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J. Biol. Chem., Vol. 280, Issue 6, 4894-4905, February 11, 2005
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From the Department of Pathology, Upstate Medical University, State University of New York, Syracuse, New York 13210
Received for publication, October 20, 2004
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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site of threonine 286. In addition, Mirk is activated through the stress signaling pathway by the mitogen-activated protein kinase kinase MKK3 (6) and interacts with p38 MAPK (7).
Mirk (minibrain-related kinase) is a member of the dyrk/minibrain family of arginine-directed serine/threonine protein kinases (8-10) and is identical to dyrk1B (11). Mirk is strongly transcriptionally up-regulated under conditions of mitogen deprivation, such as occurs when myoblasts are transferred from growth to differentiation medium. In particular when IGF-1 is eliminated (2, 3) there is an enrichment of active Mirk kinase in G0, the cell cycle stage in which the initial steps of differentiation occur. Whereas Mirk protein levels are very low in dividing myoblasts, they are increased at least 10-fold when myoblasts commit to terminal differentiation and are maintained at elevated levels in mature muscle cells (3). This unusual cluster of characteristics suggests the following model. Mirk levels increase under conditions favorable for terminal differentiation when myoblasts arrest in G0; Mirk aids in maintaining G0 arrest, and elevated amounts of Mirk protein are further activated by cellular stresses to mediate myoblast differentiation.
In this report we have investigated whether Mirk mediates myoblast differentiation solely by maintaining G0 arrest or whether Mirk has an additional role as a transcriptional activator in muscle differentiation. Mirk is also found in normal hepatocytes, and we have shown (6) that Mirk activates the hepatocyte transcription factor HNF1
by phosphorylation. In the myoblast system, depletion of Mirk by RNA interference strongly blocked expression of the myogenic regulatory factor myogenin (3), suggesting that Mirk might control myogenin expression. In the current study, we demonstrate that Mirk functions as a transcriptional activator of MEF2C, a known mediator of myogenin expression, by decreasing the nuclear concentration of class II HDACs that function as MEF2C inhibitors. Mirk phosphorylates these HDACs at a conserved serine within their nuclear localization signal.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Affymetrix Gene Chips—The MOE430A chip (mouse expression) was utilized. The chips contain 22,690 unique probe identifier oligonucleotide sequence sets. Data were analyzed by using the Microarray Suite 4.0 and, for a more stringent program, the Robust Multi-Chip Analysis program (Gene Traffic, Iobion) to identify genes exhibiting at least a 2-fold reduction, comparing the RNA populations treated with RNAi to Mirk to those treated with the vector control. 12,433 probe sets were identified in the RNAi population and 12,718 in the vector control. 193 transcripts were decreased, and 86 transcripts were increased.
Materials—Antibodies to myogenin, MEF2C, MyoD, and -tubulin were from Santa Cruz Biotechnology. Antibody to the N terminus of HDAC9 from ABGENT was used to detect MITR. Rabbit polyclonal antibody to a unique sequence at the C terminus of Mirk was raised as described (2). Polyvinylidene difluoride transfer paper Immobilon-P was purchased from Millipore. PLUS reagent and Lipofectamine were from Invitrogen; all radioactive materials were purchased from PerkinElmer Life Sciences; ECL reagents were from Amersham Biosciences, and tissue culture reagents were from Mediatech (Fisher). Alexa Fluor 594 (highly cross-adsorbed) secondary antibody conjugates were purchased from Molecular Probes. All other reagents were from Sigma.
Cell Culture—C2C12 mouse myoblasts and NIH3T3 cells were obtained from the ATCC and cultured according to their recommendations. C2C12 cells were maintained in growth medium (GM; Dulbecco's modified Eagle's medium, 4 mM L-glutamine, 4.5 g/liter glucose, containing 20% fetal bovine serum) and induced to undergo differentiation by switching to differentiation medium (Dulbecco's modified Eagle's medium containing 2% horse serum). NIH3T3 cells were maintained in Dulbecco's modified Eagle's medium, 4 mM L-glutamine, 4.5g/liter glucose, containing 7% bovine calf serum. Cells were used for experiments only from passages 3 to 10 from our frozen stocks.
