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Division of Cancer Gene Regulation, Research Section of Disease Control, Institute for Genetic Medicine, Hokkaido University, Kita-ku, Kita 15 Nishi 7, Sapporo 060-0815, Japan
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Abstract |
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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The human MKL1 gene encodes a 931-amino acids protein (Ma et al. 2001; Mercher et al. 2001), which shares the homology with two related factors, MKL2 (also named MRTF-B) (Wang et al. 2002; Selvaraj & Prywes 2003) and myocardin (Wang et al. 2001), in the N-terminal MKL homology domain, basic region, glutamine-rich region, SAP domain, and coiled-coil (leucine zipper-like) domain. In contrast to myocardin, which is specifically expressed in heart and smooth muscle cells, MKL1 is expressed in most human tissues (Mercher et al. 2001; Wang et al. 2002; Du et al. 2004). Recently, MKL1 has been shown to function as a transcriptional coactivator of serum response factor (SRF), and it mediates signals from small GTPase RhoA to SRF (Cen et al. 2003; Miralles et al. 2003).
BSAC, a mouse homolog of MKL1, was identified as an inhibitor of TNF-
–induced apoptosis in murine embryonic fibroblasts (MEFs) prepared from TRAF2 and TRAF5 double-knockout mouse (Sasazaki et al. 2002). Additionally, MKL1 was also shown to be involved in the differentiation of skeletal myoblast cells or smooth muscle cells (Selvaraj & Prywes 2003; Du et al. 2004). These facts indicate that MKL1 is an important factor in the regulation of cell growth, differentiation, and apoptosis.
Small ubiquitin-related modifier (SUMO) is a modifier protein that is structurally related to ubiquitin, and there are three types of SUMO proteins in mammals; SUMO-1/PIC1/UBL1/sentrin/GMP1, SUMO-2/SMT3a, and SUMO-3/SMT3b (Yeh et al. 2000). SUMO is covalently conjugated to target proteins by a system analogous to the ubiquitin conjugation system. SUMO is activated by E1 enzyme (SAE1/UBA2 heterodimer) in an ATP dependent manner, is transferred to E2 enzyme UBC9, and then attaches to 
-amino groups of lysine residues in a minimal sumoylation motif (
KXE/D; is a hydrophobic amino acid residue and X represents any residue) present in the SUMO target (Yeh et al. 2000; Melchior et al. 2003). Recently, three classes of E3 ligase, such as PIAS, RanBP2, and Pc2, used for sumoylation, have been reported (Johnson & Gupta 2001; Kahyo et al. 2001; Pichler et al. 2002; Kagey et al. 2003). While E3 ligase is not absolutely essential for conjugation of SUMO, unlike ubiquitination, it is shown to enhance the sumoylation of specific target proteins (Melchior et al. 2003; Muller et al. 2004). Sumoylation is a reversible process and there are several mammalian SUMO-specific proteases, which are designated as the SENP-family of protein (Melchior et al. 2003).
In contrast to ubiquitination, sumoylation does not target a protein for degradation but may affect its localization, stability, and activity with crucial implication for several cellular processes (Hay 2005). Notably, the activity of several transcription factors such as p53 (Gostissa et al. 1999; Rodriguez et al. 1999), c-Jun (Muller et al. 2000), androgen receptor (Poukka et al. 2000), and LEF1/TCF (Sachdev et al. 2001; Yamamoto et al. 2003) is modulated by conjugation to SUMO.
In this study, we identified UBC9 as a novel binding partner of MKL1 by yeast two-hybrid screening and demonstrated that MKL1 is sumoylated both in vivo and in vitro. In addition, serum stimulation or co-expression of a constitutively active form of RhoA induced sumoylation of MKL1, suggesting that RhoA activation of MKL1 is a signal for MKL1 sumoylation. The major sites for sumoylation were mapped to lysine residues at positions 499, 576, and 624 located around the coiled-coil domain. It was also discovered that mutation at the three modification sites significantly enhances the transcriptional activity of MKL1, and the covalent attachment of SUMO-1 by gene fusion induces a reduction in MKL1 activity, thereby indicating that sumoylation represses the transcriptional activity of MKL1. Sumoylation of MKL1 also down-regulates the SRF-dependent transcription without affecting MKL1-SRF interaction. The findings of this study provide a novel mechanism of negative regulation of the MKL1 transcriptional activity by sumoylation.
