. C. Buehring. 1999. Defective DNA repair in cells with human T-cell leukemia bovine leukemia viruses: role of tax gene. J. Natl. Cancer Inst. 91:933-942.
Roux, P. P., and J. Blenis. 2004. ERK and p38 MAPK-activated protein kinases: a family of protein kinases with diverse biological functions. Microbiol. Mol. Biol. Rev. 68:320-344.
Semmes, O. J., and K. T. Jeang. 1996. Localization of human T-cell leukemia virus type 1 Tax to subnuclear compartments that overlap with interchromatin speckles. J. Virol. 70:6347-6357.
Smith, M. R., and W. C. Greene. 1990. Identification of HTLV-I tax trans-activator mutants exhibiting novel transcriptional phenotypes. Genes Dev. 4:1875-1885.
Stade, K., C. S. Ford, C. Guthrie, and K. Weis. 1997. Exportin 1 (Crm1p) is an essential nuclear export factor. Cell 90:1041-1050.
Suzuki, T., H. Hirai, T. Murakami, and M. Yoshida. 1995. Tax protein of HTLV-1 destabilizes the complexes of NF-kappaB and IkappaB-a and induces nuclear translocation of NF-kappaB for transcriptional activation. Oncogene 10:1199-1207.
Suzuki, T., T. Narita, M. Uchida-Toita, and M. Yoshida. 1999. Down-regulation of the INK4 family of cyclin-dependent kinase inhibitors by Tax protein of HTLV-1 through two distinct mechanisms. Virology 259:384-391.
Uhlik, M., L. Good, G. T. Xiao, E. W. Harhaj, E. Zandi, M. Karin, and S. C. Sun. 1998. NF-kappaB-inducing kinase and IkappaB kinase participate in human T-cell leukemia virus I Tax-mediated NF-kappaB activation. J. Biol. Chem. 273:21132-21136.
Wen, W., J. L. Meinkoth, R. Y. Tsien, and S. S. Taylor. 1995. Identification of a signal for rapid export of proteins from the nucleus. Cell 82:463-473.
Xiao, G., E. W. Harhaj, and S. C. Sun. 2000. Domain-specific interaction with the IB kinase (IKK) regulatory subunit IKK is an essential step in Tax-mediated activation of IKK. J. Biol. Chem. 275:34060-34067.
Yamaoka, S., H. Inoue, M. Sakurai, T. Sugiyama, M. Hazama, T. Yamada, and M. Hatanaka. 1996. Constitutive activation of NF-kB is essential for transformation of rat fibroblasts by the human T-cell leukemia virus type I Tax protein. EMBO J. 15:873-887.
Yin, M. J., L. B. Christerson, Y. Yamamoto, Y. T. Kwak, S. C. Xu, F. Mercurio, M. Barbosa, M. H. Cobb, and R. B. Gaynor. 1998. HTLV-I Tax protein binds to MEKK1 to stimulate IkappaB kinase activity and NF-kappaB activation. Cell 93:875-884.
Yoshida, M. 2001. Multiple viral strategies of HTLV-1 for dysregulation of cell growth control. Annu. Rev. Immunol. 19:475-496(Michael L. Gatza and Susa)
ABSTRACT
Human T-cell leukemia virus type 1 Tax is a predominantly nuclear viral oncoprotein that colocalizes with cellular proteins in nuclear foci known as Tax speckled structures (TSS). Tax is also diffusely distributed throughout the cytoplasm, where it interacts with and affects the functions of cytoplasmic cellular proteins. Mechanisms that regulate the distribution of Tax between the cytoplasm and nucleus remain to be identified. Since Tax has been shown to promote genome instability by perturbing cell cycle progression and DNA repair mechanisms following DNA damage, we examined the effect of genotoxic stress on the subcellular distribution and interacting partners of Tax. Tax localization was altered in response to various forms of cellular stress, resulting in an increase in cytoplasmic Tax and a decrease in Tax speckled structures. Concomitantly, colocalization of Tax with sc35 (a TSS protein) decreased following stress. Tax translocation required the CRM1 nuclear export pathway, and a transient interaction between Tax and CRM1 was observed following stress. These results suggest that the subcellular distribution of Tax and the interactions between Tax and cellular proteins respond dynamically to cellular stress. Changes in Tax distribution and interacting partners are likely to affect cellular processes that regulate cellular transformation.
INTRODUCTION
Human T-cell leukemia virus type 1 (HTLV-1) has been etiologically linked to the development of a rare but rapidly progressing and fatal malignancy, adult T-cell leukemia (ATL), as well as to chronic conditions including tropical spastic paraparesis/HTLV-1-associated myelopathy. The HTLV-1 genome encodes several proteins that contribute to infection and oncogenesis; however, Tax is the major viral oncoprotein (17). Tax has been shown to transcriptionally activate the promoter, located within the 5' long terminal repeat, and to regulate the transcriptional activity of cellular promoters by serving as a transcriptional cofactor for the CREB, NF-B, and SRF pathways (61). In addition to interacting with and altering the activity of cellular transcription factors, Tax has been shown to increase the genomic instability (36) and mutation frequency (40-42) of the host cell, in part by repressing DNA repair mechanisms (28, 40, 49) and dysregulating cell cycle progression (17).
Pleiotropic functions of Tax, which are propagated through interactions with cellular proteins, have been shown to contribute to cellular transformation. Tax is predominantly a nuclear protein that localizes, along with a number of cellular proteins, to heterogeneous nuclear foci known as Tax speckled structures (TSS). These structures contain a variety of cellular proteins, including transcription factors, splicing cofactors, and DNA damage recognition and cell cycle regulatory proteins (4, 19, 51, 55). Tax has been reported to interact with more than 20 cellular proteins, including a number of cytoplasmic proteins, such as MEKK1, MAD1, CBP, RelA, and IB kinase subunits, as well as other nuclear proteins that are not found in TSS, including p16INK4a and p15INK4b (3, 26, 58, 60). Interactions of Tax with these proteins have profound effects on normal host cell processes and in many cases have been shown to be essential for or to enhance cellular transformation.