Plasmids—Plasmids encoding either wild-type or kinase-inactive YF Mirk had been generated previously (2). The mouse myogenin promoter reporter construct mgnp224-luciferase (-184 to +48 from the start site) and the myogenin expression plasmid in pEMSV were from S. J. Tapscott, Fred Hutchison Cancer Center. Expression plasmids for human MEF2C and MEF2D in pCDNA1 and mouse MyoD in pEMSV, and GST-HDAC5 amino acids 1-283, were the gifts of E. N. Olson, University of Texas Southwestern Medical Center. The MITR expression plasmid was from D. Sparrow, The National Institute for Medical Research, London, UK. The Myf5 expression plasmid in pEMSV was from B. Winter, Technische Universitat Braunschweig. 3xMEF2-luciferase and gal4-luciferase were the kind gifts of Dr. Zhenguo Wu, Hong Kong University of Science and Technology. Wild-type HDAC5 (pGEX-HDAC5-1-283) and wild-type MITR were mutated by site-directed mutagenesis (Gene-Editor system, Promega). All mutant constructs were sequenced to confirm the mutated sequence.
Transfection and Luciferase Assay—Cells were transfected in 12-well plates for 6 h with Lipofectamine and PLUS reagent (Invitrogen) according to the supplier's manual. Transfected cells were lysed with Cell Culture Lysis Reagent and assayed for luciferase activity (relative luciferase unit) with luciferase assay reagent (Promega) in a Turner Designs Luminometer. The same lysates were diluted and assayed for -galactosidase activity to normalize transfection efficiency. Data shown are the means of triplicate measurements.
Cell Fractionation—5 x 105 C2C12 cells were plated in 60-mm dishes overnight and transfected with the indicated constructs. After 24 h, the cells were trypsinized, washed with ice-cold PBS, and resuspended in 150 µl of Buffer A (10 mM HEPES, pH 7.4, 1 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol) containing 0.5% Nonidet P-40 and protease inhibitor mixture (Sigma P 8340). The cell suspension was kept on ice for 10 min with occasional vortexing and monitored microscopically until >90% of the cells were lysed. The nuclei were pelleted at 800 x g for 10 min. The supernatant containing the cytoplasmic fraction was centrifuged at 16,000 x g for 10 min to remove any unlysed cells. The nuclear pellet was washed with 300 µl of Buffer A without Nonidet P-40 and centrifuged at 800 x g for 10 min. The cytoplasmic and nuclear fractions were solubilized in hot Laemmli sample buffer before analysis by SDS-PAGE.
Localization of MITR Constructs—C2C12 myoblasts were plated overnight in Lab-Tek 2-well chamber slides (1 x 105 cells per well) and then transfected with FLAG-MITR (1 µg of plasmid DNA) using Lipofectamine and PLUS reagent in Dulbecco's modified Eagle's medium. After 24 h of expression in GM, cells were washed with PBS, fixed in 4% paraformaldehyde for 5 min, washed with PBS, permeabilized with 0.1% Triton in PBS (PBST) for 5 min, and blocked with 10% normal goat serum in PBST for 30 min. The cells were incubated for 45 min with a 1:1000 dilution of either rabbit antibody to the N terminus of HDAC-9 or murine monoclonal antibody to the FLAG epitope, each diluted in 10% normal goat serum in PBST. After three PBS washes, the cells were incubated for 30 min with a 1:1000 dilution of goat anti-mouse antibody or goat anti-rabbit antibody conjugated to Alexa Fluor 594 (Molecular Probes). Nuclei were detected by incubation for 5 min with a 2 ng/ml solution of 4',6'-diamidino-2-phenylindole hydrochloride in PBST. Slides were mounted with Biomedia Gel/Mount. Monochrome fluorescence images were obtained at x400 using a Diagnostic Instruments SPOT RT camera mounted on a Nikon Eclipse E50i fluorescent microscope. SPOT RT software version 4.09 was used to pseudocolor and merge the images. Final figures were arranged using Adobe Photoshop version 7.0.
Northern Analysis for Myogenin mRNA—Total RNA was prepared by the RNeasy protocol (Qiagen) and analyzed for mRNA composition by microarray analysis. 4 µg of the remaining total RNA from each cell lysate was electrophoresed in a 1.1% agarose-formaldehyde gel, transferred to nylon membranes by downward capillary transfer, and cross-linked by baking in an oven. The membranes were hybridized to a 32P-labeled 1-kb EcoRI fragment of the myogenin cDNA construct. The probes were labeled with 32P by random priming (Promega). The blot was hybridized overnight at 68 °C with at least 107 cpm of the labeled probe, washed twice at room temperature for 15 min with 1x SSC, 0.1% SDS, and then washed for 20 min at 65 °C in 0.2x SSC, 0.1% SDS and autoradiographed.