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Results |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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In order to identify proteins that could be involved in the regulation of MKL1, we performed yeast two-hybrid screening with a mouse embryonic fibroblast cDNA library using the N-terminal region of MKL1 (amino acids 1-630) as a bait. Several MKL1-interacting proteins were identified from a screening of 
1 x 105 yeast transformants. DNA sequencing analysis revealed that one of the positive clones that specifically interacted with MKL1 was identical to UBC9 (Fig. 1A). UBC9 is an E2-conjugating enzyme required for the covalent modification of the SUMO proteins (Yeh et al. 2000).
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SUMO modification of MKL1 in vivo and in vitro
Since UBC9 has been demonstrated to be an E2 conjugating enzyme used in sumoylation (Yeh et al. 2000), we sought to examine whether MKL1 is a substrate for the SUMO-1 conjugation. In order to demonstrate the SUMO-1 modification of MKL1 in mammalian cells, an immunoprecipitation assay was performed. 293T cells were transiently transfected with FLAG-tagged MKL1 along with T7-tagged SUMO-1, the cell lysates were immunoprecipitated with anti-FLAG antibody, and the resulting immunoprecipitates were subjected to Western blotting with anti-T7 antibody or anti-FLAG antibody. As shown in Fig. 2A, three bands with molecular mass higher than that of intact MKL1, by 20-60 kDa, were detected by the anti-FLAG antibody when FLAG-MKL1 and T7-SUMO-1 were co-expressed; these slow migration bands were also detected by the anti-T7 antibody at a position that corresponded to the same molecular mass. These results indicated that MKL1 is modified with three molecules of SUMO-1 in vivo.
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To further examine whether MKL1 is indeed a substrate for SUMO-1 modification, we performed an in vitro sumoylation assay using MKL1 proteins that had been purified from 293T cells that expressed GST-fused MKL1 as a substrate. The MKL1 proteins were incubated with various combinations of recombinant E1 (SAE1/UBA2 heterodimer) and E2 (UBC9) along with SUMO-1 (GG), a mature form of SUMO-1. Analysis of the reaction products by Western blotting with anti-MKL1 antibody revealed more slowly migrating MKL1 bands only when E1 and E2 as well as SUMO-1 (GG) were present (Fig. 2C). These data strongly suggest that MKL1 is a substrate for SUMO-1 modification and that UBC9, which was identified as a binding partner of MKL1 by yeast two-hybrid screening, is an essential factor for MKL1 sumoylation.
In addition, we examined the possibility of MKL1 also being conjugated to SUMO-2 and SUMO-3, two other isoforms of SUMO (Yeh et al. 2000). As shown in Fig. 2D, SUMO-2- or SUMO-3-conjugated MKL1 was detected when either T7-SUMO-2 or T7-SUMO-3 was co-expressed with FLAG-MKL1 in 293T cells (Fig. 2D). However, the amount of SUMO-2- or SUMO-3-conjugated MKL1 was much lower than that of SUMO-1-conjugated MKL1, while the same amounts of expression plasmids of T7-tagged SUMOs were transfected into the cells and each isoform was expressed at a similar level. This data suggests that SUMO-1 is more efficiently conjugated to MKL1 as compared to SUMO-2 and SUMO-3.
RhoA activation enhances sumoylation of MKL1
Recent experiments have shown that MKL1 is activated by serum stimulation via RhoA-actin signal, and it stimulates SRF-dependent transcription (Cen et al. 2003; Miralles et al. 2003). Therefore, we tested the possibility of serum- or RhoA-induced activation of MKL1 affecting MKL1 sumoylation. First, the effect of serum stimulation, which activates RhoA, on sumoylation of MKL1 was examined. Serum-starved 293T cells that had been transfected with FLAG-tagged MKL1 along with T7-tagged SUMO-1 were stimulated with 20% fetal bovine serum (FBS) for the indicated periods. The cell lysates were immunoprecipitated with anti-FLAG antibody and the resulting immunoprecipitates were subjected to Western blotting with anti-T7 antibody or anti-FLAG antibody. As shown in Fig. 3A (upper panel), the sumoylated form of MKL1 was increased approximately threefold by the serum stimulation within 30 min, and no decrease in the level of the sumoylated form was observed within 2 h. Additionally, we examined the expression pattern of the endogenous junB gene, one of the target genes of MKL1-SRF signal (Selvaraj & Prywes 2004), in serum-stimulated 293T cells using reverse transcription-PCR method (Fig. 3A, lower panel). The level of the junB transcript increased within 30 min, reached a maximum at 90 min, and then decreased, falling to the prestimulation level by 120 min. These data indicated that the kinetics of serum induced MKL1 sumoylation is not correlated with that of the induction of junB gene by serum stimulation.
