Skip Navigation

This Article
Right arrow Abstract Freely available
Right arrow Print PDF (140K) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (81)
Right arrowRequest Permissions
Right arrow Commercial Re-use Guidelines
for Open Access NAR Content
Google Scholar
Right arrow Articles by Yang, C. C.
Right arrow Articles by Prody, C. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yang, C. C.
Right arrow Articles by Prody, C. A.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Nucleic Acids Research Pages 4771-4777  

Interaction of myocyte enhancer factor 2 (MEF2) with a mitogen-activated protein kinase, ERK5/BMK1
Introduction
Materials And Methods
   Accession nos
   Cloning of MEF2D
   Construction of a rat aorta cDNA library and yeast two hybrid screening
   Glutathione S-transferase (GST)-pull down assay
   Co-immunoprecipitation between MEF2 and ERK5/BMK1
   In vitro kinase assay
   Cell culture and reporter assays
Results
   ERK5/BMK1 interacts with the N-terminus of MEF2D in a yeast two hybrid screen
   The N-terminus of MEF2 mediates the interaction between MEF2 and ERK5 in vitro
   MEF2 co-immunoprecipitates with ERK5 in vivo
   ERK5/BMK1 phosphorylates MEF2D and MEF2C in vitro
   The transcriptional activity of MEF2A and MEF2C, but not MEF2D is enhanced by ERK5/BMK1
Discussion
Acknowledgement
References

Interaction of myocyte enhancer factor 2 (MEF2) with a mitogen-activated protein kinase, ERK5/BMK1

Interaction of myocyte enhancer factor 2 (MEF2) with a mitogen-activated protein kinase, ERK5/BMK1

Chih-Chin Yang1,4,5, Olga I. Ornatsky2, John C. McDermott3, Tony F. Cruz1,4 and Catherine A. Prody1,5,*

1Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada, 2Department of Biology, Faculty of Pure and Applied Science, York University, Toronto, Ontario M3J 1P3, Canada, 3Department of Biological Sciences, University of Durham, South Road, Durham DH1 3LE, UK, 4Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada and 5Division of Cardiovascular Research, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada

Received April 27, 1998; Revised and Accepted September 3, 1998

DDBJ/EMBL/GenBank accession nos AJ005424 and AJ005425

ABSTRACT

Myocyte enhancer factor 2 (MEF2) has been implicated in the complex hierarchical regulation of muscle-specific gene expression and differentiation. While the MyoD family members are able to initiate the skeletal muscle differentiation program, whether MEF2 is sufficient in directing skeletal muscle differentiation is still controversial. Furthermore, how MEF2 transactivates its target genes is not fully understood. It has been suggested that the interactions of MEF2 with other factors modify its transcriptional activity. Therefore, the identification of MEF2-interacting factors may be important in understanding the mechanism by which MEF2 activates its target genes. In this study, a mitogen-activated protein kinase (MAP kinase), ERK5/BMK1 was found to interact with MEF2 in a yeast two hybrid screen. The interaction was confirmed by a glutathione S-transferase-pull down assay and a co-immunoprecipitation study indicating that endogenous ERK5 and MEF2 interact with each other in vivo. The interacting domain of MEF2 was mapped to the N-terminus which contains the highly conserved MADS and MEF2 domains. Functionally, ERK5/BMK1 was able to phosphorylate MEF2 in vitro. Furthermore, when cotransfected with ERK5/BMK1, the transactivation capacity of MEF2 was enhanced. These results suggest that the functions of MEF2 could be regulated through ERK5/BMK1.

INTRODUCTION

Molecular mechanisms regulating skeletal muscle development have been extensively studied (reviewed in 1). Proteins of the basic helix-loop-helix (bHLH) myogenic regulators (MyoD, MRF4, Myf5 and myogenin) have been demonstrated as key regulators of myogenesis with the ability to activate the skeletal muscle differentiation program (reviewed in 1). Conversely, relatively little is known about smooth and cardiac muscle development. The presence of myocyte enhancer factor 2 (MEF2) sites in the regulatory regions of many muscle-specific genes that are expressed in skeletal, cardiac and smooth muscle cells, suggests that it may be a common component of the differentiation programs in all three muscle lineages (reviewed in 2). In fact, by generating a loss-of-function of the D-MEF2 gene in Drosophila embryos, undetectable levels of D-MEF2 protein and mRNA correlated with the failure of differentiation of visceral, somatic and cardiac muscle (3,4). Additionally, microinjection of a dominant-negative form of MEF2A inhibited the myogenic conversion of C3H10T1/2 fibroblasts by MyoD and also the differentiation of C2C12 myoblasts (5). Moreover, in mice homozygous for a null mutation of MEF2C, cardiac morphogenesis was inhibited (6).

MEF2 was originally characterized as having a DNA-binding activity recognizing an A/T-rich element in the promoters of many skeletal muscle-specific genes (7). There are four MEF2 genes, referred to as MEF2A, -B, -C and -D, identified in vertebrate species while there is a single gene, D-MEF2, found in Drosophila (reviewed in 2,8; 10,10). MEF2 factors belong to a superfamily of transcription factors that have a highly conserved region termed the MADS domain, named after the yeast MCM1, plant homeotic genes agamous and deficiens and human serum response factor (SRF). The MADS domain is located at the N-terminus (the first 56 amino acids) of MEF2. Immediately following the MADS domain, there is a MEF2 domain (29 amino acids) which is unique to MEF2 factors. One feature of many MADS domain proteins is their propensity to make homo- and heterotypic protein-protein interactions which modulate their activity. For example, MCM1 acts in concert with the [alpha]1 repressor or [alpha]2 activator to repress or activate cell-type specific genes in [alpha]-type yeast, respectively. Also, modulation of SRF by ternary complex factors contributes to the transcriptional activation of the c-fos gene through the serum response element (reviewed in 8 and therein). Both MADS and MEF2 domains of MEF2C have been shown to be responsible for DNA binding and protein-protein interactions (11). The transactivation domain of these proteins has been mapped to the C-terminus of MEF2A, MEF2C and MEF2D (11-13).

While the MyoD family is a key regulator of skeletal myogenesis, whether MEF2 itself is sufficient in directing skeletal muscle differentiation is still controversial (2,14). How MEF2 transactivates its target genes is not clear. There are suggestions that the transcriptional activity of MEF2 is modified by the recruitment of other factors. For example, MEF2 has been shown to bind to the bHLH region of myogenin and cooperatively activate expression of muscle-specific genes (15). Furthermore, direct association of p300/CBP [CREB (cAMP-responsive element binding protein) binding protein] and the MADS domain of MEF2C was shown to potentiate transcriptional activity of MEF2C (16). Recently, MEF2C was shown to interact with p38 (a member of the MAP kinase family) and the phosphorylation of MEF2C by p38 enhanced its transcription activity (17). Since all of these examples require protein-protein interactions to regulate MEF2 activity, the identification of other MEF2-interacting factors may be important in fully understanding the mechanism by which MEF2 activates its target genes.