TSS composition and Tax protein interactions are dynamic, and changes in these interactions due to cellular or other environmental cues are likely to enhance the oncogenic properties of Tax. The ability of Tax to shuttle between the nucleus and cytoplasm may contribute to its oncogenic activity by facilitating changes in interacting partners under certain conditions (6). Nuclear localization and nuclear export sequences (NES) have been identified within the Tax protein, and these sequences are believed to modulate the nucleocytoplasmic shuttling of Tax (1, 52). However, neither the condition(s) that regulates the cycling of Tax between the nucleus and the cytoplasm nor the mechanism of Tax export from the nucleus has been identified. Here we report that Tax translocates to the cytoplasm in response to genotoxic and cellular stress. This translocation results in altered interactions between Tax and cellular proteins. We further demonstrate that Tax translocation following stress requires the CRM1 (chromosome region maintenance 1) nuclear export pathway and that functional mutations in the Tax nuclear export sequence inhibit cytoplasmic translocation following stress. This study provides the first evidence that the localization and interacting partners of Tax are regulated in response to stress and identifies a mechanism and domain within Tax that is required for this translocation. Changes in Tax localization and its interactions with cellular proteins in response to genotoxic and cellular stresses may contribute to or enhance the oncogenic activity of Tax.
MATERIALS AND METHODS
Cell lines, plasmids, and transfections. Clonal rat embryo fibroblasts that stably express Tax (CREF-Tax) (27) and 293 cells were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum. C81-66, an HTLV-1-infected human T-cell line (51), and HuT102, an HTLV-1-producing lymphocyte line (ATCC, Manassas, VA), were maintained in RPMI medium containing 10% fetal bovine serum. All cells were grown at 37°C in 5% CO2. pCMV-Tax, pCMV-M33, and pCMV-M47 (52) have been previously described and were transfected into 293 cells by use of Fugene 6 (Roche, Indianapolis, IN) as described by the manufacturer.
Antibodies. Anti-Tax antibodies (Tab170 and 586) were previously described (AIDS Research and Reference Reagent Program, Germantown, MD). Anti-CRM1 antibodies (H-300 and C-20) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-sc35 antibody was purchased from BD-Pharmacia (San Diego, CA). Alexa-Fluor 594-conjugated goat anti-rabbit and Alexa-Fluor 488-conjugated goat anti-mouse secondary antibodies were purchased from Molecular Probes (Eugene, OR). Anti--tubulin and horseradish peroxidase-conjugated secondary antibodies were purchased from Sigma-Aldrich (St. Louis, MO).
Cellular stress. For UV irradiation, medium was removed from plates and cells were washed once with phosphate-buffered saline. A Stratalinker instrument (Stratagene, La Jolla, CA) was used to deliver 30 J/m2 of UV-C irradiation. The original medium was then replaced, and the cells were allowed to recover for a specified length of time. For ionizing radiation, cells were grown on 60-mm2 plates, exposed to 10 grays of radiation, and allowed to recover for a specified length of time. Sorbitol, anisomycin, sodium arsenite, hydrogen peroxide, SB203580, SP600125, and PD098059 were added directly to either DMEM or RPMI medium at the concentrations shown in Table 1. Cells were incubated in the presence of these agents for the specified lengths of time. For heat shock, cells were incubated at 42°C for 90 min.
Cell synchronization. Cells were synchronized in G0 phase as previously described (35). In short, CREF cells were seeded into 100-mm dishes at a density of 1 x 106 cells/ml. The cells were allowed to reach confluence and maintained at 100% confluence for 48 h. Cells were released from arrest by splitting them 1:10 into new 100-mm dishes with fresh medium.
Propidium iodide staining. Cells (1 x 106) were resuspended in 2 ml of 0.9% NaCl and then fixed with 5 ml of 95% ethanol. Fixed cells were incubated at room temperature for at least 30 min and stored at 4°C. For staining, the cell pellet was resuspended in 0.5 ml of 50-μg/ml propidium iodide (Sigma-Aldrich, St. Louis, MO) and 0.1 ml of 1-mg/ml RNase A (Sigma-Aldrich, St. Louis, MO). A flow cytometer (Epic Profile; Coulter, Colorado) was used to analyze cell cycle distribution. The percentage of cells in each phase of the cell cycle was determined using ModFit (Verity, Topsham, ME).
Cellular extracts. Whole-cell extracts were prepared as previously described (9, 45) with a few modifications. Briefly, 1 x 107 cells were resuspended in 20 ml of ice-cold buffer C (20 mM HEPES [pH 7.9], 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride [PMSF], 0.5 mM dithiothreitol [DTT]) plus 0.1% NP-40, incubated on ice for 10 min, and centrifuged for 10 min at 10,000 rpm at 4°C. The supernatant was diluted with 80 μl of ice-cold buffer D (20 mM HEPES [pH 7.9], 20% glycerol, 50 mM KCl, 0.2 mM EDTA, 0.5 mM PMSF, 0.5 mM DTT) per 1 x 107 cells.
Immunofluorescent staining. CREF-Tax cells were seeded on ethanol-washed coverslips and grown to approximately 50% confluence before exposure to genotoxic conditions. C81-66 and HuT102 cells were grown in suspension, exposed to genotoxic conditions, collected by centrifugation (1,000 rpm for 10 min), washed and resuspended in phosphate-buffered saline, and then cytospun (Shandon Cytospin3) onto ethanol-washed coverslips (1,000 rpm for 1 min). The cells were washed once with PEM buffer {80 mM potassium PIPES [piperazine-N,N'-bis(2-ethanesulfonic acid)] [pH 6.8], 5 mM EGTA [pH 7.0], 2 mM MgCl2} and fixed by incubation in 5% formaldehyde diluted in PEM buffer for 30 min at 4°C. To remove excess formaldehyde, cells were washed three times in PEM buffer and permeabilized by incubation in PEM buffer containing 0.5% Triton X-100 for 30 min at room temperature. Immunofluorescent staining was performed by incubation with primary antibody diluted in 2.5 to 5% bovine serum albumin containing TBS plus 0.1% Tween 20 (TBS-T) for a minimum of 3 h at room temperature. Excess antibody was removed by washing cells three times in TBS-T. Cells were incubated in the dark with a fluorophore-conjugated secondary antibody diluted in TBS-T for 40 min at room temperature. Excess antibody was removed by washing the coverslips three times with TBS-T. The cells were counterstained with DAPI (4',6'-diamidino-2-phenylindole) (Sigma-Aldrich, St. Louis, MO) to visualize the nucleus and mounted on slides using Slow-Fade antifade mounting medium (Molecular Probes, Eugene, OR). Cells were visualized with a Zeiss AxioPlan2 microscope by use of a CoolSnap HQ charge-coupled-device camera and analyzed using MetaView MetaMorph software. For deconvolved images (see Fig. 4), an Applied Precision microscope with SoftWoRx image restoration software was utilized.