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Identification of SUMO acceptor sites
In order to map the lysine residue required for sumoylation, we generated a series of MKL1 deletion mutants (Fig. 4A, left panel) and assayed for SUMO-1 modification. 293T cells were transiently transfected with FLAG-tagged MKL1 deletion mutants along with or without T7-tagged SUMO-1, and the cell lysates were subjected to Western blotting with anti-FLAG antibody. The mutants MKL1 (1-630), MKL1 (471-931), and MKL1 (471-630) were strongly sumoylated; however, the other mutants, MKL1 (1-470), MKL1 (631-931), and MKL1 (
471-630) could not be modified by SUMO-1 (Fig. 4A, right panel). These results indicate that the lysine residues required for sumoylation are located in the region of amino acids 471-630.
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KXE/D; is a hydrophobic amino acid residue and X represents any residue) (Yeh et al. 2000; Melchior et al. 2003) (Fig. 4B, left panel). In order to test whether these lysine residues function as acceptor sites for sumoylation, we created substitution mutants in which lysine was changed to similarly charged arginine residue; the mutants were as follows: single mutants (K499R, K576R, and K724R), double mutants (K499/576R, K499/624R, and K576/624R), and triple mutant (K499/576/624R). The expression vectors of the lysine-substitution mutants of MKL1 were transfected into 293T cells along with or without T7-tagged SUMO-1, and the cell lysates were subjected to Western blotting with anti-FLAG antibody. As shown in Fig. 4B (right panel), single or double mutants of MKL1 were modified to a lesser extent by SUMO-1 in comparison with the wild-type protein. In the triple mutant MKL1 (K499/576/624R), the SUMO-1-conjugated form of MKL1 was completely abolished. The loss of SUMO-1-conjugation in the triple mutant was further confirmed by immunoprecipitation assay and in vitro sumoylation assay. The results of both the assays indicated that the K499/576/624R mutant could not be conjugated with SUMO-1 (Fig. 4C,D). Although MKL1 contains two additional sequences that are perfectly matched with the consensus sumoylation motif around Lys-356 (LKQE) and Lys-722 (LKKE) located outside the region of amino acids 471-630, MKL1 (K356R) and MKL1 (K722R), the substitution mutants of these two lysine residues were sumoylated to a similar extent as the wild-type MKL1 (data not shown). This suggests that these two potential sumoylation sites are not involved in MKL1 sumoylation. These results strongly indicate that three lysine residues, Lys-499, Lys-576, and Lys-624, are the major sites for sumoylation in MKL1. Since the three lysine residues are mapped around the coiled-coil domain of MKL1, which is required for forming homo- or hetero-dimer (Cen et al. 2003; Miralles et al. 2003; Du et al. 2004), it was suspected that replacement of the lysine residues might alter the dimerization. In order to examine this possibility, we performed a co-immunoprecipitation assay, and it was revealed that the K499/576/624R mutant could form the homodimer as well as the wild-type (data not shown). This result indicated that mutation of the three lysine residues does not affect the self-association of MKL1, and the abolishment of sumoylation in the K499/576/624R mutant is a direct effect of loss of sumoylation sites.
Sumoylation does not affect the subcellular localization and the protein stability of MKL1
Since SUMO proteins are known to affect cellular localization of target proteins (Hay 2005), we hypothesized that the sumoylation of MKL1 may be responsible for regulating its subcellular localization. To address this question, we transfected HeLa cells with expression vectors for green fluorescent protein (GFP)-tagged wild-type or K499/576/624R mutant of MKL1. As shown in Fig. 5A, the GFP-MKL1 K499/576/624R fusion was predominantly localized in nucleus, like wild-type protein, 36 h after transfection. Thus, it appears that mutation of the sumoylation sites does not affect the subcellular localization of MKL1.