The yeast two hybrid system has proven to be a powerful tool in identifying protein-protein interactions and subsequently to isolate cDNAs encoding proteins that interact with a protein of interest (reviewed in 18). In this study, we screened a smooth muscle cDNA library using the yeast two hybrid system and identified several MEF2-interacting proteins. One of them carries a partial cDNA encoding a rat homologue of human ERK5 (or BMK1) which was cloned independently by two groups and was shown to belong to the MAP kinase family by sequence analysis (19,20). MEF2 proteins were found to co-immunoprecipitate with ERK5 in vivo. The MADS and MEF2 domains of MEF2 were sufficient to mediate the interaction. We found that ERK5/BMK1 was capable of phosphorylating MEF2D and MEF2C in vitro. Functionally, the transcriptional activity of MEF2C and MEF2A was enhanced when co-expressed with ERK5/BMK1 while the transcriptional activity of MEF2D was not affected.

MATERIALS AND METHODS

Accession nos

DDBJ/EMBL/GenBank accession nos are AJ005424 for ERK5/BMK1 and AJ005425 for MEF2D.

Cloning of MEF2D

Total RNAs were extracted from the hearts of neonatal Wistar rats using Trizol reagent (Gibco-BRL) according to the manufacturer's instructions. First strand cDNAs were synthesized using reverse transcriptase (Superscript II, Gibco-BRL) and oligo(dT) as primers. MEF2D-specific synthetic oligonucleotides (5[prime]-CTCTCTGGCACTAAGGACCC-3[prime] and 5[prime]-ACCATCACTTTAATGTCCAAGTATC-3[prime]) were designed according to the published nucleotide sequence of mouse MEF2D and used to amplify MEF2D cDNA by the polymerase chain reaction (PCR). The PCR products were cloned into pBluescript-SK(+) (Stratagene) and both the sense and anti-sense strands were sequenced by the dideoxy chain termination method (accession no. AJ005425).

Construction of a rat aorta cDNA library and yeast two hybrid screening

A neonatal rat aorta cDNA library was constructed using [lambda]-ACT vectors as described previously (21,22). The yeast two hybrid system including yeast strain Y190 and Y187, pAS1-CYH, pSE1111 (SNF1) and [lambda]-ACT vectors was a generous gift ofS. Elledge (Department of Biochemistry and Human and Molecular Genetics, Baylor College of Medicine, TX). Library screening was described in detail in the report by Durfee et al. (21). Briefly, pAS1-CYH vector harboring a fragment of rat MEF2D cDNA encoding amino acids 19-167 (pAS1-CYH-MEF2D) was used to transform Y190. pAS1-CYH-MEF2D-bearing Y190 yeast were then transformed with the constructed library and plated on selection medium without tryptophan, leucine and histidine and with 25 mM 3-amino 1,2,4-triazole (3-AT) (Sigma). Histidine-positive (His+) colonies were then assayed for [beta]-galactosidase (Lac-Z) activity. To determine the specificity of interactions, colonies positive for both reporters, His+ and Lac-Z+, were plated on a 20 mM cycloheximide (Sigma)-containing plate to select against pAS1-CYH-bearing yeast and obtain cDNA-containing Y190 yeast. cDNA-containing Y190 yeast were then mated with Y187-containing partial p53, lamin C (Clontech), SNF1 or MEF2D fused with the GAL4-DNA binding domain. The resulting diploids were plated on selection medium without tryptophan, leucine and histidine and with 25 mM 3-AT and the His and Lac-Z activity were determined. cDNAs encoding proteins which interacted with MEF2D but not with the p53, lamin C and SNF1 were defined as capable of interacting with MEF2D specifically.

Glutathione S-transferase (GST)-pull down assay

Partial cDNAs encoding amino acids 1-86 of MEF2D, 87-507 of MEF2D, 1-86 of MEF2C and 87-465 of MEF2C, and full length cDNA of MEF2D and MEF2C were cloned into pGex-2T vector (Pharmacia) and transformed into Escherichia coli strain, BL21(DE3)-pLys-S (Novagen) to produce GST fusion proteins as instructed (Pharmacia). ERK5 (2 µg) in pBluescriptSK(-) (kindly provided by J. E. Dixon; 20) was linearized with SalI and in vitro translated and labeled with [35S]methionine in a 50 µl reaction according to the manufacturer's instructions (Promega). Ten microliters of in vitro translated ERK5 products were incubated with 90 µl binding buffer (50 mM Tris-HCl pH 7.6/150 mM NaCl/5 mM EDTA/0.1% NP-40/protease inhibitors aprotinin, leupeptin, pepstatin A, 10 µg/ml each) and 2-3 µg of GST or GST-MEF2 fusion proteins immobilized on agarose beads. The mixtures were shaken for 1 h at room temperature. The beads were then washed three times with binding buffer and six times with washing buffer (50 mM Tris-HCl pH 7.6/450 mM NaCl/5 mM EDTA/1% NP-40). Proteins retained on beads were boiled in 10 µl of SDS sample buffer (New England Biolabs) and subjected to 7.5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The presence of labeled ERK5 proteins was detected by autoradiography.

Co-immunoprecipitation between MEF2 and ERK5/BMK1

Immunoprecipitation and immunoblotting for endogenous MEF2 and ERK5 were performed in primary culture of rat myotubes. Hind leg muscles of 2-day-old rats were aseptically collected, minced and digested overnight with 50 mg/ml trypsin (Worthington Biochemical Corp.). The muscles were dispersed by pipetting. Collagenase (Worthington Biochemical Corp.) was pre-warmed at 37°C and added to a final concentration of 0.1%. After 30 min incubation at 37°C, cells were collected by centrifugation, washed once and resuspended in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum (FCS). Cells were preplated for 1 h to decrease fibroblast content, remaining myoblasts were seeded onto gelatin-coated 100 mm dishes. Confluent monolayers of myoblasts were induced to form myotubes by switching medium to DMEM with 5% horse serum for 3 days. Prominent contracting myotubes were collected for immunoprecipitation. Goat anti-ERK5 (20 µl; SC-1284, Santa Cruz, CA), or 2 µl of rabbit anti-MEF2A or anti-MEF2D antiserum (kindly provided by R. Prywes, Columbia University, New York) were added to the lysate prepared from a 100 mm plate. The mixtures were shaken gently at 4°C for 1 h. Thirty microliters of 20% protein A-conjugated agarose beads (for MEF2) (Pharmacia) or 20 µl of protein G-plus agarose beads (Santa Cruz) were used to collect the immune complexes which were then washed and boiled in 40 µl SDS buffer. Anti-MEF2 (1 µl) and anti-ERK5 (20 µl) precipitate and 40 µg of myotubes lysate were analysed on 10% SDS-PAGE. Proteins were then transferred to nitrocellulose and probed with anti-ERK5, anti-MEF2A or anti-MEF2D antiserum.

