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Previous Article | Next Article 
Molecular and Cellular Biology, February 2004, p. 1033-1043, Vol. 24, No. 3
0270-7306/04/$08.00+0 DOI: 10.1128/MCB.24.3.1033-1043.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Department of Hematology and Oncology, Graduate School of Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113-8655,1 Department of Hematology, Dokkyo University School of Medicine, Tochigi 321-0207,2 Department of Molecular Immunology, Institute of Development, Aging, and Cancer, Tohoku University, Sendai 980-0872,3 Department of Cell Biology, The Cancer Institute, Japanese Foundation for Cancer Research, Tokyo 170-0012, Japan,4 Institute of Molecular and Cell Biology, National University of Singapore, Singapore 117609, Singapore5
Received 17 March 2003/ Returned for modification 8 May 2003/ Accepted 31 October 2003
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ABSTRACT |
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INTRODUCTION |
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AML1 regulates the transcription of various genes that are important in hematopoiesis (27, 36, 37, 48). Furthermore, it has been revealed that AML1 can cause neoplastic transformation when overexpressed in fibroblasts, suggesting a potential role for AML1 in promoting cellular proliferation (17). From analyses of mice lacking AML1, it was shown that AML1 plays an important role in liver-derived hematopoiesis and angiogenesis (30, 38).
Previously, the regulatory mode of AML1 functions through signal transduction pathways was investigated, and it was revealed that AML1 is phosphorylated with dependence on the activation of extracellular signal-regulated kinase (ERK) (40). ERK-dependent phosphorylation enhances the transcriptional activity of AML1, and mutations of the phosphorylation sites reduce the transforming capacity of AML1 in fibroblasts (40). These results indicate that the functions of AML1 are potentially regulated by ERK-induced phosphorylation, which is activated by cytokine and growth factor stimuli. However, the mechanism by which the transcriptional ability of AML1 is activated by phosphorylation has not been clear.
A growing number of proteins have been shown to interact with AML1 to modify its function (2, 10, 12, 19, 21, 28, 44). Here, we have investigated whether these protein-protein interactions are affected by phosphorylation of AML1, and we found that the corepressor mSin3A is released from phosphorylated AML1, whereby the transcriptional activity of phosphorylated AML1 is activated. Furthermore, we demonstrated that mSin3A is a key regulator for the intranuclear localization and stability of AML1. This study reveals a previously unappreciated role of the corepressor mSin3A as a regulator of AML1.
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MATERIALS AND METHODS |
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(181-210)SA, was created by PCR from S249/266A with the insertion of a BglII site to join the fragments. The carboxy-terminal deletion mutant, AML1 1-288, was generated as described previously (17). Cell culture and DNA transfection. COS-7 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS). HEL cells were cultured in RPMI 1640 supplemented with 10% FCS. P19 mouse embryonal carcinoma cells were cultured as described previously (40). COS-7 and P19 cells were transfected with expression plasmids using SuperFect Transfection Reagent (Qiagen Inc.). Mouse embryonic fibroblasts from normal and PEBP2ß-deficient mice (26) were transfected with expression plasmids by using Effectene Transfection Reagent (Qiagen Inc.).
Immunoprecipitation and Western blotting. COS-7 cells were cultured for 30 to 35 h after transfection in DMEM containing 10% FCS and then transferred to DMEM containing 0.1% FCS and incubated for 12 to 15 h. They were then either not treated or treated for 5 min with 10% FCS plus 100 ng of recombinant human epidermal growth factor (EGF) (Sigma) per ml and harvested in phosphate-buffered saline supplemented with 1 mM EDTA, 1.5 mg of iodoacetamide/ml, 0.2 mM phenylmethylsulfonyl fluoride, 0.1 trypsin inhibitory unit/ml of aprotinin, 25 mM ß-glycerophosphate, and 0.5% Triton X-100. These cell lysates were precleared with protein G-Sepharose (Pharmacia), mixed with the anti-mSin3A antibody K-20 (Santa Cruz), and rotated for 2 h; this was followed by recovery of mSin3A on protein G-Sepharose beads. The beads were washed four times with the lysis buffer. Immunoprecipitates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting with the anti-FLAG M2 monoclonal antibody (Sigma).