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B, sumoylation has been shown to protect against ubiquitination of the same lysine residue, and thereby protect IB against ubiquitin-mediated degradation (Desterro et al. 1998). We thus tested the role of sumoylation on the stability of MKL1. 293T cells were transfected with expression vectors for FLAG-tagged wild-type or K499/576/624R of MKL1 and then treated with cycloheximide, an inhibitor of protein synthesis, for the indicated period. As shown in Fig. 5B, mutation of the sumoylation sites did not significantly affect the stability of MKL1 protein. These results indicate that sumoylation is not involved in the regulation of the subcellular localization and the protein stability of MKL1. Sumoylation induces transcriptional repression of MKL1
In order to evaluate the effect of MKL1 sumoylation on its transcriptional function, we determined the transcriptional activity of the MKL1 substitution mutants that are fused to GAL4 DNA-binding domain. 293T cells were transfected with the expression plasmid of GAL4-fused MKL1 substitution mutants along with pG5-Luc reporter plasmid that contains five GAL4 binding sites and major late promoter of adenovirus upstream of the luciferase gene, and the luciferase activities were measured. As shown in Fig. 6A, the luciferase activity of cells expressing GAL4-fused single mutants (K499R, K576R, and K624R) and GAL4-fused double mutants (K499/576R, K499/624R, and K576/624R) were two- to threefold and four- to sixfold higher, respectively, than that of the cells expressing GAL4-wild-type-MKL1. Moreover, GAL4-fused triple mutant (K499/576/624R), which lacks all the sumoylation sites, increased the luciferase activity by approximately 15-fold as compared to GAL4-wild-type-MKL1. These data strongly suggest that sumoylation is required for repression of the basal transcriptional activity of MKL1.
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Sumoylation of MKL1 regulates SRF-dependent transcription
Since MKL1 has been shown to be a transcriptional coactivator of SRF (Sasazaki et al. 2002; Wang et al. 2002; Cen et al. 2003; Miralles et al. 2003), we finally investigated the effect of MKL1 sumoylation on SRF-dependent transcriptional activation. 293T cells were transiently transfected with either T7-tagged-wild-type MKL1 or T7-tagged MKL1 (K499/576/624R) along with pSRE-Luc, which contains three tandem copies of the serum response element (SRE) and the Herpes simplex virus thymidine kinase promoter upstream of luciferase, and the luciferase activities were measured. The cells expressing MKL1 (K499/576/624R) displayed approximately twofold higher luciferase activity as compared to cells expressing wild-type MKL1 (Fig. 7A). We also examined, by co-immunoprecipitation assay, whether sumoylation of MKL1 influences the MKL1-SRF interaction. 293T cells were transiently transfected with either FLAG-tagged-wild-type MKL1 or FLAG-tagged MKL1 (K499/576/624R) along with T7-tagged SRF, and the cell lysates were immunoprecipitated with anti-FLAG antibody. The resulting immunoprecipitates were subjected to Western blotting with anti-T7 antibody or anti-FLAG antibody. As shown in Fig. 7B, both wild-type MKL1 as well as MKL1 (K499/576/624R) associated with SRF with similar efficiency.
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Discussion |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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In this study, we performed yeast two-hybrid screening and identified UBC9 as an MKL1-binding protein. UBC9 is a component of the sumoylation pathway and acts at the second step in the process of conjugating the SUMO to its target proteins (Yeh et al. 2000). Consistent with this function, MKL1 was found to be modified in vivo by three isoforms of SUMO (SUMO-1, SUMO-2, and SUMO-3). Additionally, in vitro sumoylation assay showed that MKL1 is conjugated with SUMO-1 in an E1 (SAE1/UBA2 heterodimer)- and E2 (UBC9)-dependent manner. Recently, three classes of E3-ligase, such as PIAS, RanBP2, and Pc2, used for sumoylation, have been identified and have been shown to enhance the sumoylation of several proteins including transcriptional regulators (Melchior et al. 2003; Muller et al. 2004; Hay 2005). Although our results showed that E3-ligase is dispensable for MKL1 sumoylation in vitro, it may be required for efficient sumoylation of MKL1 in vivo. Further identification and characterization of E3-ligase for sumoylation of MKL1 is required for understanding the detailed mechanism of the modification.