In vitro kinase assay

ERK5 was cloned into the pREP10 vector (Invitrogen) and tagged with a hemagglutinin (HA) epitope, YPYDVPDYA. The resulting construct, pREP10-HA-ERK5, was used to transfect COS-7 cells cultured in [alpha]-MEM with 10% FCS in 60 mm plates. Forty-eight hours after transfection, cells were treated with or without 200 mM H2O2 for 5 min and lysed with 0.5 ml of TEN buffer (40 mM Tris-HCl, pH 7.5/10 mM EDTA/150 mM NaCl) plus 0.1% Triton X-100. Monoclonal anti-HA antibodies (4 µg) (BABCO, CA) were added into the cell extract. After 1 h of shaking at 4°C, the immune complexes were precipitated with 20 µl protein G-plus agarose beads (Santa Cruz) and washed with phosphate-buffered saline (PBS)/1% Triton X-100 six times. MEF2D and MEF2C (23) cDNAs were subcloned into the pET15b vector (Novagen) with a 6× histidine (6×His) tag fused at the N-terminus, expressed in E.coli strain BL21(DE3)-pLysS, and purified with nickel-conjugated beads according to the manufacturer's instructions (Novagen). Purified 6×His-MEF2D (2 µg) or 6×His-MEF2C (1 µg) were added into a reaction mixture containing immunoprecipitated ERK5, kinase assay buffer (10 mM MgCl2/150 mM NaCl/10 mM Tris-HCl, pH 7.4/0.1% DTT/protease inhibitors aprotinin, leupeptin, pepstatin A, 10 µg/ml each), and 2 µCi of [[gamma]-32P]ATP. The mixture was shaken at 30°C for 20 min. Phosphorylation of MEF2 proteins was analyzed by SDS-PAGE and autoradiography.

Cell culture and reporter assays

HeLa cells were cultured in DMEM supplemented with 10% FCS. For the reporter assay, 1 µg of an appropriate chloramphenicol acetyltransferase (CAT) reporter and 0.5 µg of pCMV-luciferase, which served as an internal control for transfection efficiency, were transfected into HeLa cells (35 mm plates) at 60% confluence by calcium phosphate coprecipitation. An additional 0.5 µg of pREP10-HA-MEF2D, pMT2-MEF2A or pMT2-MEF2C (23), and/or 1 µg of pREP10-HA-ERK5 were co-transfected. Empty vector pREP10-HA was used to keep the total amount of DNA constant for each transfection. There were three CAT reporter constructs used in this study: the embryonic myosin heavy chain (MHC) promoter fused with CAT (PE102CAT), two copies of the muscle creatine kinase MEF2-binding sites inserted in a concatemerized orientation at the -102 position of MHC promoter (PE102CAT-2×MEF2), or two copies of mutated MEF2-binding sites inserted at the -102 position of MHC promoter (PE102CAT-2×MEF2mt) (23). Twenty-four hours after transfection, cells were serum-starved for 24 h and then treated with 10% FCS for 4 h before harvest. An aliquot of 0.5 ml of TEN buffer (40 mM Tris-HCl pH 7.5/10 mM EDTA/150 mM NaCl) was used to harvest the cells. The CAT assay was performed as described (24) with minor modifications. After five rounds of freeze-thaw, 100 µl of centrifuged cell extract was added to 150 µl of reagent mixture (final concentration: 1 mM chloramphenicol/80 mM Tris-HCl, pH 7.5) plus 1 µCi of 3H-labeled acetyl-coenzyme A (NEN). After 30 min of incubation at 37°C, 5 ml of organic scintillation fluid was carefully overlaid and samples were incubated for 1 h before being quantitated by liquid scintillation counting. Transfection efficiency was measured by luciferase assay (25). The CAT activities were normalized by transfection efficiency. The relative CAT activities were calculated based on the activity of sample transfected with PE102CAT-2×MEF2 (as 1-fold).

RESULTS

ERK5/BMK1 interacts with the N-terminus of MEF2D in a yeast two hybrid screen

A neonatal rat aorta cDNA library was constructed using [lambda]-ACT vectors. The phage library, which has 2.6 million independent recombinants was converted into a plasmid library for yeast two hybrid screening (11,11). Each cDNA was fused to the GAL4-activation domain. Three constructs containing the N-terminal region of rat MEF2D were prepared for library screening. As shown in Table 1, the construct covering the complete MADS and MEF2 domain (Fig. 1a) of MEF2D (amino acids 1-175) autoactivated the Lac-Z reporter in yeast strain Y190. While amino acids 10-105 resulting from further deletion at both the N- and C-terminus still activated the reporter, amino acids 19-167 (named as pAS1-CYH-MEF2D) abolished the autoactivation activity. Moreover, the bHLH region of myogenin, which is known to interact with the MADS domain (15), can still mildly interact with amino acids 19-167 of MEF2D (data not shown). This result suggested that pAS1-CYH-MEF2D may retain at least part of the protein-protein interaction ability. pAS1-CYH-MEF2D was then used to screen the library. 1.7 million recombinants were screened and >50 cDNAs were found to encode proteins interacting specifically with the N-terminus of MEF2D (Table 1). One of the clones carries a partial cDNA (accession no. AJ005424) encoding a rat homologue of human ERK5/BMK1, which was cloned independently by two groups (19,20). It has been shown by sequence analysis that ERK5/BMK1 belongs to the MAP kinase family (19,20). A full length ERK5/BMK1 cDNA was kindly provided by J. E. Dixon and was used for the following studies.

   a-b

   c

Figure 1. ERK5 interacts with MEF2 in vitro and in vivo. (a) The schematic structure of MEF2D. (b) ERK5 associates with MEF2 through the N-terminal region which contains the MADS and MEF2 domains. Ten microliters of 35S-labeled ERK5 (lane 1) were mixed with 2-3 µg of GST (lane 2) or MEF2-GST (lanes 3-8) fusion proteins immobilized on GSH-agarose beads. After intensive washing, retained proteins were analyzed by SDS-PAGE and autoradiography. The arrow indicates the position of 110 kDa ERK5. (c) MEF2 co-immunoprecipitates with ERK5. Anti-MEF2D ([alpha]MEF2D-ip), anti-MEF2A ([alpha]MEF2A-ip) and anti-ERK5 ([alpha]ERK5-ip) immunoprecipitates obtained from the lysate of rat myotubes were subjected to 10% SDS-PAGE analysis, transferred to nitrocellulose membrane and blotted with anti-MEF2D (blot: [alpha]MEF2D, lanes 1-3), anti-MEF2A (blot: [alpha]MEF2A, lanes 4-6) and anti-ERK5 (blot: [alpha]ERK5, lanes 7-10) antiserum. The arrows indicate the positions of MEF2D, MEF2A and ERK5.

Table 1. Results of the yeast two hybrid screening using partial MEF2D
pAS1-CYH construct containing MEF2D Autoactivation of Lac-Z reporter Recombinants screened Number of His+ and Lac-Z+ Interaction specific to MEF2D
Amino acids 1-175 yes N.A. N.A. N.A.
Amino acids 10-105 yes N.A. N.A. N.A.
Amino acids 19-167 no 1.7 million 247 >50
The top two constructs (amino acids 1-175 and 10-105) activated the Lac-Z reporter in Y-190 and therefore were not used in the library screening. N.A., not assayed.