Immunoprecipitation. Whole-cell extracts were prepared from 293 cells (1.5 x 107) that had been transfected with either pCMV-Tax or pCMV-M33. The extracts were incubated with either anti-Tax (568) or anti-CRM1 (H-300) antibodies diluted in incubation buffer (20 mM HEPES [pH 7.9], 75 mM KCl, 2.5 mM MgCl2, 1 mM DTT, 0.1% NP-40, 0.5 mM PMSF, 1 μg/ml aprotinin, 1 μg/ml pepstatin, 2 μg/ml leupeptin, 1 mM sodium orthovanadate) overnight at 4°C on a rotator. Thirty microliters of a 50% protein G-bead slurry (Upstate, Lake Placid, NY) was added to the mixture, and the mixture was incubated for 90 min at 4°C. Beads were collected by centrifugation, washed five times with 500 μl of incubation buffer, and resuspended in 100 μl sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample buffer. Samples were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10%), transferred to a nitrocellulose membrane, and incubated with either anti-Tax (Tab170) or anti-Crm1 (C-20) antibodies. The membrane was then incubated with the appropriate horseradish peroxidase-conjugated secondary antibody.
Leptomycin B treatment. Leptomycin B (LMB) (LC Laboratories, Woburn, MA) was resuspended in absolute ethanol and added at the described concentrations directly to the medium (DMEM) containing CREF-Tax cells. The cells were incubated at 37°C for 1 h and then exposed to the specified genotoxic conditions.
CHX treatment. Cycloheximide (CHX) (Sigma-Aldrich, St. Louis, MO) was resuspended in absolute ethanol and added at the described concentrations directly to the medium (DMEM) containing CREF-Tax cells. The cells were incubated for the specified lengths of time and then exposed to the described genotoxic conditions.
RESULTS
Genotoxic stress and cellular stress alter the subcellular localization of Tax. Tax is known to shuttle between the nucleus and cytoplasm; however, the mechanisms and cellular conditions that regulate Tax distribution remain unknown. Since Tax has been shown to promote genome instability in response to DNA damage (20, 28), we examined the effects of genotoxic stress on the subcellular distribution and cellular interacting partners of Tax. Immunofluorescent microscopy was used to examine the effects of UV irradiation on the subcellular distribution of Tax in asynchronously growing CREF-Tax, C81-66, and HuT102 cells. Consistent with previous reports (4, 6, 51), Tax was located predominantly in nuclear TSS in the absence of genotoxic stress in each of these cell lines (Fig. 1A, C, and E). However, UV irradiation (30 J/m2) followed by a 30-minute recovery period resulted in increased cytoplasmic Tax and decreased TSS (Fig. 1B, D, and F). Ten minutes after UV irradiation, most CREF-Tax cells (95.7% ± 0.8%) demonstrated Tax localization predominantly in TSS (Fig. 1G). The percentage of cells expressing TSS decreased until approximately 30 min after UV irradiation, when most cells (94.3% ± 1.4%) demonstrated predominantly cytoplasmic Tax and a significant decrease (>1 standard deviation from the mean of 85 TSS/cell) in the number of TSS (Fig. 1G). Predominantly cytoplasmic Tax distribution was maintained until approximately 8 h after UV irradiation, after which TSS had re-formed in the majority (72.9% ± 6.9%) of cells (Fig. 1G). The temporal and spatial distributions of Tax were consistent in each cell line tested, and the rate of translocation was consistent with those of other proteins known to undergo nuclear export following UV irradiation (10). To confirm these results, immunoblot analysis of nuclear and cytoplasmic fractions of unirradiated and UV-irradiated CREF-Tax cells showed a 2.9-fold increase in cytoplasmic Tax and a 50% decrease in nuclear Tax following UV irradiation (data not shown).
Lymphocytes are susceptible to a variety of genotoxic and cellular stresses, including oxidative and osmotic stress; various forms of DNA damage, including single- and double-stranded DNA breaks; and induction of bulky DNA adducts (2, 8, 16, 25, 30, 48). To determine whether genotoxic and cellular stresses other than UV irradiation could induce nucleocytoplasmic shuttling of Tax, Tax-expressing cells (CREF-Tax, C81-66, and HuT102) were exposed to ionizing radiation, which induces double-stranded DNA breaks; hydrogen peroxide, which induces oxidative damage; or sodium arsenite, which induces clastogenic mutations and aneuploidy (Table 1). In a manner similar to that of UV irradiation, each of these genotoxic stressors increased the amount of cytoplasmic Tax and decreased the number and intensity of nuclear TSS (Table 1).
Since multiple DNA-damaging agents altered Tax localization in similar ways, the effects of other types of cellular stress on Tax distribution were examined (Table 1). The effects of sorbitol, which causes osmotic stress, of anisomycin, an inhibitor of protein translation and activator of mitogen-activated protein kinase (MAPK) signaling pathways, or of heat shock, a destabilizer of tertiary protein conformation on Tax localization, were similar to the effects observed following UV and ionizing irradiation (Table 1). Cytoplasmic localization of Tax was stress dependent, because agents that do not induce stress (diluted media, ethanol, and MAPK-specific inhibitors SB203580, SP600125, and PD098059) did not affect the subcellular distribution of Tax. These results demonstrate that Tax localization is affected by genotoxic and cellular stress and that altered Tax localization is a consistent outcome following exposure to each form of stress examined.
Translation inhibition does not affect stress-induced Tax redistribution. The decrease in nuclear Tax and accumulation of cytoplasmic Tax observed following stress could result from Tax export out of the nucleus or from localized Tax degradation in the nucleus with concomitant Tax synthesis in the cytoplasm. To determine if new translation contributed to the appearance of Tax in the cytoplasm, CREF-Tax cells were treated with the translational inhibitor CHX for 30 minutes prior to either mock or UV irradiation, and Tax localization was examined by immunofluorescent microscopy. CHX treatment alone did not affect Tax localization (compare Fig. 2A [94.8% ± 2.2% cells express TSS] and C [94.3% ± 1.2% cells express TSS]). Importantly, CHX treatment did not reduce the accumulation of Tax in the cytoplasm of UV-irradiated cells (compare Fig. 2B and D). Consistent with our previous results (Fig. 1), very few (6.9% ± 2.3% mock-treated [Fig. 2B] versus 7.1% ± 1.2% CHX-treated [Fig. 2D]) UV-irradiated cells expressed TSS (Fig. 2E). These results indicate that new protein synthesis is not required for Tax to accumulate in the cytoplasm following stress and suggest that cytoplasmic Tax results from Tax transport out of the nucleus after stress.