The small GTPase RhoA is known to activate SRF-dependent transcription independent of ternary complex factor (TCF) pathway (Hill et al. 1995). It has been recently reported that MKL1 appears to mediate RhoA-SRF signal in HeLa and NIH3T3 cells, because dominant negative MKL1 blocks RhoA activation of SRE reporter genes (Cen et al. 2003; Miralles et al. 2003). In NIH3T3 cells, it was found that MKL1 is sequestered into the cytoplasm by binding to G-actin under a serum-starved condition and it translocates to the nucleus after serum stimulation, because actin polymerization induced by serum-activated RhoA causes G-actin depletion (Miralles et al. 2003; Posern et al. 2004). However, the other reports showed that subcellular localization of MKL1 is not affected by serum stimulation (Selvaraj & Prywes 2003; Du et al. 2004). This suggests that the induction of nuclear localization of MKL1 may not be a common mechanism of RhoA activation of MKL1. Our data showed that the sumoylation of MKL1 is increased by serum stimulation and co-expression of constitutively active form of RhoA in 293T cells. This suggests that RhoA activation of MKL1 is a signal for sumoylation. It has been reported that sumoylation of STAT1 or Smad4 in vivo is enhanced by treatment with interferon-
or transforming growth factor-ß (TGF-ß), respectively (Ungureanu et al. 2003; Ohshima & Shimotohno 2003). In the case of Smad4, it was demonstrated that the enhancement of Smad4 sumoylation is caused by TGF-ß-dependent induction of PIASxß, an E3-ligase for Smad4 (Ohshima & Shimotohno 2003). Like Smad4, RhoA signal might induce the expression of a specific E3-ligase that enhances the sumoylation of MKL1. Alternatively, since MKL1 is reported to be phosphorylated by serum stimulation dependent on Rho activity (Miralles et al. 2003), it is also speculated that Rho-dependent phosphorylation might lead to sumoylation by increasing the interaction with UBC9 or reducing the interaction with the SUMO-specific proteases of the SENP-family. Although our results of the reporter gene analysis indicated that sumoylation represses the transcriptional activity of MKL1, the physiological role of sumoylation-dependent transcriptional inactivation is unknown. The result of RT-PCR analysis showed that the kinetics of junB mRNA induction is not correlated with that of the serum induced sumoylation of MKL1, suggesting that the transcriptional repression of MKL1 caused by sumoylation is not involved in the down-regulation of junB mRNA induction. However, it has been known that in some cases, the effect of sumoylation on the transcription is influenced by the promoter context (Hay 2005). To evaluate the physiological role of transcriptional repression of MKL1 induced by sumoylation, it is required to examine the effect of sumoylation on the induction of other MKL1 target genes.
A large number of proteins that are involved in transcriptional regulation are modified by SUMO. In particular, a role for sumoylation in the negative regulation of transcription appears to be emerging (Hay 2005). For example, sumoylation of the transcription factors Elk-1 (Yang et al. 2003), Sp3 (Ross et al. 2002), c-Jun (Muller et al. 2000), c-Myb (Bies et al. 2002), and C/EBP (Subramanian et al. 2003) results in a down-regulation of their transactivation functions. This is consistent with our findings that the transcriptional activity of MKL1 is repressed by sumoylation. However, the mechanism of sumoylation-dependent transcriptional repression of MKL1 is not entirely clear. Our results showed that the sumoylation dose not affect subcellular localization and protein stability of MKL1. It has been recently demonstrated that in Histone H4 (Shiio & Eisenman 2003) or the two transcriptional regulators, Elk-1 (Yang & Sharrocks 2004) and p300 (Girdwood et al. 2003), sumoylation promotes the interaction with histone deacthylase (HDAC) and HDAC-dependent transcriptional repression. Our preliminary experiments showed that the transcriptional activity of wild-type MKL1 is increased approximately twofold by treatment with HDAC inhibitor Trichostatin A (TSA), but the activity of MKL1 (K499/576/624R) mutants is not affected by TSA treatment (data not shown). This suggests that HDAC is involved in the sumoylation-dependent transcriptional repression of MKL1. It remains to be determined whether sumoylation of MKL1 directly recruits the HDAC.
It has been demonstrated SRF activates genes involved in smooth muscle differentiation and proliferation by recruiting specific cofactors, such as MKL family coactivators (MKL1 and Myocardin), and ternary complex factors of the ETS-domain family, respectively (Mack & Hinson 2005). Interestingly, when smooth muscle cells are stimulated with growth factors such as PDGF, the SRF-dependent expression of smooth muscle genes is repressed (Itoh et al. 2001; Wang et al. 2004). Since MKL1-SRF signal is known to be important for regulating the expression of smooth muscle genes, rather than growth response genes such as c-fos and egr-1, it is suggested that serum-induced sumoylation of MKL1 might contribute for the growth factor-induced repression of smooth muscle genes. To test this possibility, and to clarify the role of MKL1 sumoylation on the regulation of smooth muscle cell growth, it is needed to examine the correlation between the sumoylation state of MKL1 and the expression of smooth muscle genes.