The N-terminus of MEF2 mediates the interaction between MEF2 and ERK5 in vitro

Both MADS and MEF2 domains of MEF2C have been shown to be responsible for DNA binding and protein-protein interactions (11). A GST-pull down assay was performed to verify that the N-terminus of MEF2 is sufficient to mediate the interaction and to determine whether full length MEF2 interacts with ERK5/BMK1. In vitro translated and 35S-labeled ERK5/BMK1 products (Fig. 1b, lane 1) were mixed with immobilized GST or GST-MEF2 fusion proteins. After extensive washing, ERK5/BMK1 proteins were retained by GST-MEF2D (amino acids 1-86), GST-MEF2D (full length), GST-MEF2C (amino acids 1-86) and GST-MEF2C (full length) fusion proteins (Fig. 1b, lanes 3, 5, 6 and 8, respectively), but not by GST, GST-MEF2D (amino acids 87-507) and GST-MEF2C (amino acids 87-465) (Fig. 1b, lanes 2, 4 and 7, respectively). This result showed that the N-terminus of MEF2 (amino acids 1-86) was sufficient for the interaction and full length MEF2 was able to interact with ERK5/BMK1 in vitro.

MEF2 co-immunoprecipitates with ERK5 in vivo

To investigate whether ERK5/BMK1 does interact with MEF2 in vivo, anti-ERK5, anti-MEF2D and anti-MEF2A antiserum were used to immunoprecipitate ERK5, MEF2D and MEF2A and their associated proteins from the lysate of primary rat myotube cultures. The precipitates were analyzed by SDS-PAGE, then transferred to nitrocellulose and blotted with anti-ERK5, anti-MEF2D or anti-MEF2A antiserum. The MEF2D, MEF2A and ERK5 proteins are expressed in myotubes since they were detected in the immunoprecipitate prepared from myotube lysate using specific antiserum (Fig. 1c: lanes 1, 4, 8 and 10, respectively). Also, MEF2D and MEF2A were detectable in the myotube lysate (lanes 3 and 6, respectively). Both MEF2A and MEF2D were detected in the anti-ERK5 precipitate (lanes 2 and 5, respectively). However, ERK5 was not detectable in the anti-MEF2A or anti-MEF2D precipitate (lanes 7 and 9). These results indicate that endogenous ERK5 proteins associate with endogenous MEF2D and MEF2A proteins and suggest a functional role for the interaction.


Figure 2. Recombinant MEF2D and MEF2C are phosphorylated by ERK5in vitro. Recombinant MEF2D proteins were incubated with anti-HA immunoprecipitate (IP) from COS without transfection and with H2O2 treatment (lane 1); anti-HA IP from COS with ERK5 (HA-tagged) transfection and with H2O2 treated (lane 2); anti-HA IP from COS without transfection (lane 3); anti-HA IP from COS with ERK5 transfection (lane 4). Recombinant MEF2C proteins were incubated with anti-HA IP from COS without transfection and with H2O2 treatment (lane 5); anti-HA IP from COS with ERK5 transfection and with H2O2 treated (lane 6). Arrows indicate the positions of MEF2D and MEF2C.

ERK5/BMK1 phosphorylates MEF2D and MEF2C in vitro

Phosphorylation of MEF2C has been shown to enhance its DNA-binding and its transactivation activity (17,26). To explore the functional aspects of the MEF2D-ERK5/BMK1 interaction, we next determined whether ERK5/BMK1 is able to phosphorylate MEF2D in vitro. Since the MADS and MEF2 domains are homologous among the four MEF2 proteins in vertebrate species and are believed to mediate protein-protein interactions, we also tested whether MEF2C can be phosphorylated by ERK5/BMK1. HA-tagged ERK5/BMK1 proteins were immunoprecipitated from transfected COS cells treated with or without H2O2 which has been shown to activate ERK5/BMK1 (27). 6×His-tagged MEF2D and MEF2C recombinant proteins were incubated with anti-HA immunoprecipitate from COS cells in the presence of [[gamma]-32P]ATP. As shown in Figure 2, anti-HA immunoprecipitate prepared from COS cells transfected with pREP10-HA-ERK5 did phosphorylate MEF2D and MEF2C (Fig. 2, lanes 2 and 6, respectively), while anti-HA immunoprecipitate from untransfected cells did not (Fig. 2, lanes 1 and 5). Note that ERK5/BMK1 immunoprecipitate prepared from COS cells in the presence of serum and without treatment of H2O2 can still phosphorylate MEF2D (Fig. 2, lane 4). This result suggests that ERK5/BMK1 could also be activated by serum.

The transcriptional activity of MEF2A and MEF2C, but not MEF2D is enhanced by ERK5/BMK1

Phosphorylation of MEF2C by a MAP kinase, p38, was shown to enhance the transcriptional activity of MEF2C (17). Whether transcriptional activities of MEF2A, MEF2C and MEF2D increase in response to activation by ERK5/BMK1 was determined by a co-transfection assay. MEF2, ERK5/BMK1 and PE102CAT, PE102CAT-2×MEF2, or PE102CAT-2×MEF2mt were transfected into HeLa cells. Since the above in vitro phosphorylation result suggested that serum activates ERK5/BMK1, cells were incubated with serum to activate ERK5/BMK1. HeLa cells were transfected as indicated in Figure 3. We found that overexpression of MEF2A and MEF2C can activate the PE102CAT-2×MEF2 reporter 10-14-fold (Fig. 3, lanes 3 and 7) over the control, which has only the PE102CAT-2×MEF2 reporter transfected (lane 1), whereas MEF2D did not activate the reporter (lane 11). Transfection of ERK5 alone did not activate the PE102CAT-2×MEF2 (lane 2). However, co-transfection of ERK5 further enhanced the activity of MEF2A and MEF2C by ~2-fold (lanes 4 and 8) but had no effect on MEF2D (lane 12). The induction of the PE102CAT-2×MEF2 reporter was dependent on the activity of MEF2A and MEF2C since there was no induction of PE102CAT and PE102CAT-2×MEF2-mt reporter (lanes 5, 6, 9 and 10).


Figure 3. ERK5/BMK1 enhances the transcriptional activity of MEF2A and MEF2C. HeLa cells were transfected with CAT reporters (PE102CAT, PE102CAT-2×MEF2 or PE102CAT-2×MEF2mt), MEF2 and/or ERK5 as indicated. Twenty-four hours after transfection, cells were serum-starved for 24 h and then treated with 10% FCS for 4 h before harvest. CAT activity from cells transfected with PE102CAT-2×MEF2 was defined as 1-fold (lane 1). Results were shown as mean ± standard deviation obtained from three independent experiments.