To confirm that CHX treatment did in fact inhibit translation, Tax expression in CREF-Tax cells was examined by immunoblot analysis in the presence and absence of CHX and UV irradiation. Total Tax protein expression remained constant following UV irradiation (Fig. 2F). Since the half-life of Tax has been reported to be 15 h (21), CHX treatment caused a decrease in Tax protein at 5 and 7 h, which confirmed the inhibition of translation by CHX in this experiment (Fig. 2G). When translation was inhibited, similar levels of Tax protein expression were observed in the presence and absence of UV irradiation (compare Fig. 2H and G). In summary, UV irradiation had no apparent effect on Tax degradation or translation, indicating that cytoplasmic accumulation of Tax following stress is due to increased Tax export rather than nuclear degradation of Tax coupled with enhanced cytoplasmic translation.
Tax localization is not affected by cell cycle progression. Tax was recently shown to interfere with the G1/S-phase cell cycle checkpoint that is typically induced in response to UV irradiation (20). Tax can also interact with and affect the activity of nuclear and cytoplasmic proteins that regulate normal cell cycle progression (26, 29, 37, 38). Therefore, we wanted to determine whether normal cell cycle progression was associated with any changes in Tax localization. To address this question, CREF-Tax cells were synchronized in the quiescent (G0) stage of the cell cycle by contact inhibition. Cells were released into the cell cycle by splitting into new plates containing sterile coverslips. At specific time points following G0-phase release, the coverslips were stained for Tax. Cells remaining on the dish were collected for propidium iodide staining and cell cycle analysis by flow cytometry. No difference in subcellular Tax distributions was observed in cells enriched in the G0/G1, S, or G2/M phases of the cell cycle (Fig. 3A to C). In each phase of the cell cycle, nearly all cells (>99%) showed a predominantly nuclear distribution of Tax, demonstrating that Tax localization is not affected during normal cell cycle progression.
Cellular stress affects the colocalization of Tax and sc35. Since Tax localization is altered in response to various forms of stress, we wanted to determine whether proteins remain associated with TSS, dissociate, or translocate with Tax to the cytoplasm. sc35, a splicing cofactor that has been reported to interact and colocalize with Tax in TSS, is commonly used as a marker of these structures (51). As previously reported for other cell lines (4, 6, 51), sc35 and Tax colocalize in asynchronous cells under normal cellular conditions (Fig. 4A to C). Consistent with previous data demonstrating the heterogeneous nature of TSS (51), Tax foci and sc35 foci that do not colocalize were readily identified (Fig. 4C).
We next examined of the effect of genotoxic stress on the colocalization of Tax and sc35. As shown in Fig. 1, Tax rapidly translocated to the cytoplasm following UV irradiation (Fig. 4E), while sc35 localization remained nuclear (Fig. 4D). As a result, a marked decrease in the number of overlapping foci was observed in the merged image following UV irradiation (Fig. 4F), suggesting a reduced interaction between Tax and sc35 following stress. These results are consistent with the previous report that heat shock disrupts the interaction between Tax and sc35 (51). When TSS re-formed 8 hours after UV irradiation (Fig. 1G), the percentage of speckles containing both Tax and sc35 (48.8% ± 4.3%) was similar to that seen in unirradiated cells (51.3% ± 0.8%). These results were consistent for each type of stress examined in this study (data not shown), suggesting that genotoxic stress and cellular stress affect both the localization of Tax and the interactions of Tax with cellular proteins.
Stress-induced changes in Tax localization are LMB sensitive. The functions and activities of many cellular and viral proteins depend upon their subcellular distributions (23). Nuclear export of protein macromolecules is regulated by interactions with specific nuclear export proteins. These interactions are controlled by NES located in the cargo protein (18, 44). A leucine-rich NES characterized by the canonical sequence LX2-3(L/I/M/V/F)X2-3LX(L/I), where X is any amino acid, was recently identified at amino acids 188 to 202 of Tax (1, 12, 57). In most cases, nuclear export of leucine-rich NES-containing proteins is regulated through an interaction with the nuclear export factor CRM1 (11, 14, 15, 53). When conjugated to green fluorescent protein, export of the Tax NES is CRM1 dependent. However, full-length Tax protein does not utilize this pathway under normal conditions, leading to speculation that the Tax NES is conditionally masked and that a conformational change in the protein, either through posttranslational modification or through protein interactions, is required to expose the NES (1).
To test the possibility that stress-induced changes in Tax localization involved a CRM1-dependent pathway, cells were pretreated with a CRM1 inhibitor (LMB) that interacts with CRM1 and prevents its binding to the NES of the cargo protein (31, 32). Proteins that require the CRM1 nuclear export pathway remain sequestered in the nucleus of LMB-treated cells. To examine the effect of LMB on Tax localization, CREF-Tax cells were pretreated with LMB or a control for 1 hour before exposure to genotoxic agents. Pretreatment of cells with an ethanol control did not affect the typical distribution of Tax in the absence (Fig. 5A) or presence (Fig. 5B) of stress. Consistent with previous studies (1), LMB alone had no obvious effect on Tax localization (Fig. 5C), suggesting that the normal distribution of Tax between the nucleus and cytoplasm of undamaged cells does not involve the CRM1 pathway. However, pretreatment of cells with LMB blocked the expected increase in cytoplasmic Tax following genotoxic stress. Instead, Tax remained in nuclear speckles (Fig. 5D). Consistent with our previous data, after a 30-min recovery period, most (99.3% ± 0.5%) unstressed and only a small percentage (7.3% ± 2.3%) of UV-irradiated CREF-Tax cells expressed TSS. In contrast, similar percentages of cells expressing TSS were observed in unirradiated (99.2% ± 0.4%) and UV-irradiated (97.8% ± 1.4%) CREF-Tax cells pretreated with LMB (Fig. 5E). Similar results were observed for each type of stress examined (data not shown), demonstrating that Tax translocation following genotoxic and cellular stress is LMB sensitive and suggesting that the CRM1 nuclear export pathway is involved in this process.