HEL cells (107) were harvested in 1% Triton lysis buffer (10 mM Tris-HCl, pH 7.4, 5 mM EDTA, 150 mM NaCl, 1% Triton-X, 10% glycerol, 10 U of aprotinin/ml, 2 mM phenylmethylsulfonyl fluoride, 25 mM ß-glycerophosphate), and the cell lysates were precleared with protein G-Sepharose. The same amount of cell lysates was mixed with rabbit polyclonal immunoglobulin G (IgG) (Santa Cruz) as a control or with anti-AML1 antibody (Ab-1; Oncogene), and rotated for 12 h; this was followed by recovery of AML1 on protein G-Sepharose beads. The beads were washed four times with the lysis buffer. Immunoprecipitates were subjected to SDS-PAGE and Western blotting with anti-mSin3A antibody.
We lysed the PEBP2ß-deficient fibroblasts with 1% Triton lysis buffer, and the cell lysates were subjected to SDS-PAGE and Western blotting with anti-FLAG M2 monoclonal antibody, anti-PEBP2ß antibody, or anti-actin monoclonal antibody (Chemicon International). The anti-PEBP2ß antibody was prepared as described elsewhere (39). To block proteolysis, lactacystin (10 mM) dissolved in dimethyl sulfoxide was added to the cell culture medium overnight.
Chromatin and nuclear matrix fractions were prepared by sequential extraction with cytoskeleton buffer and digestion buffer followed by 0.25 M ammonium sulfate extraction in a biochemical-fractionation assay as described previously (47). To detect lamin B1 localized in the nuclear matrix, we used anti-lamin B1 antibody (M-20) (Santa Cruz).
Transcriptional-response assays. Luciferase assays were performed as described previously (8). Briefly, reporter and expression plasmids were transfected into P19 or COS-7 cells by a SuperFect protocol. P19 cells were harvested and subjected to the luciferase assay after 30 to 36 h of transfection. COS-7 cells were starved in DMEM containing 0.1% FCS with or without lactacystin, harvested, and subjected to the luciferase assay after 12 h of transfection. The data were normalized using the internal control of transfection efficiency, as described previously (8).
Immunofluorescence and in situ nuclear matrix isolation. COS-7 cells transfected with expression plasmids were fixed in 3.7% formaldehyde in phosphate-buffered saline. They were treated with the anti-FLAG and anti-mSin3A antibodies and then incubated with Texas-red-conjugated donkey anti-mouse IgG and fluorescein isothiocyanate-conjugated donkey anti-rabbit IgG (Jackson Immuno Research Laboratories, Inc.), respectively, as secondary antibodies, as described previously (8, 16). In situ nuclear matrices were prepared as described elsewhere (46, 47).
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RESULTS |
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It has been shown that AML1 associates with the mSin3A corepressor (21). Consistent with these observations, we found that FLAG-tagged AML1 is coimmunoprecipitated with mSin3A by the anti-mSin3A antibody when it is overexpressed together with PEBP2ß in COS-7 cells (Fig. 1A, lane 1). When COS-7 cells were cotransfected with FLAG-tagged AML1 and PEBP2ß plus ERK and stimulated with EGF, we observed shifted bands of AML1 mirroring its phosphorylation (40) (Fig. 1A, lane 4). Significantly, the amount of AML1 coimmunoprecipitated with mSin3A was markedly reduced upon phosphorylation (Fig. 1A, lane 2). The total amount of AML1 was constant regardless of overexpression of ERK combined with EGF stimulation (Fig. 1A, lanes 3 and 4). These results suggest that AML1 dissociates from mSin3A when it is phosphorylated by ERK.