The t(1; 22) chromosomal rearrangement in acute megakaryoblastic leukemia results in the fusion of the RBM15 and MKL1 genes (Ma et al. 2001; Mercher et al. 2001). The RBM15-MKL1 fusion protein contains the entire sequence of MKL1 protein, because the chromosome 22 breakpoint of the t(1; 22) is located in 60 bp upstream of the first ATG of MKL1 gene, and also the open reading frame of MKL1 is fused inframe with the coding sequence of RBM15. Recent reports have shown that the RBM15-MKL1 fusion strongly enhanced the MKL1 function, particularly by activating the c-fos reporter gene that contains SRE, despite the significantly lower levels of expression of the chimeric protein compared to normal MKL1 (Cen et al. 2003). RBM15 alone had no effect on the expression of c-fos reporter (Cen et al. 2003). This suggests that it does not regulate SRE but rather affects the activity of MKL1 when the two are linked due to the t(1; 22) translocation. These results suggest that these leukemias may be caused in part by the inappropriate activation of MKL1 and SRF target genes, including c-fos. It is not known how the fusion with RBM15 causes the up-regulation of the transcriptional activity of MKL1. Nonetheless, it is conceivable that MKL1 sumoylation and sumoylation-dependent transcriptional repression might be inhibited in the fusion protein by the structural change of MKL1. In order to test this possibility, a detailed analysis of the sumoylation state of RBM15-MKL1 fusion protein in the megakaryoblastic leukemia cells is required in future studies.
Our findings provide a novel mechanism of the negative regulation of MKL1-SRF signaling by sumoylation. A more detailed understanding of the regulatory mechanism of MKL1-dependent transcription by sumoylation may provide not only critical information on cell growth, differentiation, and apoptosis but also new therapeutic approaches for the MKL1-mediated pathological conditions including leukemias and other cancers.
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Experimental procedures |
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Human embryonic kidney 293T cells and human cervical cancer HeLa cells were cultured in Dulbecco's modified Eagle's medium (Nissui, Tokyo, Japan) supplemented with 10% fetal bovine serum (Invitrogen) at 37 °C in 5% CO2 atmosphere. Transfections were performed either by standard calcium phosphate precipitation method or by using Perfectin (Gene Therapy Systems, San Diego, CA, USA) according to the manufacturer's protocol.
Plasmid construction
The mammalian expression vectors, pCGT-T7-SUMO-1 and pcDNA3-FLAG-SUMO-1 (GG), and the yeast expression vector pGLex were gifted by Dr H. Ariga and Dr T. Taira of Hokkaido University. The mammalian expression vector pCMV-3 x FLAG was constructed by inserting the fragment encoding 3 x FLAG tag into pcDNA3.1(+) (Invitrogen), and the mammalian expression vector pCMV-GST was constructed by inserting the coding sequence of glutathione S-transferase into pcDNA3.1(+). The plasmid clone containing the human MKL1 cDNA (clone number KIAA1438) was obtained from Kazusa DNA Research Institute (Chiba, Japan). The coding region of MKL1 was amplified by PCR, and the resulting fragment was digested by restriction enzymes and subcloned into pCMV-3 x FLAG, pCMV-GST, pCI-neo-3 x T7 (gifted by Dr H. Sasajima of Hokkaido University), pCMX-Gal4 (gifted by Dr K. Umesono of Kyoto University) or pEGFP-C2 (Clontech). Deletion mutants or substitution mutants of MKL1 were generated by a PCR-based method. Expression vectors of SUMO-1-fused MKL1 protein were generated by inserting the fragment encoding SUMO-1 (1-96) into pCMX-GAL4-MKL1 and pCMX-GAL4-MKL1 (K499/576/624R). pLexA-MKL1 (1-630) was constructed by fusing the MKL1 (1-630) coding sequence in-frame with the LexA DNA-binding domain encoded in the vector pGLex. cDNAs encoding human SAE1, human UBA2, mouse SRF, mouse SUMO-2, mouse SUMO-3, and mouse RhoA were obtained by reverse transcriptase polymerase chain reaction (RT-PCR) using total RNA from HeLa cells or NIH3T3 cells. Coding region of UBC9 was amplified by PCR using UBC9 cDNA as a template, obtained from the positive clone isolated by the yeast two-hybrid screening. The resulting fragments were digested by restriction enzymes, subcloned into pBluescript KS(-) (Stratagene) or pGEM (Promega), and the DNA sequences were confirmed. Bacterial expression vectors, pGST-SAE1, pGST-UBA2, pGST-UBC9, and pGST-SUMO-1 (GG), were constructed by subcloning the fragments containing respective cDNAs into pGEX6P1 vector (Amersham Bioscience). The mammalian expression vectors of T7-tagged SUMO-2 or SUMO-3 were constructed by replacing the SUMO-1 coding sequence of pCGT-T7-SUMO-1 by cDNA encoding SUMO-2 or SUMO-3. The mammalian expression vector of SRF was constructed by inserting the coding sequence of SRF into pCI-neo-3x T7. RhoA V14, the constitutively active form of RhoA, was generated by PCR-based method using pBS-RhoA as a template. The mammalian expression vector of RhoA V14 was constructed by inserting the PCR product into pCI-neo-6x Myc that had been generated by inserting a fragment containing six copies of the Myc epitope into the pCI-neo mammalian expression vector (Promega).