The Tax NES is required for stress-induced nucleocytoplasmic translocation. We next wanted to map the region of Tax responsible for its redistribution following genotoxic or cellular stress. Leucine or isoleucine residues at specific amino acid positions within the NES are required for its function, since a mutation within the Tax NES (L200A) resulted in exclusive nuclear localization (1). The Tax M33 mutant (52) contains two mutations within the Tax NES (S199A and L200S), which we predicted would eliminate NES function. We used this mutant to determine whether the Tax NES was required for redistribution of Tax following UV irradiation. Since we do not have a cell line that stably expresses the Tax M33 mutant, these studies were performed using transient expression. Wild-type Tax was first transiently expressed in 293 cells to determine whether the effect of UV irradiation on Tax localization was similar to that previously observed in cell lines that stably expressed Tax. Consistent with our previous results (Fig. 1), transfection of a Tax expression vector into 293 cells resulted in predominant nuclear localization in TSS (85.3% ± 1.6% of Tax-expressing cells displayed TSS; Fig. 6A and G). Thirty minutes after UV irradiation, fewer cells displayed TSS (20.5% ± 1.8%), and a significant increase in cytoplasmic Tax was observed (Fig. 6B and G). In contrast, the percentages of 293 cells transfected with the M33 Tax mutant that expressed TSS were similar in the presence (81.4% ± 2.2%; Fig. 6D) and absence (81.5% ± 1.5%; Fig. 6C) of UV irradiation. The localization of another Tax mutant which does not contain mutations in the NES, M47 (L319R, L320S), was similar to that of wild-type Tax (Fig. 6E and F). That is, most (82.8% ± 2.1%) unstressed and fewer (27.0% ± 4.1%) UV-irradiated 293 cells expressing the M47 Tax mutant demonstrated TSS (Fig. 6G). These results suggest that the NES regulates Tax nuclear export in response to genotoxic stress.
Tax and CRM1 interact following stress. As previously discussed, the Tax NES may be conditionally masked. Revealing the NES may require changes in protein interactions or posttranslational modifications (1). The CRM1 nuclear export pathway relies on a direct interaction between CRM1 and the NES of the cargo protein (14, 15, 53), which, together with the transport cofactor RanGTP, form a trimeric complex that is exported through the nuclear pore in an energy-dependent manner (18, 44). To examine the potential interaction of Tax with CRM1 in response to cellular stress, whole-cell lysates were prepared from Tax-expressing 293 cells that were either undamaged, UV irradiated (30 J/m2, 20-min recovery), or treated with sorbitol (850 mM for 35 min) or sodium arsenite (1 mM for 35 min). These lysates were immunoprecipitated using anti-CRM1 (Fig. 7A) or anti-Tax (Fig. 7B) antibodies and subsequently immunoblotted for Tax and CRM1. No interaction between Tax and CRM1 was detected in undamaged cells (Fig. 7A and B, lanes 1); however, a complex containing Tax and CRM1 was detected following stress (Fig. 7A and B, lanes 2 to 4). Similar results were observed when CREF-Tax cell extracts were used (data not shown).
To determine whether the Tax NES itself was responsible for the interaction between Tax and CRM1, similar experiments were performed using extracts from 293 cells transfected with a Tax M33 expression vector. As predicted, no interaction between the NES-defective M33 mutant and CRM1 was detected in the absence (Fig. 7A and B, lanes 5) or presence (Fig. 7A and B, lanes 6 to 8) of stress. These results, combined with results from the LMB and Tax mutant localization experiments, demonstrate that the CRM1 nuclear export pathway is involved in Tax nucleocytoplasmic translocation following cellular stress and confirm that the Tax NES is required for this process. Since Tax and CRM1 interact after, but not before, stress, a change in protein conformation, interactions, or posttranslational modifications is likely to make the Tax NES accessible to CRM1.
Tax dissociates from sc35 before interacting with CRM1. We previously showed that Tax interacts with CRM1 following stress. To determine whether Tax dissociates from TSS before interacting with CRM1 or whether the interaction between Tax and CRM1 facilitates the separation of Tax from TSS, we examined the composition of Tax speckles in response to LMB treatment and UV irradiation. If Tax dissociates from TSS before interacting with CRM1, then colocalization of Tax and sc35 should not be seen following LMB treatment and UV irradiation. Alternatively, since LMB blocks the ability of CRM1 to interact with Tax, if an interaction between Tax and CRM1 is required to dissociate Tax from TSS, then Tax should remain associated with sc35 following UV irradiation.
CREF-Tax cells were pretreated with 10 nM LMB for 1 h, exposed to 30 J/m2 of UV irradiation or mock treated, and allowed to recover for 30 min. As expected, in the presence of LMB both sc35 (Fig. 8D) and Tax (Fig. 8E) were retained in nuclear speckles following UV irradiation. In contrast to unirradiated cells, which showed extensive colocalization of these two proteins (Fig. 8C), UV-irradiated cells showed significantly less colocalization of Tax and sc35 (Fig. 8F). UV irradiation induced a significant increase (P < 0.05) in the number of nuclear speckles containing only Tax (22.0% ± 1.4% unirradiated; 41.4% ± 1.7% irradiated) or only sc35 (18.3% ± 0.7% unirradiated; 51.1% ± 1.1% UV irradiated) and a significant decrease (P < 0.05) in the number of speckles containing both Tax and sc35 (59.7% ± 2.1% unirradiated; 7.5% ± 0.6% UV irradiated) (Fig. 8G). These results, together with the observation that M33 and sc35 do not colocalize following stress (Fig. 6D), suggest that stress causes Tax to be released from TSS, which allows it to interact with CRM1, and is required for nuclear export of Tax.
DISCUSSION
The pleiotropic HTLV-1 Tax protein is known to interact with a number of cellular proteins to affect a variety of cellular processes. Understanding how these interactions and functions are regulated within the cell remains a subject of intense interest. This study examined the effects of genotoxic and cellular stress on the subcellular distribution of Tax. The dramatic redistribution of Tax in response to various forms of stress is characterized by a transient increase in cytoplasmic Tax and a decrease in the number and intensity of TSS (Fig. 1). Mutation of the NES in the carboxy terminus of Tax (M33) demonstrated that this sequence is required for nuclear export. We further demonstrated that Tax utilizes the CRM1 nuclear export pathway to facilitate nucleocytoplasmic translocation in response to stress by demonstrating an interaction between Tax and CRM1 that is present only in response to stress (Fig. 7). Pretreatment of CREF-Tax cells with the CRM1-specific inhibitor LMB inhibited the cytoplasmic translocation of Tax in response to stress, confirming that changes in Tax localization following genotoxic and cellular stress occur through the CRM1 nuclear export pathway (Fig. 5).
It was previously suggested that the Tax NES is not accessible and that posttranslational modification of Tax, or changes in its protein partners, may be required to expose the NES (1). We demonstrated that Tax and sc35 fail to colocalize (Fig. 8) following UV irradiation. This result was confirmed using the NES-defective Tax mutant, M33, which also failed to colocalize with sc35 following UV irradiation (Fig. 6). These results suggest that in response to stress, Tax dissociates from TSS without requiring an interaction with CRM1. However, the release of Tax from TSS does allow Tax to interact with CRM1, which then facilitates the nuclear export of Tax following exposure to genotoxic or cellular stress.