DISCUSSION

Skeletal muscle differentiation is regulated by the bHLH myogenic regulators, MyoD, MRF4, Myf5 and myogenin (reviewed in 1). Overexpression of any one of these bHLH myogenic factors was able to initiate myogenesis in a variety of non-muscle cell types (reviewed in 28). Although MEF2 was indispensable for the development of visceral, somatic and cardiac muscle in Drosophila (3), most reports so far indicate that overexpression of MEF2 is insufficient to convert fibroblasts into differentiated muscle cells (2). It is possible that the functions of MEF2 are regulated through protein-protein interaction and/or post-translational modification. Regulation of activity of MEF2 through protein-protein interactions has been reported. For example, MEF2C has been shown to bind to the bHLH region of myogenin and cooperatively activate expression of muscle-specific genes (15). In addition, MEF2 associates with the ligand-bound thyroid hormone receptor and synergistically activates the [alpha]-cardiac myosin heavy-chain gene (29). p300/CBP binds to the MADS domain of MEF2C and potentiates the transcriptional activity of MEF2C (16). With regard to the role of post-translational modification in the regulation of MEF2, casein kinase II phosphorylates MEF2C and increases its DNA binding activity (26). A recent study searching for p38-interacting proteins by the yeast two hybrid screen led to the finding that p38 interacted with, phosphorylated, and activated MEF2C (17).

In this study, we found that ERK5/BMK1 interacted with amino acids 19-167 of MEF2D in a yeast two hybrid screen. Since the N-terminus of MEF2D contains the MADS and MEF2 domains which are homologous among the four MEF2 proteins, the N-terminus of MEF2 may mediate the interaction and therefore other MEF2 proteins may also interact with ERK5/BMK1 proteins. Indeed, the N-terminus of MEF2D and MEF2C was sufficient to interact with ERK5/BMK1 and the full length of MEF2D and MEF2C proteins were capable of interacting with ERK5/BMK1 (Fig. 1b). Furthermore, a co-immunoprecipitation study showed that endogenous MEF2D and MEF2A proteins were present in the anti-ERK5 immunoprecipitate, although ERK5 proteins were not detectable in the anti-MEF2 immunoprecipitate (Fig. 1c). There are several possible reasons for the failure of observing ERK5 in the anti-MEF2 immunoprecipitate. Firstly, it is possible that the amount of ERK5 associated with MEF2 is below the detection limit of anti-ERK5 antiserum since the absolute amount of ERK5 in myotubes could be much less than that of MEF2 and, therefore, anti-MEF2 immunoprecipitate may contain only a small portion of the ERK5-associating MEF2. Our observation that MEF2D and MEF2A proteins, but not ERK5, can be detected in the myotube lysate (data not shown) also supports this notion. Secondly, it is also possible that MEF2:ERK5 interaction masks the epitope on MEF2 recognized by the anti-MEF2 antiserum. Nevertheless, the co-immunoprecipitation results show that endogenous MEF2D and MEF2A interact with endogenous ERK5 in vivo.

A functional role for the MEF2-ERK5 interaction was tested by determining whether MEF2D and MEF2C can be phosphorylated and activated by ERK5/BMK1. Although both the recombinant MEF2D and MEF2C proteins were found to be phosphorylated by ERK5/BMK1 in vitro (Fig. 2), the transcriptional activity of MEF2C was enhanced by ERK5/BMK1 in HeLa cells; however, the activity of MEF2D was not affected (Fig. 3). MEF2A was included in the CAT-reporter assay and found to act similarly to MEF2C. It has been reported previously that the endogenous MEF2D expressed in myoblasts (30) and also in HeLa cells (23) is transcriptionally inactive, supporting the idea that MEF2D may be regulated differently compared with the other MEF2 proteins. In addition, overexpression of MEF2D weakly stimulates a reporter gene with oligomerized MEF2 sites whereas MEF2C and MEF2A potently activate transcription (23 and Fig. 3). Therefore, the transcriptional activity of MEF2D may be subject to a regulatory mechanism different from the one governing the regulation of MEF2A and MEF2C.

Since two library screening approaches (17 and our data) indicate that MEF2 interacts with two members of the MAP kinase family, MEF2 could be an important physiological target of ERK5/BMK1 and p38 MAP kinases. In this study, ERK5/BMK1 was isolated from a library made from aorta, which contains mainly smooth muscle cells. Indeed, Abe et al. (27) detected the expression of endogenous ERK5/BMK1 in cultured rat vascular smooth muscle cells. Moreover, the addition of H2O2 and sorbital to cultured cells stimulated the activity of ERK5/BMK1. Recently, Abe et al. (31) also showed that c-Src is required for oxidative stress-mediated activation of BMK1. Therefore, MEF2 could be a candidate for mediation of stress signals from activated ERK5/BMK1 in vascular smooth muscle cells.

Phosphorylation at Ser59 of MEF2C (26) and at Ser103 of SRF (32), a member of the MADS-domain family, were shown to increase their DNA-binding affinity in vitro. During the preparation of this manuscript, Kato et al. (33) reported that ERK5/BMK1 regulated serum-induced expression of c-Jun through MEF2C. While the direct association between ERK5/BMK1 and MEF2 was not addressed by Kato et al., our study shows that MEF2 proteins physically interact with and are phosphorylated by ERK5/BMK1. In two reports (17,33), Ser387 of MEF2C was phosphorylated by p38 or ERK5/BMK1, respectively. Furthermore, this phosphorylation was critical for the activation of MEF2C. Interestingly, this Ser387 is conserved between MEF2C and MEF2A, but not MEF2D which is unresponsive to ERK5/BMK1 (33). Therefore, the results of our reporter assays suggest that both MEF2A and MEF2C could respond to ERK5/BMK1 via phosphorylation of this conserved serine, while phosphorylation of MEF2D at an unknown site by ERK5/BMK1 is insufficient for activation.

In summary, our data indicate that ERK5/BMK1 can target several members of the MEF2 family through the highly conserved N-terminus of MEF2 and this interaction can result in phosphorylation and modulation of the transactivation properties of MEF2A and MEF2C.

ACKNOWLEDGEMENT

This study was supported by research grant PG12351 to C.A.P. from the Medical Research Council of Canada.

REFERENCES

1. Olson,E.N. (1990) Genes Dev., 4, 1454-1461.

2. Olson,E.N., Perry,M. and Schulz,R.A. (1995) Dev. Biol., 172, 2-14. MEDLINE Abstract

3. Lilly,B., Zhao,B., Ranganayakulu,G., Paterson,B.M., Schulz,R.A. and Olson,E.N. (1995) Science, 267, 688-693. MEDLINE Abstract

4. Bour,B.A., O'Brien,M.A., Lockwood,W.L., Goldstein,E.S., Bodmer,R., Taghert,P.H., Abmayr,S.M. and Nguyen,H.T. (1995) Genes Dev., 9, 730-741.