In Vitro Kinase Assay—GST-Mirk or GST-YF-Mirk preparations were incubated for 5 min at 30 °C with 20 µl of kinase buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 0.5 mM dithiothreitol), containing 50 µM cold ATP plus 5 µCi of [
-32P]ATP and 1 µg of purified recombinant GST-HDAC5 protein as substrate, and then analyzed by PAGE and autoradiography.
Glutathione S-Transferase Fusion Proteins—Mirk and YF-Mirk were subcloned into the pGEX-6P1 vector (Amersham Biosciences), and the HDAC5 constructs were subcloned into the pGEX 4T1 vector (Amersham Biosciences), and both were expressed and purified as described.2
Metabolic Labeling—C2C12 cells were incubated for 6 h in serum-free low phosphate Dulbecco's modified Eagle's medium (9:1 ratio phosphate-free medium/normal medium) with 150 µCi of [32P]orthophosphate/2 ml of medium.
Immunodetection—Following treatment as indicated and washing two times with PBS, cells were lysed in hot SDS-PAGE sample buffer (50 mM Tris, pH 6.8, 2% SDS, 10% glycerol), boiled for 5 min, and vortexed vigorously. Protein determinations were made using Coomassie protein assay reagent from Pierce. Depending upon the experiment, 30-50 µg of cell lysate were blotted onto polyvinylidene difluoride membranes after separation on SDS-PAGE. The blots were blocked in 5% milk in TBST (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 0.1% Tween 20) for 1 h at room temperature and incubated for 1-2 h at room temperature with primary antibody in TBST buffer, 3% milk, and proteins were subsequently detected by enhanced chemiluminescence.
Band Analysis—Immunoblots were scanned using a Lacie Silver-scanner, and densitometry was performed using the IP Lab Gel program (Scanalytics).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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We first determined whether endogenous Mirk could up-regulate myogenin expression by transfecting a myogenin promoter reporter construct into growing C2C12 myoblasts. The cells were then switched to differentiation medium for 24 h to induce the up-regulation of Mirk (3). The cells were not sorted in this experiment, and the transfection efficiency was at best 30%, so some background was detected due to the residual endogenous Mirk activity. However, a clear difference was observed. The myogenin promoter reporter construct was activated 4-fold in differentiating C2C12 cells that had up-regulated endogenous Mirk expression as part of their differentiation program (Fig. 2). However, when the myogenin reporter construct was co-transfected with an expression plasmid for RNAi to Mirk to knockdown endogenous Mirk levels and cells were then cultured in differentiation medium, little increase in myogenin reporter activity was observed. Therefore, reduction of endogenous Mirk levels blocked the activation of the myogenin promoter.
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and phosphorylates HNF1 at a residue within its CBP-binding domain (6). We postulated that Mirk would also phosphorylate, and thus activate, one of the transcription factors that mediate transcription of the myogenin gene. We co-transfected a myogenin promoter reporter construct together with expression plasmids for either Mirk, kinase-inactive YF-Mirk, or the vector, and we then performed parallel experiments with the Mirk constructs in the presence of expression constructs for MEF2C, MEF2D, MyoD, Myf5, or combinations. Mirk activated the myogenin promoter reporter construct about 2-fold, whereas YF-Mirk had no effect (Fig. 3A, vector lanes). When either MEF2C or MEF2D were co-expressed with wild-type Mirk, there was a further 2-fold increase in reporter activity. MEF2D, and to a lesser extent MEF2C, increased myogenin reporter expression when co-expressed with kinase-inactive YF-Mirk, probably because each factor complexed with endogenous transcription factors. However, MEF2C clearly activated the myogenin reporter in a Mirk kinase-dependent manner. In contrast, MyoD increased the activity of the myogenin reporter construct about 10-fold, but expression of Mirk did not alter this activation (Fig. 3B). When MyoD and MEF2C were co-expressed, the stimulatory effect of Mirk was seen again (Fig. 3C). As in the case with MyoD, Myf5 increased the activity of the myogenin reporter in a Mirk-independent manner (Fig. 3D). However, when MEF2C was co-expressed with Myf5, the large activation of the myogenin reporter that was seen was further enhanced by expression of wild-type Mirk but not kinase-inactive Mirk. These data, taken together, demonstrated that Mirk functioned by activating MEF2C.