Tax has been shown to be posttranslationally ubiquitinated and phosphorylated, and these modifications of Tax and/or other TSS proteins are likely to regulate Tax localization. Posttranslational phosphorylation or ubiquitination has been shown to regulate nuclear export of other cellular proteins through the CRM1 pathway (5, 7, 13, 24, 39, 47). Sumoylation and ubiquitination of Tax at specific residues have recently been shown to play an integral role in regulating the subcellular distribution and functions of Tax (33, 43). It is also possible that posttranslational modification of one or more Tax-interacting proteins in response to stress may cause them to dissociate from Tax and enable Tax to interact with CRM1. The specific role of these mechanisms in regulating the nuclear export of Tax remains to be determined.
Genotoxic and cellular stressors, including each of those utilized in this study (Table 1), have been shown to induce MAPK signaling pathways (10, 46, 50). Tax was not able to translocate to the cytoplasm in the presence of the MAPK inhibitors (Table 1). Since many other proteins known to undergo nucleocytoplasmic shuttling in response to stress require the activity of these pathways (10), the activation of MAPK signaling may be important for regulating Tax localization in response to stress. However, additional studies will be required to identify the pathway and mechanisms responsible for this effect.
Although a number of studies have demonstrated that Tax functions both in the nucleus and in the cytoplasm, its cytoplasmic functions in particular remain to be fully elucidated. Cytoplasmic Tax has been shown to modulate the activity of a number of cellular proteins and pathways. In particular, Tax has been shown to interact with and target the retinoblastoma protein for proteasomal degradation and to enhance activation of the NF-B pathway through a variety of mechanisms, including activation of upstream kinases, interaction with and targeting of NF-B inhibitors for proteasomal degradation, and direct interaction with the NF-B transcription factor p65/RelA (3, 22, 54, 56, 60). Tax mutants that are deficient for cytoplasmic functions display a reduced transformation capacity (59), and the Tax M33 mutant, which is defective for nuclear export, is 50% less effective at repressing DNA repair than wild-type Tax (34). Therefore, transient cytoplasmic localization of Tax may interfere with the host response to genotoxic or cellular stress and have important implications for cellular transformation.
Tax-expressing cells do not efficiently repair DNA damage by either the nucleotide excision or the base excision DNA repair pathways (28, 49). In addition, Tax expression is associated with defects in cell cycle checkpoints (20). Together, these effects are believed to enhance a cell's propensity to accumulate mutations. Since lymphocytes are exposed to genotoxic and cellular stress on a regular basis (2, 8, 16, 25, 30, 48), HTLV-1-infected lymphocytes are likely to be susceptible to the introduction of mutations due to unrepaired DNA lesions. Indeed, Tax-expressing cells demonstrate higher gene amplification levels and higher mutation frequency than normal cells (36, 42). HTLV-1-infected cells and cells isolated from ATL patients also contain a large number of chromosomal abnormalities, including deletions, duplications, and translocations (17). Collectively, these results suggest that unrepaired DNA damage in Tax-expressing cells may contribute to cellular transformation and that changes in Tax localization due to genotoxic and cellular stress may enhance this process.
In summary, this study demonstrates that exposure of Tax-expressing cells to genotoxic or other forms of stress alters the subcellular distribution of Tax. Altered Tax localization may contribute to the enhanced mutation frequency observed in Tax-expressing cells by affecting DNA repair mechanisms and/or cell cycle progression and may ultimately play an important role in generating chromosomal abnormalities characteristic of HTLV-1 Tax-transformed cells and lymphocytes from ATL patients. Future studies to specifically address the biological consequences of Tax localization will likely reveal important insights into cellular transformation by this viral oncoprotein.
ACKNOWLEDGMENTS
We thank Ian Cushman (Duke University) and Mary Moore (Baylor College of Medicine) for their insights into nuclear export as well as Catherine Gatza (Baylor College of Medicine) and the members of the Marriott laboratory for helpful suggestions and editorial comments. We also acknowledge the AIDS Research and Reference Reagent Program for providing anti-Tax antibodies.
This study was supported, in part, by U.S. Public Service grant CA-77371 from the National Cancer Institute, National Institutes of Health, awarded to S.J.M. M.L.G. is supported in part by National Institutes of Health Training Grant CA-09197 and a Sigma Xi Grant-in-Aid of Research award.
REFERENCES
Alefantis, T., K. Barmak, E. W. Harhaj, C. Grant, and B. Wigdahl. 2003. Characterization of a nuclear export signal within the human T cell leukemia virus type I transactivator protein Tax. J. Biol. Chem. 278:21814-21822.
Andreoli, C., P. Leopardi, S. Rossi, and R. Crebelli. 1999. Processing of DNA damage induced by hydrogen peroxide and methyl methanesulfonate in human lymphocytes: analysis by alkaline single cell gel electrophoresis and cytogenetic methods. Mutagenesis 14:497-504.
Azran, I., K. T. Jeang, and M. Aboud. 2005. High levels of cytoplasmic HTLV-1 Tax mutant proteins retain a Tax-NF-kappaB-CBP ternary complex in the cytoplasm. Oncogene 24:4521-4530.
Bex, F., A. McDowall, A. Burny, and R. Gaynor. 1997. The human T-cell leukemia virus type 1 transactivator protein Tax colocalizes in unique nuclear structures with NF-B proteins. J. Virol. 71:3484-3497.
Bex, F., K. Murphy, R. Wattiez, A. Burny, and R. B. Gaynor. 1999. Phosphorylation of the human T-cell leukemia virus type 1 transactivator Tax on adjacent serine residues is critical for Tax activation. J. Virol. 73:738-745.
Burton, M., C. D. Upadhyaya, B. Maier, T. J. Hope, and O. J. Semmes. 2000. Human T-cell leukemia virus type 1 Tax shuttles between functionally discrete subcellular targets. J. Virol. 74:2351-2364.
Chiari, E., I. Lamsoul, J. Lodewick, C. Chopin, F. Bex, and C. Pique. 2004. Stable ubiquitination of human T-cell leukemia virus type 1 Tax is required for proteasome binding. J. Virol. 78:11823-11832.
Courtemanche, C., A. C. Huang, I. Elson-Schwab, N. Kerry, B. Y. Ng, and B. N. Ames. 2004. Folate deficiency and ionizing radiation cause DNA breaks in primary human lymphocytes: a comparison. FASEB J. 18:209-211.