Yeast two-hybrid screening
The Saccharomyces cerevisiae strain L40 was transformed with pLexA-MKL1 (1-630) and the mouse embryonic fibroblast MATCHMAKER cDNA library (Clontech). These were then plated on to media lacking tryptophan, leucine, and histidine and supplemented with 5 mM 3-amino-1,2,4-triazole (Sigma). Approximately 1 x 105 colonies were screened for growth in the absence of histidine and lacZ expression. The activity of ß-galactosidase was determined with X-gal as substrate. Plasmid DNAs derived from double positive clones were extracted from yeasts, and nucleotide sequences were determined.
Immunoprecipitation and Western blotting
293T cells were transiently transfected with expression plasmid as indicated. Forty-eight hours after transfection, the cells were washed with ice-cold phosphate-buffered saline (PBS) and harvested. The cells were then either lyzed with RIPA buffer containing 10 mM Tris-HCl (pH 7.5), 0.15 M NaCl, 1% NP-40, 0.1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, Complete Protease Inhibitor Cocktail (Roche Applied Science), and 10 mM N-ethylmaleimide (Sigma) for sumoylation analysis, or with IP buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% Triton X-100, 10% glycerol, and Complete Protease Inhibitor Cocktail for co-immunoprecipitation assay. The lysates were incubated on ice for 30 min, and the cell debris was removed by centrifugation at 13 000 g for 20 min. The resulting supernatants were incubated with anti-FLAG M2 affinity gel (Sigma) for 2 h at 4 °C, and the immunocomplex that was produced was washed five times with the same buffer. SDS-sample buffer (100 mM Tris-HCl (pH 6.8), 20% glycerol, 4% SDS, 5% 2-mercaptoethanol, and 0.001% bromophenol blue) was added to the beads, and the samples were boiled. In order to detect endogenous MKL1 protein, the cell lysates from HeLa cells were immunoprecipitated with anti-MKL1 (anti-BSAC) rabbit polyclonal antibody (Sasazaki et al. 2002) (gifted by Dr H. Nakano of Juntendo University) or nonspecific rabbit IgG bound to protein A-agarose (Roche). For Western blotting, whole cell lysates and immunoprecipitates were separated by SDS-PAGE and transferred on to Hybond ECL nitrocellulose membranes (Amersham Biosciences). The membranes were immunoblotted with the following antibodies: anti-T7 tag monoclonal antibody (Novagen), anti-FLAG (M2) monoclonal antibody (Sigma), anti-Myc monoclonal antibody (Clontech), anti-SUMO-1 (anti-GMP1) monoclonal antibody (zymed), and anti-MKL1 (anti-BSAC) rabbit polyclonal antibody. The bound primary antibodies were incubated with horseradish peroxidase-conjugated antibody against mouse or rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) and detected by ECL Western blotting detection reagents (Amersham Biosciences). Band images were detected by a LAS 1000 mini system (Fuji Film, Kanagawa, Japan) and densitometric analyses were performed using Image Gauge (Fuji Film). Proteins were normalized against the level of actin protein.