5. Ornatsky,O.I., Andreucci,J.J. and McDermott,J.C. (1997) J. Biol. Chem., 272, 33271-33278. MEDLINE Abstract

6. Lin,Q., Schwarz,J., Bucana,C. and Olson,E.N. (1997) Science, 276, 1404-1407. MEDLINE Abstract

7. Gossett,L.A., Kelvin,D.J., Sternberg,E.A. and Olson,E.N. (1989)Mol. Cell. Biol., 9, 5022-5033. MEDLINE Abstract

8. Shore,P. and Sharrocks,A.D. (1995) Eur. J. Biochem., 229, 1-13. MEDLINE Abstract

9. Nguyen,H.T., Bodmer,R., Abmayr,S.M., McDermott,J.C. and Spoerel,N.A. (1994) Proc. Natl Acad. Sci. USA, 91, 7520-7524. MEDLINE Abstract

10. Lilly,B., Galewsky,S., Firulli,A.B., Schulz,R.A. and Olson,E.N. (1994) Proc. Natl Acad. Sci. USA, 91, 5662-5666. MEDLINE Abstract

11. Molkentin,J.D., Black,B.L., Martin,J.F. and Olson,E.N. (1996)Mol. Cell. Biol., 16, 2627-2636. MEDLINE Abstract

12. Wong,M.W., Pisegna,M., Lu,M.F., Leibham,D. and Perry,M. (1994)Dev. Biol., 166, 683-695. MEDLINE Abstract

13. Martin,J.F., Miano,J.M., Hustad,C.M., Copeland,N.G., Jenkins,N.A. and Olson,E.N. (1994) Mol. Cell. Biol., 14, 1647-1656. MEDLINE Abstract

14. Kaushal,S., Schneider,J.W., Nadal-Ginard,B. and Mahdavi,V. (1994) Science, 266, 1236-1240. MEDLINE Abstract

15. Molkentin,J.D., Black,B.L., Martin,J.F. and Olson,E.N. (1995) Cell, 83, 1125-1136. MEDLINE Abstract

16. Sartorelli,V., Huang,J., Hamamori,Y. and Kedes,L. (1997) Mol. Cell. Biol., 17, 1010-1026. MEDLINE Abstract

17. Han,J., Jiang,Y., Li,Z., Kravchenko,V.V. and Ulevitch,R.J. (1997) Nature, 386, 296-299. MEDLINE Abstract

18. Fields,S. and Sternglanz,R. (1994) Trends Genet., 10, 286-292. MEDLINE Abstract

19. Lee,J.D., Ulevitch,R.J. and Han,J. (1995) Biochem. Biophys. Res. Commun., 213, 715-724. MEDLINE Abstract

20. Zhou,G., Bao,Z.Q. and Dixon,J.E. (1995) J. Biol. Chem., 270, 12665-12669. MEDLINE Abstract

21. Durfee,T., Becherer,K., Chen,P.L., Yeh,S.H., Yang,Y., Kilburn,A.E., Lee,W.H. and Elledge,S.J. (1993) Genes Dev., 7, 555-569.

22. Elledge,S.J., Mulligan,J.T., Ramer,S.W., Spottswood,M., Davis and RW (1991) Proc. Natl Acad. Sci USA, 88, 1731-1735. MEDLINE Abstract

23. Ornatsky,O.I. and McDermott,J.C. (1996) J. Biol. Chem., 271, 24927-24933. MEDLINE Abstract

24. Sleigh,M.J. (1986) Anal. Biochem., 156, 251-256.

25. Elsholtz,H.P., Lew,A.M., Albert,P.R. and Sundmark,V.C. (1991)J. Biol. Chem., 266, 22919-22925. MEDLINE Abstract

26. Molkentin,J.D., Li,L. and Olson,E.N. (1996) J. Biol. Chem., 271, 17199-17204. MEDLINE Abstract

27. Abe,J., Kusuhara,M., Ulevitch,R.J., Berk,B.C. and Lee,J.D. (1996) J. Biol. Chem., 271, 16586-16590. MEDLINE Abstract

28. Edmondson,D.G. and Olson,E.N. (1993) J. Biol. Chem., 268, 755-758. MEDLINE Abstract

29. Lee,Y., Nadal-Ginard,B., Mahdavi,V. and Izumo,S. (1997) Mol. Cell. Biol., 17, 2745-2755. MEDLINE Abstract

30. Breitbart,R.E., Liang,C.S., Smoot,L.B., Laheru,D.A., Mahdavi,V. and Nadal-Ginard,B. (1993) Development, 118, 1095-1106. MEDLINE Abstract

31. Abe,J., Takahashi,M., Ishida,M., Lee,J.D. and Berk,B.C. (1997)J. Biol. Chem., 272, 20389-20394. MEDLINE Abstract

32. Rivera,V.M., Miranti,C.K., Misra,R.P., Ginty,D.D., Chen,R.H., Blenis,J. and Greenberg,M.E. (1993) Mol. Cell. Biol., 13, 6260-6273. MEDLINE Abstract

33. Kato,Y., Kravchenko,V.V., Tapping,R.I., Han,J., Ulevitch,R.J. and Lee,J.D. (1997) EMBO J., 16, 7054-7066. MEDLINE Abstract

*To whom correspondence should be addressed at present address: Skye Pharmatech, Inc., 6354 Viscount Road, Mississauga, Ontario L4V 1H3, Canada. Tel: +1 905 677 1944; Fax: +1 905 677 1674; Email: cprody@globalserve.net

This page is run by Oxford University Press, Great Clarendon Street, Oxford OX2 6DP, as part of the OUP Journals
Comments and feedback: www-admin{at}oup.co.uk
Last modification: 30 Sep 1998
Copyright©Oxford University Press, 1998.
Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Mol. Cell. Biol.Home page
R. L. S. Perry, C. Yang, N. Soora, J. Salma, M. Marback, L. Naghibi, H. Ilyas, J. Chan, J. W. Gordon, and J. C. McDermott
Direct Interaction between Myocyte Enhancer Factor 2 (MEF2) and Protein Phosphatase 1{alpha} Represses MEF2-Dependent Gene Expression
Mol. Cell. Biol., June 15, 2009; 29(12): 3355 - 3366.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
M. Du, R. L. S. Perry, N. B. Nowacki, J. W. Gordon, J. Salma, J. Zhao, A. Aziz, J. Chan, K. W. M. Siu, and J. C. McDermott
Protein Kinase A Represses Skeletal Myogenesis by Targeting Myocyte Enhancer Factor 2D
Mol. Cell. Biol., May 1, 2008; 28(9): 2952 - 2970.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
H. Morimoto, K. Kondoh, S. Nishimoto, K. Terasawa, and E. Nishida
Activation of a C-terminal Transcriptional Activation Domain of ERK5 by Autophosphorylation
J. Biol. Chem., December 7, 2007; 282(49): 35449 - 35456.
[Abstract] [Full Text] [PDF]

Home page
 PhysiologyHome page
B. C. W. Groenendijk, K. Van der Heiden, B. P. Hierck, and R. E. Poelmann
The Role of Shear Stress on ET-1, KLF2, and NOS-3 Expression in the Developing Cardiovascular System of Chicken Embryos in a Venous Ligation Model
Physiology, December 1, 2007; 22(6): 380 - 389.
[Abstract] [Full Text] [PDF]