Dignam, J. D., R. M. Lebowitz, and R. G. Roeder. 1983. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11:1475-1489.
Ducret, C., S. M. Maira, A. Dierich, and B. Wasylyk. 1999. The net repressor is regulated by nuclear export in response to anisomycin, UV, and heat shock. Mol. Cell. Biol. 19:7076-7087.
Elfgang, C., O. Rosorius, L. Hofer, H. Jaksche, J. Hauber, and D. Bevec. 1999. Evidence for specific nucleocytoplasmic transport pathways used by leucine-rich nuclear export signals. Proc. Natl. Acad. Sci. USA 96:6229-6234.
Fischer, U., J. Huber, W. C. Boelens, I. W. Mattaj, and R. Luhrmann. 1995. The HIV-1 Rev activation domain is a nuclear export signal that accesses an export pathway used by specific cellular RNAs. Cell 82:475-483.
Fontes, J. D., J. M. Stawhecker, N. D. Bills, R. E. Lewis, and S. H. Hinrichs. 1993. Phorbol esters modulate the phosphorylation of human T-cell leukemia virus type I Tax. J. Virol. 67:4436-4441.
Fornerod, M., M. Ohno, M. Yoshida, and I. W. Mattaj. 1997. CRM1 is an export receptor for leucine-rich nuclear export signals. Cell 90:1051-1060.
Fukuda, M., S. Asano, T. Nakamura, M. Adachi, M. Yoshida, M. Yanagida, and E. Nishida. 1997. CRM1 is responsible for intracellular transport mediated by the nuclear export signal. Nature 390:308-311.
Gabbianelli, R., C. Nasuti, G. Falcioni, and F. Cantalamessa. 2004. Lymphocyte DNA damage in rats exposed to pyrethroids: effect of supplementation with vitamins E and C. Toxicology 203:17-26.
Gatza, M. L., C. Chandhasin, R. I. Ducu, and S. J. Marriott. 2005. Impact of transforming viruses on cellular mutagenesis, genome stability, and cellular transformation. Environ. Mol. Mutagen. 45:304-325.
Gorlich, D. 1998. Transport into and out of the cell nucleus. EMBO J. 17:2721-2727.
Haoudi, A., R. C. Daniels, E. Wong, G. Kupfer, and O. J. Semmes. 2003. Human T-cell leukemia virus-I tax oncoprotein functionally targets a subnuclear complex involved in cellular DNA damage-response. J. Biol. Chem. 278:37736-37744.
Haoudi, A., and O. J. Semmes. 2003. The HTLV-1 tax oncoprotein attenuates DNA damage induced G1 arrest and enhances apoptosis in p53 null cells. Virology 305:229-239.
Hemelaar, J., F. Bex, B. Booth, V. Cerundolo, A. McMichael, and S. Daenke. 2001. Human T-cell leukemia virus type 1 Tax protein binds to assembled nuclear proteasomes and enhances their proteolytic activity. J. Virol. 75:11106-11115.
Hirai, H., T. Suzuki, J.-I. Fujisawa, J. Inoue, and M. Yoshida. 1994. Tax protein of human T-cell leukemia virus type I binds to the ankyrin motifs of inhibitory factor kappaB and induces nuclear translocation of transcription factor NF-kappaB proteins for transcriptional activation. Proc. Natl. Acad. Sci. USA 91:3584-3588.
Hood, J. K., and P. A. Silver. 1999. In or out Regulating nuclear transport. Curr. Opin. Cell Biol. 11:241-247.
Ishida, N., T. Hara, T. Kamura, M. Yoshida, K. Nakayama, and K. I. Nakayama. 2002. Phosphorylation of p27Kip1 on serine 10 is required for its binding to CRM1 and nuclear export. J. Biol. Chem. 277:14355-14358.
Jaloszynski, P., M. Kujawski, M. Wasowicz, R. Szulc, and K. Szyfter. 1999. Genotoxicity of inhalation anesthetics halothane and isoflurane in human lymphocytes studied in vitro using the comet assay. Mutat. Res. 439:199-206.
Jin, D. Y., F. Spencer, and K. T. Jeang. 1998. Human T cell leukemia virus type 1 oncoprotein Tax targets the human mitotic checkpoint protein MAD1. Cell 93:81-91.
Kao, S. Y., F. J. Lemoine, and S. J. Marriott. 2001. p53-independent induction of apoptosis by the human T cell leukemia virus type I Tax protein following UV irradiation. Virology 291:292-298.
Kao, S. Y., and S. J. Marriott. 1999. Disruption of nucleotide excision repair by the human T-cell leukemia virus type 1 Tax protein. J. Virol. 73:4299-4304.
Kehn, K., C. L. Fuente, K. Strouss, R. Berro, H. Jiang, J. Brady, R. Mahieux, A. Pumfery, M. E. Bottazzi, and F. Kashanchi. 2005. The HTLV-I Tax oncoprotein targets the retinoblastoma protein for proteasomal degradation. Oncogene 24:525-540.
Kim, C. H., and N. D. Vaziri. 2005. Hypertension promotes integrin expression and reactive oxygen species generation by circulating leukocytes. Kidney Int. 67:1462-1470.
Kudo, N., N. Matsumori, H. Taoka, D. Fujiwara, E. P. Schreiner, B. Wolff, M. Yoshida, and S. Horinouchi. 1999. Leptomycin B inactivates CRM1/exportin 1 by covalent modification at a cysteine residue in the central conserved region. Proc. Natl. Acad. Sci. USA 96:9112-9117.
Kudo, N., B. Wolff, T. Sekimoto, E. P. Schreiner, Y. Yoneda, M. Yanagida, S. Horinouchi, and M. Yoshida. 1998. Leptomycin B inhibition of signal-mediated nuclear export by direct binding to CRM1. Exp. Cell Res. 242:540-547.
Lamsoul, I., J. Lodewick, S. Lebrun, R. Brasseur, A. Burny, R. Gaynor, and F. Bex. 2005. Exclusive ubiquitination and sumoylation on overlapping lysine residues mediate NF-B activation by the human T-cell leukemia virus Tax oncoprotein. Mol. Cell. Biol. 25:10391-10406.
Lemoine, F. J., S. Y. Kao, and S. J. Marriott. 2000. Suppression of DNA repair by HTLV-I Tax correlates with Tax transactivation of PCNA gene expression. AIDS Res. Hum. Retrovir. 16:1623-1627.
Lemoine, F. J., and S. J. Marriott. 2001. Accelerated G1 phase progression induced by the human T cell leukemia virus type I (HTLV-I) Tax oncoprotein. J. Biol. Chem. 276:31851-31857.