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To confirm that AML1 and mSin3A form a complex in hematopoietic cells, we investigated the interactions between endogenous AML1 and mSin3A in HEL cells, a human erythroleukemia-derived cell line. Endogenous mSin3A is not immunoprecipitated by normal rabbit polyclonal IgG in HEL cells (Fig. 1C, lane 1). On the other hand, the precipitate by the anti-AML1 antibody (Ab-1) contains endogenous mSin3A (Fig. 1C, lane 2). In the previous study, the anti-AML1 antibody (Ab-1) was shown to immunoprecipitate endogenous AML1 in HEL cells (21). These results indicate that mSin3A binds to AML1 in hematopoietic cells.
Transcriptional activity of unphosphorylated AML1 is recovered by addition of HDAC inhibitor. In a previous study, it was shown that ERK-dependent phoshphorylation of AML1 can potentiate its transcriptional ability (40). However, the identities of regulatory proteins that repress the transcriptional activity of unphosphorylated AML1 have not been well characterized. Given the interaction between AML1 and mSin3A, it might be that mSin3A plays an important role in the repression of AML1 transcriptional activity. Histone deacetylases (HDACs) that are recruited by mSin3A mediate transcriptional repression by rendering the nearby chromatin inaccessible to transcriptional activators through deacetylation of histone proteins (32, 42). Trichostatin A (TSA), a specific HDAC inhibitor, has been shown to relieve transcriptional repression by AML1, suggesting a role for mSin3A-HDAC in the repressor activity of AML1 (21). Therefore, we investigated whether unphosphorylated AML1 can be derepressed when mSin3A-HDAC is inhibited by TSA in a transcriptional-response assay.
When AML1 and ERK were cotransfected into P19 cells with the Tww-tk-Luc reporter that can be activated by AML1, we observed a fivefold induction of transcriptional activity compared to overexpression of ERK alone (Fig. 2A, lane 2). On the other hand, when S249/266A was coexpressed with ERK, only 1.5-fold induction of the transcriptional activity was observed (Fig. 2A, lane 3), which is consistent with our previous report. Upon treatment with TSA, both wild-type AML1 and S249/266A cotransfected with ERK induced 2.5-fold transcriptional activity compared to overexpression of ERK alone, and the reduced transcriptional activity of S249/266A in the absence of TSA was restored to a level similar to that of wild-type AML1 (Fig. 2B, lanes 2 and 3). These results suggest that the transcriptional activity of unphosphorylated AML1 is suppressed by the interaction with mSin3A-HDAC.
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FLAG-tagged AML1, PEBP2ß, and ERK were coexpressed in COS-7 cells, and the cells were cultured with or without EGF. They were stained with the anti-FLAG and anti-mSin3A antibodies. Both AML1 and endogenous mSin3A were localized predominantly in the nucleus regardless of EGF stimulation (data not shown). The AML1 mutants, S249/266A and S249/266E, are also located in the nucleus (data not shown). Next, we analyzed the cells in in situ nuclear-matrix preparations (46, 47). Staining the nuclear matrix with anti-FLAG and anti-mSin3A antibodies revealed that AML1 and mSin3A were colocalized in the nuclear matrix without EGF stimulation (Fig. 3A to C). When we stimulated the transfected cells with EGF, mSin3A was detected in the nuclear matrix (Fig. 3E). Under these conditions, however, AML1 was absent from the nuclear matrix, where no colocalization signal of AML1 and mSin3A was detected (Fig. 3D and F). Subsequently, we investigated the intranuclear localization of S249/266A and S249/266E. As shown in Fig. 3, S249/266A and mSin3A were colocalized in the nuclear matrix (Fig. 3G to I), whereas S249/266E was neither detected in the nuclear matrix nor colocalized with mSin3A (Fig. 3J to L). These results indicate that AML1 and mSin3A are colocalized in the nuclear matrix when AML1 is not phosphorylated and that AML1 translocates from the nuclear matrix upon phosphorylation.