Home page
 DevelopmentHome page
M. J. Potthoff and E. N. Olson
MEF2: a central regulator of diverse developmental programs
Development, December 1, 2007; 134(23): 4131 - 4140.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
G. B. Atkins and M. K. Jain
Role of Kruppel-Like Transcription Factors in Endothelial Biology
Circ. Res., June 22, 2007; 100(12): 1686 - 1695.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
S. Gregoire, L. Xiao, J. Nie, X. Zhang, M. Xu, J. Li, J. Wong, E. Seto, and X.-J. Yang
Histone Deacetylase 3 Interacts with and Deacetylates Myocyte Enhancer Factor 2
Mol. Cell. Biol., February 15, 2007; 27(4): 1280 - 1295.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Y. Wang, B. Su, and Z. Xia
Brain-derived Neurotrophic Factor Activates ERK5 in Cortical Neurons via a Rap1-MEKK2 Signaling Cascade
J. Biol. Chem., November 24, 2006; 281(47): 35965 - 35974.
[Abstract] [Full Text] [PDF]

Home page
 Eukaryot CellHome page
A. W. Truman, S. H. Millson, J. M. Nuttall, V. King, M. Mollapour, C. Prodromou, L. H. Pearl, and P. W. Piper
Expressed in the Yeast Saccharomyces cerevisiae, Human ERK5 Is a Client of the Hsp90 Chaperone That Complements Loss of the Slt2p (Mpk1p) Cell Integrity Stress-Activated Protein Kinase
Eukaryot. Cell, November 1, 2006; 5(11): 1914 - 1924.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
W.-h. Feng and S. C. Kenney
Valproic Acid Enhances the Efficacy of Chemotherapy in EBV-Positive Tumors by Increasing Lytic Viral Gene Expression.
Cancer Res., September 1, 2006; 66(17): 8762 - 8769.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
R. E. Schweppe, T. H. Cheung, and N. G. Ahn
Global Gene Expression Analysis of ERK5 and ERK1/2 Signaling Reveals a Role for HIF-1 in ERK5-mediated Responses
J. Biol. Chem., July 28, 2006; 281(30): 20993 - 21003.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
K. Kondoh, K. Terasawa, H. Morimoto, and E. Nishida
Regulation of nuclear translocation of extracellular signal-regulated kinase 5 by active nuclear import and export mechanisms.
Mol. Cell. Biol., March 1, 2006; 26(5): 1679 - 1690.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. Gregoire, A. M. Tremblay, L. Xiao, Q. Yang, K. Ma, J. Nie, Z. Mao, Z. Wu, V. Giguere, and X.-J. Yang
Control of MEF2 Transcriptional Activity by Coordinated Phosphorylation and Sumoylation
J. Biol. Chem., February 17, 2006; 281(7): 4423 - 4433.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
S. J. Sohn, D. Li, L. K. Lee, and A. Winoto
Transcriptional Regulation of Tissue-Specific Genes by the ERK5 Mitogen-Activated Protein Kinase
Mol. Cell. Biol., October 1, 2005; 25(19): 8553 - 8566.
[Abstract] [Full Text] [PDF]

Home page
 Genes Dev.Home page
O. Nakagawa, M. Arnold, M. Nakagawa, H. Hamada, J. M. Shelton, H. Kusano, T. M. Harris, G. Childs, K. P. Campbell, J. A. Richardson, et al.
Centronuclear myopathy in mice lacking a novel muscle-specific protein kinase transcriptionally regulated by MEF2
Genes & Dev., September 1, 2005; 19(17): 2066 - 2077.
[Abstract] [Full Text] [PDF]

Home page
 GeneticsHome page
P. W. Baker, K. K. K. Tanaka, N. Klitgord, and R. M. Cripps
Adult Myogenesis in Drosophila melanogaster Can Proceed Independently of Myocyte Enhancer Factor-2
Genetics, August 1, 2005; 170(4): 1747 - 1759.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
L.-M. Sturla, C. W. Cowan, L. Guenther, R. C. Castellino, J. Y.H. Kim, and S. L. Pomeroy
A Novel Role for Extracellular Signal-Regulated Kinase 5 and Myocyte Enhancer Factor 2 in Medulloblastoma Cell Death
Cancer Res., July 1, 2005; 65(13): 5683 - 5689.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
X. Carvajal-Vergara, S. Tabera, J. C. Montero, A. Esparis-Ogando, R. Lopez-Perez, G. Mateo, N. Gutierrez, M. Parmo-Cabanas, J. Teixido, J. F. San Miguel, et al.
Multifunctional role of Erk5 in multiple myeloma
Blood, June 1, 2005; 105(11): 4492 - 4499.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
C. Grey, A. Mery, and M. Puceat
Fine-tuning in Ca2+ homeostasis underlies progression of cardiomyopathy in myocytes derived from genetically modified embryonic stem cells
Hum. Mol. Genet., May 15, 2005; 14(10): 1367 - 1377.
[Abstract] [Full Text] [PDF]

Home page
 Eukaryot CellHome page
S. H. Millson, A. W. Truman, V. King, C. Prodromou, L. H. Pearl, and P. W. Piper
A Two-Hybrid Screen of the Yeast Proteome for Hsp90 Interactors Uncovers a Novel Hsp90 Chaperone Requirement in the Activity of a Stress-Activated Mitogen-Activated Protein Kinase, Slt2p (Mpk1p)
Eukaryot. Cell, May 1, 2005; 4(5): 849 - 860.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
J. C. Barros and C. J. Marshall
Activation of either ERK1/2 or ERK5 MAP kinase pathways can lead to disruption of the actin cytoskeleton
J. Cell Sci., April 15, 2005; 118(8): 1663 - 1671.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
S. Gregoire and X.-J. Yang
Association with Class IIa Histone Deacetylases Upregulates the Sumoylation of MEF2 Transcription Factors
Mol. Cell. Biol., March 15, 2005; 25(6): 2273 - 2287.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
J. W. Edmunds and L. C. Mahadevan
MAP kinases as structural adaptors and enzymatic activators in transcription complexes
J. Cell Sci., September 1, 2004; 117(17): 3715 - 3723.
[Abstract] [Full Text] [PDF]

Home page
 J BiochemHome page
K. Kuida and D. M. Boucher
Functions of MAP Kinases: Insights from Gene-Targeting Studies
J. Biochem., June 1, 2004; 135(6): 653 - 656.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
Z. Raviv, E. Kalie, and R. Seger
MEK5 and ERK5 are localized in the nuclei of resting as well as stimulated cells, while MEKK2 translocates from the cytosol to the nucleus upon stimulation
J. Cell Sci., May 1, 2004; 117(9): 1773 - 1784.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Q. Zheng, G. Yin, C. Yan, M. Cavet, and B. C. Berk
14-3-3{beta} Binds to Big Mitogen-activated Protein Kinase 1 (BMK1/ERK5) and Regulates BMK1 Function
J. Biol. Chem., March 5, 2004; 279(10): 8787 - 8791.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
L. Liu, J. E. Cavanaugh, Y. Wang, H. Sakagami, Z. Mao, and Z. Xia
ERK5 activation of MEF2-mediated gene expression plays a critical role in BDNF-promoted survival of developing but not mature cortical neurons
PNAS, July 8, 2003; 100(14): 8532 - 8537.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
H. Akazawa and I. Komuro
Roles of Cardiac Transcription Factors in Cardiac Hypertrophy
Circ. Res., May 30, 2003; 92(10): 1079 - 1088.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
D. M. Cox, M. Du, M. Marback, E. C. C. Yang, J. Chan, K. W. M. Siu, and J. C. McDermott
Phosphorylation Motifs Regulating the Stability and Function of Myocyte Enhancer Factor 2A
J. Biol. Chem., April 18, 2003; 278(17): 15297 - 15303.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. J. Sohn, B. K. Sarvis, D. Cado, and A. Winoto
ERK5 MAPK Regulates Embryonic Angiogenesis and Acts as a Hypoxia-sensitive Repressor of Vascular Endothelial Growth Factor Expression
J. Biol. Chem., November 1, 2002; 277(45): 43344 - 43351.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. Buschbeck, J. Eickhoff, M. N. Sommer, and A. Ullrich
Phosphotyrosine-specific Phosphatase PTP-SL Regulates the ERK5 Signaling Pathway
J. Biol. Chem., August 9, 2002; 277(33): 29503 - 29509.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
C. P. Regan, W. Li, D. M. Boucher, S. Spatz, M. S. Su, and K. Kuida
Erk5 null mice display multiple extraembryonic vascular and embryonic cardiovascular defects
PNAS, July 9, 2002; 99(14): 9248 - 9253.
[Abstract] [Full Text] [PDF]