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in a kinase-dependent manner and that Mirk phosphorylated HNF1 at Ser-247 (6). We scanned MEF2C for residues conserved between human and mouse that were similar to the amino acids around the site phosphorylated by Mirk in HNF1 and other substrates (4, 5).2 No consensus sequences were detected in MEF2C, suggesting that Mirk activated MEF2C in an indirect manner. We tested this hypothesis by expressing MEF2C in C2C12 cells with either wild-type Mirk or kinase-inactive YF-Mirk for 24 h, then culturing cells in medium containing [32P]orthophosphate for 6 h, and immunoprecipitating the labeled MEF2C (Fig. 3E). Western blotting after autoradiography demonstrated that equal amounts of MEF2C were immunoprecipitated, but no difference in 32P labeling of the protein was seen between cells expressing ectopic wild-type Mirk and those expressing kinase-inactive YF-Mirk. Endogenous kinases phosphorylated MEF2C, but the abundant ectopic Mirk did not. In contrast, wild-type Mirk strongly phosphorylated co-expressed HNF1, whereas this phosphorylation was greatly diminished when kinase-inactive YF Mirk was co-expressed with HNF1 (data not shown). Because Mirk activates the transcriptional activity of MEF2C in a kinase-dependent manner (Fig. 3A), Mirk must phosphorylate one of the many factors that associate with MEF2, either to release its inhibition, such as class II histone deacetylases (HDACs), or to potentiate its activation, such as nuclear histone acetyltransferases. Mirk Opposes Transcriptional Inhibition Caused by HDACs—In myoblasts, HDACs associate with muscle regulatory factors such as myogenin and MyoD, whereas in differentiating myotubes HDACs have lost association with these regulatory factors and either bind preferentially to the retinoblastoma protein or are excluded from the nucleus (for a review see Ref. 17). Deacetylase inhibitors such as sodium butyrate are commonly used to enhance differentiation. However, if added to myoblasts just as they are signaled to differentiate by a switch from growth medium to differentiation medium, deacetylase inhibitors have been observed to do the opposite, to block myogenesis and to block the induction of myogenin (18, 19). It has been proposed that the release of HDACs from association with muscle regulatory factors at the start of differentiation can be blocked by the addition of a deacetylase inhibitor (20). We tested whether elevated levels of Mirk in stable C2C12 transfectants (3) would prevent a deacetylase inhibitor from blocking the release of HDACs from MEF2C in transcriptional complexes at the myogenin promoter and thus enhance the transcription of myogenin. C2C12 cells that had been stably transfected with Mirk and control transfectants were transferred to differentiation medium in the presence of an increasing concentration of the histone deacetylase inhibitor sodium butyrate, and the induction of myogenin was measured by Western blotting. Higher levels of myogenin protein were observed in the presence of overexpressed Mirk (Fig. 4A). Thus Mirk diminished the effect of the deacetylase inhibitor and restored some myogenin transcription, with the greatest effect seen at the lower levels of sodium butyrate. The hypothesis that Mirk antagonized the function of endogenous histone deacetylases was next tested directly.
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We next tested whether MITR would antagonize myogenin reporter activation by Mirk. Increasing concentrations of MITR inhibited the activation of the myogenin reporter (Fig. 4D), as has been reported by many investigators. The highest concentration of MITR expression plasmid tested inhibited the activity of the myogenin reporter 3-fold (Fig. 4D). Wild-type Mirk partially blocked the transcriptional inhibition caused by MITR, whereas no such effect was seen when kinase-inactive Mirk was co-expressed (Fig. 4D). Thus Mirk activated the myogenin promoter reporter by blocking, in a kinase-dependent manner, the transcriptional repression caused by the class II HDAC MITR.