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(181-210), which generates the double mutant, (181-210)SA. We introduced FLAG-tagged (181-210)SA, together with PEBP2ß, into COS-7 cells and assessed the interaction with mSin3A by a coimmunoprecipitation assay. The expression levels of wild-type AML1 and (181-210)SA were almost the same when they were overexpressed with PEBP2ß (Fig. 4A, lanes 5 and 6). Consistent with the previous report, (181-210)SA was hardly coimmunoprecipitated with mSin3A by the anti-mSin3A antibody, in contrast to wild-type AML1 (Fig. 4A, lanes 2 and 3). Next, we confirmed the intranuclear localization of (181-210)SA. FLAG-tagged wild-type AML1 or (181-210) SA was introduced into COS-7 cells, and the cells were stained with the anti-FLAG and anti-mSin3A antibodies. Both forms of AML1 were visualized exclusively in the nucleus by whole-cell staining (Fig. 4B and E). Wild-type AML1 was consistently colocalized with mSin3A, whereas (181-210)SA showed altered nuclear distribution, which does not coincide with that of mSin3A (Fig. 4D and G). In in situ nuclear matrix preparations, wild-type AML1 and mSin3A were colocalized in the nuclear matrix (Fig. 4H to J). In contrast, (181-210)SA was not detected in the nuclear matrix, where mSin3A was invariably located (Fig. 4K to M). The data we obtained indicate that targeting of AML1 to the nuclear matrix is dependent on the interaction with mSin3A, which suggests that AML1 is tethered to the nuclear matrix by mSin3A. These results are consistent with our findings that phosphorylation-induced release from mSin3A translocates AML1 from the nuclear matrix.
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(181-210)SA is also undetectable in the nuclear-matrix fraction, again suggesting the requirement for mSin3A interaction in nuclear-matrix targeting of AML1
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In embryonic fibroblasts from normal mice, we detected endogenous PEBP2ß (Fig. 8A, lane 1). On the other hand, PEBP2ß was not detected in PEBP2ß-deficient fibroblasts (Fig. 8A, lane 2). First, we examined whether AML1 would undergo ERK-mediated phosphorylation and subsequent proteolytic degradation in these cells. We cotransfected wild-type AML1 and ERK into PEBP2ß-deficient fibroblasts and stimulated the cells with EGF for 5 min. The cell lysates were analyzed by Western blotting. After 5 min of EGF stimulation, both phosphorylated and unphosphorylated AML1 were observed by Western blotting (Fig. 8B, lane 2). However, when we harvested the transfected cells after 30 min of EGF stimulation, there was a marked reduction in the amount of AML1 such that both forms of AML1 were barely detected (Fig. 8B, lane 3). When S249/266A was transfected into these cells together with ERK, followed by stimulation with EGF, neither the mobility shift nor the decrease in the amount of S249/266A was observed, regardless of EGF stimulation (Fig. 8B, lanes 4 to 6). These results suggest that AML1 is phosphorylated upon the activation of ERK in PEBP2ß-deficient fibroblasts and that phosphorylated AML1 is degraded with the lapse of time after EGF stimulation.
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Recapitulating phosphorylation-dependent degradation of AML1 in PEBP2ß-deficient cells, we subsequently tested whether mSin3A can protect AML1 from degradation in these cells. We transfected wild-type AML1 or (181-210)SA into the cells without EGF stimulation. As shown in Fig. 8D, expression of (181-210)SA was much lower than that of wild-type AML1 regardless of lactacystin treatment in PEBP2ß-deficient fibroblasts. These results suggest that mSin3A plays a key role in protecting AML1 from proteasome-mediated degradation and provide us with a potential mechanism for phosphorylation-induced degradation of AML1.