Home page
 JCBHome page
J. Li, M. Puceat, C. Perez-Terzic, A. Mery, K. Nakamura, M. Michalak, K.-H. Krause, and M. E. Jaconi
Calreticulin reveals a critical Ca2+ checkpoint in cardiac myofibrillogenesis
J. Cell Biol., July 8, 2002; 158(1): 103 - 113.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
A. Esparis-Ogando, E. Diaz-Rodriguez, J. C. Montero, L. Yuste, P. Crespo, and A. Pandiella
Erk5 Participates in Neuregulin Signal Transduction and Is Constitutively Active in Breast Cancer Cells Overexpressing ErbB2
Mol. Cell. Biol., January 1, 2002; 22(1): 270 - 285.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
M. Li, D. A. Linseman, M. P. Allen, M. K. Meintzer, X. Wang, T. Laessig, M. E. Wierman, and K. A. Heidenreich
Myocyte Enhancer Factor 2A and 2D Undergo Phosphorylation and Caspase-Mediated Degradation during Apoptosis of Rat Cerebellar Granule Neurons
J. Neurosci., September 1, 2001; 21(17): 6544 - 6552.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
E. A. Miska, E. Langley, D. Wolf, C. Karlsson, J. Pines, and T. Kouzarides
Differential localization of HDAC4 orchestrates muscle differentiation
Nucleic Acids Res., August 15, 2001; 29(16): 3439 - 3447.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
Z. A. Quinn, C.-C. Yang, J. L. Wrana, and J. C. McDermott
Smad proteins function as co-modulators for MEF2 transcriptional regulatory proteins
Nucleic Acids Res., February 1, 2001; 29(3): 732 - 742.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
J. E. Cavanaugh, J. Ham, M. Hetman, S. Poser, C. Yan, and Z. Xia
Differential Regulation of Mitogen-Activated Protein Kinases ERK1/2 and ERK5 by Neurotrophins, Neuronal Activity, and cAMP in Neurons
J. Neurosci., January 15, 2001; 21(2): 434 - 443.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
H. G. Kasler, J. Victoria, O. Duramad, and A. Winoto
ERK5 Is a Novel Type of Mitogen-Activated Protein Kinase Containing a Transcriptional Activation Domain
Mol. Cell. Biol., November 15, 2000; 20(22): 8382 - 8389.
[Abstract] [Full Text]

Home page
 J. Biol. Chem.Home page
A. G. Ridgeway, S. Wilton, and I. S. Skerjanc
Myocyte Enhancer Factor 2C and Myogenin Up-regulate Each Other's Expression and Induce the Development of Skeletal Muscle in P19 Cells
J. Biol. Chem., January 7, 2000; 275(1): 41 - 46.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
S.-H. Yang, A. Galanis, and A. D. Sharrocks
Targeting of p38 Mitogen-Activated Protein Kinases to MEF2 Transcription Factors
Mol. Cell. Biol., June 1, 1999; 19(6): 4028 - 4038.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
M. J. Marinissen, M. Chiariello, M. Pallante, and J. S. Gutkind
A Network of Mitogen-Activated Protein Kinases Links G Protein-Coupled Receptors to the c-jun Promoter: a Role for c-Jun NH2-Terminal Kinase, p38s, and Extracellular Signal-Regulated Kinase 5
Mol. Cell. Biol., June 1, 1999; 19(6): 4289 - 4301.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Y. Kato, M. Zhao, A. Morikawa, T. Sugiyama, D. Chakravortty, N. Koide, T. Yoshida, R. I. Tapping, Y. Yang, T. Yokochi, et al.
Big Mitogen-activated Kinase Regulates Multiple Members of the MEF2 Protein Family
J. Biol. Chem., June 9, 2000; 275(24): 18534 - 18540.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
W. Sun, K. Kesavan, B. C. Schaefer, T. P. Garrington, M. Ware, N. L. Johnson, E. W. Gelfand, and G. L. Johnson
MEKK2 Associates with the Adapter Protein Lad/RIBP and Regulates the MEK5-BMK1/ERK5 Pathway
J. Biol. Chem., February 9, 2001; 276(7): 5093 - 5100.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Y. Tamir and E. Bengal
Phosphoinositide 3-Kinase Induces the Transcriptional Activity of MEF2 Proteins during Muscle Differentiation
J. Biol. Chem., October 27, 2000; 275(44): 34424 - 34432.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
F. Dong, J. S. Gutkind, and A. C. Larner
Granulocyte Colony-stimulating Factor Induces Erk5 Activation, Which Is Differentially Regulated by Protein-tyrosine Kinases and Protein Kinase C. REGULATION OF CELL PROLIFERATION AND SURVIVAL
J. Biol. Chem., March 30, 2001; 276(14): 10811 - 10816.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
N. V. Kumar and L. R. Bernstein
Ten ERK-related Proteins in Three Distinct Classes Associate with AP-1 Proteins and/or AP-1 DNA
J. Biol. Chem., August 17, 2001; 276(34): 32362 - 32372.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. E. Dunn, A. R. Simard, R. Bassel-Duby, R. S. Williams, and R. N. Michel
Nerve Activity-dependent Modulation of Calcineurin Signaling in Adult Fast and Slow Skeletal Muscle Fibers
J. Biol. Chem., November 21, 2001; 276(48): 45243 - 45254.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
J. Lu, T. A. McKinsey, R. L. Nicol, and E. N. Olson
Signal-dependent activation of the MEF2 transcription factor by dissociation from histone deacetylases
PNAS, April 11, 2000; 97(8): 4070 - 4075.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Print PDF (140K) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (81)
Right arrowRequest Permissions
Right arrow Commercial Re-use Guidelines
for Open Access NAR Content
Google Scholar
Right arrow Articles by Yang, C. C.
Right arrow Articles by Prody, C. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yang, C. C.
Right arrow Articles by Prody, C. A.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?