Mirk Phosphorylates HDAC5 at a Site Conserved in Class II HDACs—Activated CaMK regulates MEF2-HDAC interactions by phosphorylating two conserved HDAC sites flanking a nuclear localization sequence near their N termini in response to calcium signaling (22, 23). These sites serve as binding sites for 14-3-3 proteins when phosphorylated by CaMK (23). In HDAC5, one CaMK site is Ser-259. We found a consensus region for Mirk phosphorylation 20 amino acids from the CaMK site at serine 279, which is conserved within all class II HDACs (Fig. 5A). To test whether Mirk phosphorylated this site, we mutated this site in HDAC5 to S279A both in the presence of the S259A mutation or with this site unmutated. Neither HDAC5-S259S/S279A nor HDAC-S259A/S279A was a substrate for Mirk. However, Mirk strongly phosphorylated both wild-type HDAC5 and HDAC5-S259A/S279S (Fig. 5B), demonstrating that Mirk phosphorylates HDAC5 at Ser-279 but not at the CaMK site of Ser-259. Mutation of serine 279 in HDAC5 to the nonphosphorylatable alanine did not prevent nuclear export in the presence of activated CaMK (22) in transient transfection experiments in COS cells. However, serine 279 is within the nuclear localization region, so we next tested whether phosphorylation of a class II HDAC by Mirk would block its import into the nucleus.
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-tubulin (Fig. 6A). There is very little endogenous Mirk in growing myoblasts, and when MITR was expressed in the absence of exogenous Mirk, about half of the MITR was found in the nucleus (Fig. 6A, 1st 2 lanes, also note the lack of endogenous Mirk). However, when wild-type MITR was co-expressed with Mirk, there was a dramatic 6-fold decrease in the amount of MITR in the nuclear fraction but no increase in the amount of cytoplasmic MITR. In sharp contrast, when MITR was co-expressed with kinase-inactive Mirk, the amount of MITR in the nucleus doubled. These data suggest the following model: Mirk phosphorylates the class II HDAC MITR within its nuclear localization domain thus decreasing the rate of its translocation into the nucleus. Exogenous kinase-dead YF-Mirk did not block MITR from entering the nucleus. In fact, both MITR and kinase-inactive YF-Mirk were enriched in the nucleus. This unusual pattern was seen in duplicate experiments and may suggest some continued association of YF-Mirk and MITR.
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When MITR was expressed in COS cells, C2 myoblasts, and 10T1/2 fibroblasts, MITR localized to discrete nuclear bodies, but in response to CaMK signaling, MITR remained in the nucleus but was evenly distributed, as determined by immunofluorescence studies (26, 27). Ectopic MITR was seen in the cytoplasm of C2 skeletal myotubes after 9 days in differentiation medium (26). In contrast to these experiments, MITR was readily detected biochemically in the cytoplasmic fractions of C2C12 skeletal myoblasts (Fig. 6, A and B). To resolve these apparent differences, we examined the location of ectopic MITR in C2C12 myoblasts by immunofluorescence analysis using the same antibody, anti-HDAC9, which readily detected MITR in the cytoplasm of fractionated cells in multiple experiments. In each case, MITR was only detected in the nucleus by immunofluorescence (Fig. 6C). We repeated this experiment using FLAG epitope-tagged MITR, and we detected the FLAG epitope in immunofluorescence experiments. Again, the majority of FLAG epitope-tagged MITR was found localized in the nucleus. MITR occasionally was detected throughout the entire cell (Fig. 6C), as expected from the fractionation experiments. This cytoplasmic and nuclear localization was observed in 7% of the cells (74 of 1053 cells examined). This low frequency prevented analysis of the effect of Mirk on MITR nuclear localization by immunofluorescence, parallel to the studies shown in Fig. 6, A and B. In our fractionation scheme a known transcription factor, MyoD, was localized only in the nuclei, and a known cytoskeletal protein, -tubulin, was found almost exclusively in the cytoplasmic fractions in multiple experiments (Fig. 6, A and B). We postulate that cytoplasmic MITR is complexed with other cellular proteins in vivo which block the epitopes recognized by the anti-HDAC9 antibody and, to a lesser extent, the FLAG epitope. In contrast cytoplasmic MITR was readily detected when samples were boiled in SDS-PAGE loading buffer before Western blotting. We have demonstrated previously that Mirk, which dimerizes to a 140-kDa form in vivo (28,) is localized in higher molecular weight complexes in vivo,uptoat least 660 kDa by analysis by fast protein liquid chromatography (7). MKK3, the activator kinase for Mirk, can readily be detected by Western blotting of these complexes but cannot be immunoprecipitated from the same complexes (7). Possibly Mirk associates with MITR or other newly synthesized HDACs in such large complexes, preventing their detection by immunofluorescence techniques.