To address the relative contributions of PEBP2ß to the stability of AML1 when AML1 is phosphorylated, we investigated whether the interaction between AML1 and PEBP2ß would change when AML1 was phosphorylated. We transfected FLAG-tagged AML1 together with ERK and PEBP2ß into COS-7 cells. When we stimulated the cells with EGF, we observed the shifted band of AML1 corresponding to the phosphorylated AML1 (Fig. 9, lane 3). When we immunoprecipitated FLAG-tagged AML1 with the anti-FLAG antibody, constant amounts of PEBP2ß were coimmunoprecipitated regardless of EGF stimulation (Fig. 9, lanes 2 and 3). These results indicate that the interaction between AML1 and PEBP2ß does not change when AML1 is phosphorylated and that the degradation of phosphorylated AML1 is not due to loss of interaction with PEBP2ß. These results argue that association with mSin3A, in addition to PEBP2ß, is required for AML1 to exist stably in cells and that loss of interaction with mSin3A plays an important role in the degradation of phosphorylated AML1.
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(181-210)SA mutants of AML1.
We found that S249/266E and (181-210)SA are dissociated from the corepressor mSin3A. These results suggest that the transcriptional activities of these mutants are higher than those of wild-type AML1. Therefore, we tested the transcriptional activities of these mutants in transcriptional-response assays.
When we cotransfected wild-type AML1, S249/266E, or (181-210)SA into P19 cells in the presence of 10% FCS with the Tww-tk-Luc reporter, there was no significant difference between the transcriptional activities of wild-type AML1 and those of the mutants (data not shown). We showed the loss of stability of S249/266E and (181-210)SA (Fig. 8C and D), and these results may explain the decrease in the transcriptional activities of the mutants. Furthermore, when transfected into COS-7 cells in the presence of 10% FCS, a significant proportion of wild-type AML1 is phosphorylated without EGF stimulation (data not shown). We therefore cotransfected wild-type AML1, S249/266E, or (181-210)SA into COS-7 cells with Tww-tk-Luc reporter and created the cells with 0.1% FCS to eliminate the effect of FCS. In these experiments, S249/266E and (181-210)SA showed higher transcriptional activities than wild-type AML1 (Fig. 10, lanes 3 and 4). These results are consistent with our findings that the mutants lose interaction with the corepressor mSin3A. Compared with S249/266E, (181-210)SA showed reduced transcriptional activities. As shown in Fig. 8C and D, the expression of (181-210)SA is lower than that of wild-type AML1 regardless of lactacystin treatment, although wild-type AML1 and S249/266E were expressed at equivalent levels in the presence of lactacystin. These results suggest that the stability of (181-210)SA is lower than that of S249/266E, and this may explain the reduction in the transcriptional activity of (181-210)SA.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We demonstrated that mSin3A binds to AML1 when it is not phosphorylated and that mSin3A is released from phosphorylated AML1 upon activation of ERK. mSin3A is known to recruit class I HDACs (13). When associated with DNA, this complex can deacetylate chromatin and silence transcription. In the previous study, we showed that the transcription of TCRß induced by AML1 is dependent on phosphorylation of AML1 and that the AML1 mutant cannot be phosphorylated, showing reduced transcriptional activity. Here, we revealed that this reduction is alleviated by treatment with TSA. This suggests that the reduced transcriptional activity of the unphosphorylated AML1 mutant is due to its interaction with mSin3A. We conclude that mSin3A suppresses AML1 transcriptional activity by linking AML1 to HDACs when AML1 is not phosphorylated and that AML1 released from mSin3A upon ERK-induced phosphorylation becomes active as a transcriptional activator. These results provide a novel mechanism by which extracellular stimuli convert inactive forms of transcription factors into active ones.
Intranuclear localization of AML1 is regulated by interaction with mSin3A. AML1 is a nuclear protein that is localized in the nuclear matrix (47). The in situ immunofluorescence study and the biochemical-fractionation assay demonstrated that wild-type AML1 translocates from the nuclear matrix, where mSin3A is located when it is phosphorylated, supporting our finding that AML1 dissociates itself from mSin3A upon phosphorylation. The deletion mutant of AML1 lacking the interaction domain with mSin3A translocates from the nuclear matrix without phosphorylation, and a mutation in the nuclear-matrix attachment site disrupts the interaction between AML1 and mSin3A. One plausible explanation for this observation is that the interaction of AML1 with mSin3A plays an important role in targeting to the nuclear matrix. This is also supported by our findings that the mutant of AML1 that mimics phosphorylated AML1 lacks its association with mSin3A and that it is released from the nuclear matrix. The previous studies showed that the carboxyl terminus of AML1 is also necessary for targeting to the nuclear matrix (3, 47). From these results, it is suggested that both the mSin3A interaction domain and the carboxyl-terminal domain of AML1 are necessary for its localization in the nuclear matrix.