Expression and purification of recombinant proteins and in vitro sumoylation assay
GST-fused SAE1, UBA2, UBC9, or SUMO-1 (GG) were expressed in E. coli strain DH5 after induction with 0.1 mM isopropyl ß-D(-)-thiogalactopyranoside (IPTG). Cells were suspended in lysis buffer (PBS containing 0.5% Triton X-100, 0.1 mM DTT, and Complete Protease Inhibitor Cocktail) and sonicated for 3 x 10 s. After centrifugation, the cell lysate was incubated with glutathione-Sepharose 4B beads (Amersham Biosciences) for 2 h at 4 °C, and the beads were washed with lysis buffer. 293T cells seeded in 10-cm dishes were transiently transfected with 20 µg of pCMV-GST-MKL1 or pCMV-GST-MKL1 (K499/576/624R) for expressing the wild-type or K499/576/624R mutant of MKL1 protein. Forty-eight hours after transfection, the cells were washed with ice-cold PBS and harvested. The cells were lyzed with IP buffer, the lysates were sonicated for 10 s, and the cell debris was removed by centrifugation. The resulting supernatants were incubated with glutathione-Sepharose 4B beads for 2 h at 4 °C, and the beads were washed five times with IP buffer. The GST-fusion proteins bound to the beads were incubated with PreScission protease (Amersham Biosciences) for 2 h at 4 °C to remove the GST, and the resulting supernatants were collected. For in vitro sumoylation assay, the purified MKL1 protein was incubated with a reaction mixture containing 50 mM Tris-HCl (pH 7.4), 2 mM DTT, 5 mM ATP, 10 mM MgCl2, SAE1 (100 ng), UBA2 (100 ng), UBC9 (200 ng), and SUMO-1-GG (500 ng) for 2 h at 30 °C, and the MKL1 protein was detected by Western blotting with anti-MKL1 antibody.
GST pull-down assay
293T cells seeded in a 10-cm dish were transfected with pCMV-3 x FLAG-MKL1 (20 µg). Forty-eight hours after transfection, the cells were washed with ice-cold PBS and lyzed in a lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% tween-20, 1 mM DTT, and Complete Protease Inhibitor Cocktail. Bacterial expressed GST or GST-UBC9 proteins (4 µg each) bound to the glutathione-Sepharose 4B beads were incubated with the cell lysate for 2 h at 4 °C. After washing with lysis buffer, the SDS-sample buffer was added to the beads, and the samples were boiled. The bound MKL1 protein was detected by Western blotting with anti-FLAG M2 monoclonal antibody.
Reverse transcription-polymerase chain reaction (RT-PCR) analysis
Total RNA was isolated from 293T cells using TRIZOL reagent (Invitrogen). Total RNA 2 µg was then reverse-transcribed using the Transcriptor First Strand cDNA synthesis Kit (Roche, Mannheim, Germany) with random hexamers. The cDNA product was amplified with specific primers: for junB, forward (5'-CCAGTCCTTCCACCTCGACGTTTACAAG-3') and reverse (5'-GACTAAGTGCGTGTTTCTTTTCCACAGTAC-3'); for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), forward (5'-AAGGCTGGGGCTCATTT-3') and reverse (5'-CCGTATTCATTGTCATACCA-3'). The thermal cycle profile consisted of an initial denaturation at 94 °C for 2 min, followed by 30 cycles consisting of a 30 s denaturation at 94 °C, a 30 s annealing of primers at 55 °C (for junB) or 51 °C (for GAPDH), and a 30 s extension at 72 °C, followed by a final 7 min extension at 72 °C. The PCR products were then electrophoresed through 1.5% agarose gels.
Analysis of subcellular localization
HeLa cells grown on glass coverslips were transfected with expression vector for GFP-tagged wild-type or K499/576/624R mutant of MKL1. Thirty-six hours after transfection, the cells were fixed with 4% paraformalehyde in phosphate-buffered saline (PBS) for 20 min at 4 °C. The cells were then covered with Slowfade anti-fade reagent (Molecular Probes) and observed under a confocal laser fluorescent microscopy (LSM510, Carl-Zeiss).
Reporter gene assay
293T cells seeded in 24-well dishes were transfected with pG5-Luc (Promega) or pSRE-Luc (Clotech) reporter plasmid and with combinations of expression plasmid of effectors as indicated, along with pRL-CMV (Promega) for normalizing transfection efficiencies. The total amount of plasmid was adjusted using each empty vector. Twenty-four hours after transfection, the cells were harvested and luciferase activities were measured using Dual-Luciferase Reporter Assay System (Promega) and LB9506 luminometer (EG & G Berthold).
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: nakagawa{at}igm.hokudai.ac.jp
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Received: 17 January 2005
Accepted: 16 May 2005
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