Decreased Abundance of Phosphomimetic MITR in the Nucleus—The results of the fractionation experiments (Fig. 6) implied that a wild-type MITR construct would accumulate in the nucleus to a greater extent than a MITR construct that was mutated to the phosphomimetic aspartic acid residue at the Mirk phosphorylation site. We tested this model by mutating MITR at Ser-243, the Mirk phosphorylation site homologous to the HDAC5 site of Ser-279 (Fig. 5A). Wild-type MITR, MITR mutated at the Mirk site to the nonphosphorylatable residue alanine (S243A), or a MITR phosphomimetic site mutant (S243D) was transiently transfected into C2C12 cells and maintained for 24 h in growth medium, and then one set of cells was switched into differentiation medium for 24 h. Each set of cells was then separated into nuclear and cytoplasmic fractions that were analyzed by Western blotting. The fractionation was effective as only the nuclear fractions contained MyoD, whereas the cytoplasmic fractions were highly enriched in -tubulin, and the amounts of either MyoD or tubulin were similar in cells expressing each construct (Fig. 7A).
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We had considered the possibility that the phosphomimetic construct might be expressed at a lower level than the wild-type construct. Parallel cultures were transfected with each construct, maintained in growth medium for 24 h, and harvested at the same time as the GM experiment shown in Fig. 7A. However, these cells were not fractionated before lysis. Western blotting demonstrated that the three constructs were expressed at similar levels, and there was no decrease in expression of MITR-S243D, which could explain the decrease in its nuclear abundance in fractionated cells (Fig. 7B). Therefore, studies using the mutant MITR constructs gave results generally similar to the co-expression studies. When MITR was phosphorylated by Mirk, its transport to the nucleus was inhibited so less MITR accumulated in the nucleus, and thus less was available to inhibit MEF2C.
Two hypotheses were considered to account for the lack of accumulation of MITR within the cytoplasm when Mirk was overexpressed (Fig. 6A). In the first hypothesis, Mirk only blocked MITR transport into the nucleus and had no effect on the proteolysis of MITR within the cytoplasm. Alternatively, the phosphorylation of MITR by Mirk could also trigger the rapid turnover of MITR within the cytoplasm. We have shown previously that Mirk can mark proteins for proteolysis. Phosphorylation by Mirk shortens the half-life of cyclin D1 in myoblasts. Mirk phosphorylates cyclin D1 at Thr-288 when cyclin D1 is bound to GSK3 (5). GSK3 itself is well known to destabilize cyclin D1 by phosphorylation at the adjacent site of Thr-286 (29).
MITR that did not reach the nucleus was rapidly turned over within the cytoplasm when cells began to differentiate (Fig. 7A). However, the hypothesis that phosphorylation of MITR at Ser-243 by Mirk would directly trigger the rapid turnover of MITR within the cytoplasm was not supported. There was no increase in the amount of MITR-S243A in the cytoplasm in either growing or differentiating myoblasts, compared with wild-type MITR. Therefore, phosphorylation by Mirk at this site was not essential for proteolysis of MITR.
Mutation of the Mirk Phosphorylation Site in MITR Does Not Alter the Capacity of MITR to Function as a Transcriptional Inhibitor—Three MITR constructs were compared for their ability to inhibit the activity of an MEF2-dependent luciferase reporter (3xMEF2-luc). MEF2C activates this reporter although the class II HDAC, HDAC4, blocks its activity in a dose-dependent manner (21). NIH3T3 cells were transiently co-transfected with the 3xMEF2-luc reporter plasmid together with an expression plasmid for MEF2C and expression plasmids for either wild-type MITR, MITR mutated at the Mirk phosphorylation site to the phosphomimetic (S243D), or MITR mutated at the Mirk phosphorylation site to the nonphosphorylatable alanine (S243A). All three constructs inhibited MEF2C to the same extent, about 6-fold (Fig. 7C, mean ± S.D. of three replicate points). Therefore, phosphorylation of MITR by Mirk did not alter the capacity of MITR to inhibit MEF2C, but instead modulated the amount of MITR which reached the nucleus.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The abundance of nuclear HDACs is regulated by nuclear localization and nuclear export signals as well as by phosphorylation of 14-3-3-binding sites. Nuclear/cytoplasmic shuttling of class II HDACs depends on a C-terminal nuclear export sequence and a nuclear localization sequence (NLS) in the N terminus (22). Microinjected HDAC4-GFP was shown previously (35) to be localized to the cytoplasm of undifferentiated C2C12 myoblasts, consistent with our observation that some of the ectopic MITR molecules are found in the cytoplasm (Fig. 6, A and B). Microinjection of full-length HDAC4 resulted in cytoplasmic localization in 30-40% of IMR90-EIA fibroblasts, whereas expression of the C-terminal fragment, which lacks the NLS as well as the MEF2 binding region, resulted in cytoplasmic localization in 90% of the cells (12). Formation of an MEF2-HDAC4 complex efficiently targets this complex to the nucleus, whereas deletion of the MEF2 NLS results in cytoplasmic retention of this complex (30).