The importance of the nuclear-matrix targeting in the function of AML1 is not clear. AML1 was shown to colocalize with a subset of hyperphosphorylated RNA polymerase II in the nuclear matrix, which suggests that the association with the nuclear matrix is necessary to support AML1 transcriptional activity (46). In that study, however, only a subset of AML1 was associated with RNA polymerase II in the nuclear matrix whereas a significant amount of AML1 proteins was not. More recently, it was shown that targeting of AML1 to the nuclear matrix is required for the initiation of DNA replication (3). Our data raise the possibility that AML1 works as an active repressor when bound to mSin3A in the nuclear matrix. Once it is phosphorylated, transcriptional repression by AML1 may be abolished. Furthermore, AML1 released from the nuclear matrix may bind to the other regulatory elements in the chromatin and activate gene transcription. Further mechanisms that dictate AML1 activation after mSin3A release remain to be elucidated.
Regulation of the stability of AML1 by phosphorylation. The previous study reported that heterodimerization with PEBP2ß protects AML1 from proteasome-mediated degradation (7). However, our data indicate that phosphorylated AML1 is degraded in a time-dependent manner in COS-7 cells that contain endogenous PEBP2ß. These results raise the possibility that some proteins other than PEBP2ß may protect unphosphorylated AML1 from degradation and that their loss of interaction may cause degradation of phosphorylated AML1. Our analyses of the AML1 mutant lacking the domain interacting with mSin3A support the ability of mSin3A to protect AML1 from degradation. These results provide a novel regulatory mechanism that governs the stability of AML1.
In PEBP2ß-deficient fibroblasts, both phosphorylated and unphosphorylated AML1 were observed by Western blotting after 5 min of EGF stimulation (Fig. 8B, lane 2), and unphosphorylated AML1 was completely degraded after 30 min of EGF stimulation (Fig. 8B, lane 3). It is supposed that unphosphorylated AML1 is phosphorylated and degraded during the 30 min after EGF stimulation. In COS-7 cells, both phosphorylated and unphosphorylated forms of AML1 are observed after 30 min of EGF stimulation (Fig. 7, lanes 4 and 6). This discrepancy is due to the fact that COS-7 cells contain endogenous PEBP2ß, which protects AML1 from degradation.
A mechanism for regulation of AML1 activation induced by its phosphorylation. Recently, it was shown that phosphorylation of the transcription factor Elk-1 in response to ERK activation induces recruitment of the mSin3A-HDAC1 complex and repression of transcription from its target promoters (45). In our study, phosphorylation of AML1 induced the release of AML1 from mSin3A and enhanced AML1 transcriptional activity. Furthermore, phosphorylation regulates the stability of AML1 via interaction with mSin3A, manifested by the fact that phosphorylated AML1 is subjected to proteasome-mediated degradation. Thus, one additional role of corepressor release from AML1 may be to set a ceiling on the degree and duration of AML1 activation in response to cytokine and growth factor stimuli.
In summary, our studies have identified a novel mechanism by which ERK-induced phosphorylation regulates AML1 function. These findings will contribute to elucidation of the leukemogenic mechanisms derived from the dysfunction of AML1.
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ACKNOWLEDGMENTS |
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This work was supported in part by grants-in-aid from the Japan Society for the Promotion of Science; the Ministry of Education, Culture, Sports, Science and Technology; and the Ministry of Health, Labor and Welfare.
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FOOTNOTES |
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