Lemoine, F. J., and S. J. Marriott. 2002. Genomic instability driven by the human T-cell leukemia virus type I (HTLV-I) oncoprotein, Tax. Oncogene 21:7230-7234.
Li, J., H. Li, and M. D. Tsai. 2003. Direct binding of the N-terminus of HTLV-1 Tax oncoprotein to cyclin-dependent kinase 4 is a dominant path to stimulate the kinase activity. Biochemistry 42:6921-6928.
Liang, M. H., T. Geisbert, Y. Yao, S. H. Hinrichs, and C. Z. Giam. 2002. Human T-lymphotropic virus type I oncoprotein Tax promotes S-phase entry but blocks mitosis. J. Virol. 76:4022-4033.
Lohrum, M. A., D. B. Woods, R. L. Ludwig, E. Balint, and K. H. Vousden. 2001. C-terminal ubiquitination of p53 contributes to nuclear export. Mol. Cell. Biol. 21:8521-8532.
Majone, F., and K. T. Jeang. 2000. Clastogenic effect of the human T-cell leukemia virus type I Tax oncoprotein correlates with unstabilized DNA breaks. J. Biol. Chem. 275:32906-32910.
Majone, F., O. J. Semmes, and K.-T. Jeang. 1993. Induction of micronuclei by HTLV-I Tax: a cellular assay for function. Virology 193:456-459.
Miyake, H., T. Suzuki, H. Hirai, and M. Yoshida. 1999. Trans-activator Tax of human T-cell leukemia virus type 1 enhances mutation frequency of the cellular genome. Virology 253:155-161.
Nasr, R., E. Chiari, M. El-Sabban, R. Mahieux, Y. Kfoury, V. Yazbeck, O. Hermine, H. De The, C. Pique, and A. Bazarbachi. 19 January 2006, posting date. Tax ubiquitylation and sumoylation control critical cytoplasmic and nuclear steps of NF-kB activation. Blood [Online.] doi:10.1182/blood-2005-09-3572.
Nigg, E. A. 1997. Nucleocytoplasmic transport: signals, mechanisms and regulation. Nature 386:779-787.
Osborn, L., S. Kunkel, and G. J. Nabel. 1989. Tumor necrosis factor alpha and interleukin 1 stimulate the human immunodeficiency virus enhancer by activation of the nuclear factor kappa B. Proc. Natl. Acad. Sci. USA 86:2336-2340.
Pearson, G., F. Robinson, T. Gibson, B. Xu, M. Karandikar, K. Berman, and M. Cobb. 2001. Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions. Endocrinol. Rev. 22:153-183.
Peloponese, J. M., Jr., H. Iha, V. R. Yedavalli, A. Miyazato, Y. Li, K. Haller, M. Benkirane, and K. T. Jeang. 2004. Ubiquitination of human T-cell leukemia virus type 1 Tax modulates its activity. J. Virol. 78:11686-11695.
Peluso, M., M. Neri, G. Margarino, C. Mereu, A. Munnia, M. Ceppi, M. Buratti, R. Felletti, F. Stea, R. Quaglia, R. Puntoni, E. Taioli, S. Garte, and S. Bonassi. 2004. Comparison of DNA adduct levels in nasal mucosa, lymphocytes and bronchial mucosa of cigarette smokers and interaction with metabolic gene polymorphisms. Carcinogenesis 25:2459-2465.
Philpott, S. M., and G. C. Buehring. 1999. Defective DNA repair in cells with human T-cell leukemia bovine leukemia viruses: role of tax gene. J. Natl. Cancer Inst. 91:933-942.
Roux, P. P., and J. Blenis. 2004. ERK and p38 MAPK-activated protein kinases: a family of protein kinases with diverse biological functions. Microbiol. Mol. Biol. Rev. 68:320-344.
Semmes, O. J., and K. T. Jeang. 1996. Localization of human T-cell leukemia virus type 1 Tax to subnuclear compartments that overlap with interchromatin speckles. J. Virol. 70:6347-6357.
Smith, M. R., and W. C. Greene. 1990. Identification of HTLV-I tax trans-activator mutants exhibiting novel transcriptional phenotypes. Genes Dev. 4:1875-1885.
Stade, K., C. S. Ford, C. Guthrie, and K. Weis. 1997. Exportin 1 (Crm1p) is an essential nuclear export factor. Cell 90:1041-1050.
Suzuki, T., H. Hirai, T. Murakami, and M. Yoshida. 1995. Tax protein of HTLV-1 destabilizes the complexes of NF-kappaB and IkappaB-a and induces nuclear translocation of NF-kappaB for transcriptional activation. Oncogene 10:1199-1207.
Suzuki, T., T. Narita, M. Uchida-Toita, and M. Yoshida. 1999. Down-regulation of the INK4 family of cyclin-dependent kinase inhibitors by Tax protein of HTLV-1 through two distinct mechanisms. Virology 259:384-391.
Uhlik, M., L. Good, G. T. Xiao, E. W. Harhaj, E. Zandi, M. Karin, and S. C. Sun. 1998. NF-kappaB-inducing kinase and IkappaB kinase participate in human T-cell leukemia virus I Tax-mediated NF-kappaB activation. J. Biol. Chem. 273:21132-21136.
Wen, W., J. L. Meinkoth, R. Y. Tsien, and S. S. Taylor. 1995. Identification of a signal for rapid export of proteins from the nucleus. Cell 82:463-473.
Xiao, G., E. W. Harhaj, and S. C. Sun. 2000. Domain-specific interaction with the IB kinase (IKK) regulatory subunit IKK is an essential step in Tax-mediated activation of IKK. J. Biol. Chem. 275:34060-34067.
Yamaoka, S., H. Inoue, M. Sakurai, T. Sugiyama, M. Hazama, T. Yamada, and M. Hatanaka. 1996. Constitutive activation of NF-kB is essential for transformation of rat fibroblasts by the human T-cell leukemia virus type I Tax protein. EMBO J. 15:873-887.
Yin, M. J., L. B. Christerson, Y. Yamamoto, Y. T. Kwak, S. C. Xu, F. Mercurio, M. Barbosa, M. H. Cobb, and R. B. Gaynor. 1998. HTLV-I Tax protein binds to MEKK1 to stimulate IkappaB kinase activity and NF-kappaB activation. Cell 93:875-884.
Yoshida, M. 2001. Multiple viral strategies of HTLV-1 for dysregulation of cell growth control. Annu. Rev. Immunol. 19:475-496(Michael L. Gatza and Susa)