In the current study we have found that the kinase Mirk/dyrk1B is essential for the transcription of myogenin and that Mirk induces myogenin transcription through indirect activation of the MEF2 transcription factor. Mirk relieves the inhibition of MEF2 by the class II HDAC MITR in a kinase-dependent manner by phosphorylating the class II HDACs on a conserved serine within the highly conserved nuclear localization sequence. Separation of C2C12 cells into nuclear and cytoplasmic fractions and Western blotting analysis demonstrated that the nuclear entry of the HDAC MITR was inhibited when MITR was co-expressed with Mirk. The higher the ratio of Mirk to MITR expressed, the more MITR was constrained to the cytoplasm. In contrast, when kinase-inactive Mirk was co-expressed, MITR was able to concentrate in the nucleus. Cell fractionation studies with mutant MITR constructs confirmed the interpretation that phosphorylation by Mirk decreased the rate of accumulation of MITR in the nucleus. A phosphomimetic construct of MITR mutated at the Mirk phosphorylation site exhibited less accumulation in the nucleus than wild-type MITR. When C2C12 cells were induced to differentiate, MITR was solely detected in the nucleus, but more wild-type MITR was found there than the phosphomimetic construct. Mirk phosphorylated class II HDACs at a conserved serine within the nuclear localization domain and inhibited accumulation of MITR in the nucleus, presumably by blocking a step in nuclear import. Another example of a serine/threonine kinase that blocks nuclear import is Akt/protein kinase B. Akt is well known for its ability to block the nuclear import of the CDK inhibitor p21 Cip1 by phosphorylating p21 at threonine 145 within its nuclear localization domain in Her-2/neu-transformed NIH3T3 cells (24).
The ability of Mirk to activate myogenin transcription complements the various other functions of Mirk during muscle differentiation. Terminal myoblast differentiation occurs when myoblasts cease cycling and become arrested in G0/G1. Myoblast growth arrest is mediated by increased expression of the CDK inhibitor p27 Kip1 and by decreased expression of the G1 cyclin, cyclin D1. However, cell cycle regulation occurs at both the transcriptional level and the post-translational level in myoblasts. Mirk functions at the post-translational level to maintain p27 protein levels while reducing the abundance of cyclin D1. Mirk phosphorylates p27 at Ser-10, thus stabilizing p27 and blocking its nuclear export and degradation (4). At the same time, Mirk acts together with GSK3 to induce cyclin D1 degradation by phosphorylating cyclin D1 at Thr-288, whereas GSK3 phosphorylates the adjacent site of Thr-286 (5). Supporting the results of these phosphorylation studies was the observation that depletion of endogenous Mirk by RNAi in differentiating myoblasts increased cyclin D1 protein and decreased p27 protein without modulating the mRNA levels of either molecule (4, 5). The current study demonstrates the multifunctional role of Mirk in myoblast differentiation. In addition to the previously established role for Mirk in facilitating cell cycle arrest, Mirk is now shown to facilitate progression of the subsequent differentiation program through transcriptional activation of the muscle regulatory factor myogenin.
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FOOTNOTES |
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To whom correspondence should be addressed: Upstate Medical University, Pathology Dept., 2303 Weiskotten Hall, 750 East Adams St., Syracuse, NY 13210. Tel.: 315-464-7138; Fax: 315-464-8419; E-mail: friedmae{at}upstate.edu.
1 The abbreviations used are: CDK, cyclin-dependent kinase; HDACs, histone deacetylases; CaMK, calcium/calmodulin-dependent protein kinase; HNF1, hepatocyte nuclear factor ; RNAi, RNA interference; MITR, MEF2-interacting transcriptional repressor; NLS, nuclear localization sequence; EGFP, enhanced green fluorescent protein; PBS, phosphate-buffered saline; GM, growth medium.
2 S. Mercer, D. Ewton, S. Lim, T. Mazur, and E. Friedman, submitted for publication.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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