1: Mol Cell Biol. 2005 May;25(9):3784-92. 

Selective regulation of c-jun gene expression by mitogen-activated protein
kinases via the 12-o-tetradecanoylphorbol-13-acetate- responsive element and
myocyte enhancer factor 2 binding sites.

Kayahara M, Wang X, Tournier C.

Faculty of Life Sciences, University of Manchester, The Michael Smith Building,
Oxford Road, Manchester M13 9PT, United Kingdom.

To further understand how the mitogen-activated protein kinase (MAPK) signaling
pathways regulate AP-1 activity, we have elucidated the physiological role of
these cascades in the regulation of c-jun gene expression. c-Jun is a crucial
component of AP-1 complexes and has been shown in vitro to be a point of
integration of numerous signals that can differentially affect its expression as
well as its transcriptional activity. Our strategy was based on the use of (i)
genetically modified fibroblasts deficient in components of the MAPK cascades
and (ii) pharmacological reagents. The results demonstrate that c-Jun
NH(2)-terminal protein kinase (JNK) is essential for a basal level of c-Jun
expression and for c-Jun phosphorylation in response to stress. In addition to
JNK, p38 MAPK or ERK1/2 and ERK5 are required for mediating UV radiation- or
epidermal growth factor (EGF)-induced c-Jun expression, respectively. Further
studies indicate that p38 MAPK inhibits the activation of JNK in response to
EGF, causing a down-regulation of c-Jun. Overall, these data provide important
insights into the mechanisms that ultimately determine the function of c-Jun as
a regulator of cell fate.

PMID: 15831482 [PubMed - indexed for MEDLINE]


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2: Biochem J. 2004 Aug 1;381(Pt 3):693-9. 

ERK5 is targeted to myocyte enhancer factor 2A (MEF2A) through a MAPK docking
motif.

Barsyte-Lovejoy D, Galanis A, Clancy A, Sharrocks AD.

School of Biological Sciences, University of Manchester, Michael Smith building,
Oxford Road, Manchester, M13 9PT, UK.

One critical component in determining the specificity, and efficiency of MAPK
(mitogen-activated protein kinase) substrate phophorylation is the presence of
distinct docking domains in the substrate proteins. Docking domains have been
shown to be important for the activities of members of the ERK
(extracellular-signal-regulated kinase), JNK (c-Jun N-terminal kinase) and p38
subfamilies of MAPKs towards their substrates. Here, we demonstrate that docking
domains also play an important role in ERK5-mediated substrate phosphorylation.
The presence of a docking domain promotes both phosphorylation of myocyte
enhancer factor, MEF2A, in vitro and its activation in vivo by ERK5. Mutational
analysis of the MEF2A docking domain demonstrates that the specificity
determinants for ERK5 are similar to those observed with members of the p38
subfamily. A docking domain recognized by ERK5 can direct ERK5 to activate
heterologous substrates. Deletion analysis demonstrates that as with other
MAPKs, it is the catalytic domain of ERK5 that recognizes the docking domain.
Our data therefore extend previous observations on other MAPKs and demonstrate
that the requirement for specific docking domains in promoting MAPK action
towards substrates is a general property of MAPKs.

PMID: 15132737 [PubMed - indexed for MEDLINE]


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3: Circ Res. 2003 Feb 7;92(2):243-51. 

Load-induced transcriptional activation of c-jun in rat myocardium: regulation
by myocyte enhancer factor 2.

Nadruz W Jr, Kobarg CB, Constancio SS, Corat PD, Franchini KG.

Department of Internal Medicine, School of Medicine, State University of
Campinas, Campinas, SP, Brazil.

The increased expression of immediate-early genes is a key feature of the
myocardial response to hypertrophic stimuli. In this study, we investigated
whether pressure overload or phenylephrine treatment stimulated myocyte enhancer
factor 2 (MEF2)-dependent transcriptional activation of c-jun in cardiac
myocytes. Western blotting and immunohistochemical analysis of rat myocardium
demonstrated that p70(MEF2) is highly expressed in the rat heart and is
predominantly located at the nuclei of cardiac myocytes. Electrophoretic
mobility shift assays of myocardial nuclear extracts revealed a consistent DNA
binding activation of MEF2 after 1 and 2 hours of pressure overload. We further
showed that pressure overload induced a progressive nuclear translocation and
activation of extracellular signal-regulated kinase 5 (ERK5).
Coimmunoprecipitation and in vitro kinase assays indicated that the activation
of ERK5 was paralleled by increased association of ERK5/p70(MEF2) and by
enhanced ability of ERK5 to phosphorylate p70(MEF2). Experiments with in vivo
transfection of the left ventricle with the c-jun promoter reporter gene showed
that pressure overload induced a consistent increase of c-jun transcriptional
activity in the rat myocardium. Rendering the MEF2 site of the c-jun plasmid
inactive by mutation abolished the load-induced activation of the c-jun promoter
reporter gene. Mutation of the MEF2 site also abolished the
phenylephrine-induced c-jun promoter activation in neonatal rat ventricular
myocytes. In addition, we demonstrated that neonatal rat ventricular myocyte
transfection with ERK5-antisense oligodeoxynucleotide inhibited the
phenylephrine-induced c-jun promoter activation. These findings identify MEF2 as
a potential regulator of c-jun transactivation and suggest that ERK5 might be an
important mediator of MEF2 and c-jun promoter activation in response to
hypertrophic stimuli in cardiac myocytes.

PMID: 12574153 [PubMed - indexed for MEDLINE]


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4: Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai). 2000;32(3):258-264. 

Signal Transduction in TNF-alpha-induced c-jun Gene Expression.

Zhao M, Liu YW, Liu AH, Zhao KS, Jiang Y.

Department of Pathophysiology and Key Laboratory for Shock and Microcirculation
of PLA, The First Military Medical University, Guangzhou 510515, China.
yjiang@public.guangzhou.gd.cn

Our previous studies demonstrated that p38 mitogen-activated protein (MAP)
kinase regulated the c-jun protein expression through phosphorylation of
transcription factors of myocyte enhancer factors 2 (MEF2) family. There was a
MEF2 binding site in the promoter of c-jun gene. Members of the MEF2 family of
trans-cription factors bound as homo- and heterodimers to this MEF2 binding
site. Here the potential role of the p38 and BMK1 MAP kinases in the regulation
of c-jun expression induced by TNF-alpha was examined. It was shown that p38 MAP
kinase up-regulated the transcription activity of MEF2A, while BMK1 MAP kinase
up-regulated not only the transcription activity of MEF2A, but also MEF2D. The
p38 and BMK1 MAP kinases had coordinated effect on the regulation of c-jun
transcription. TNF-alpha induced the formation of MEF2A/MEF2D hete-rodimer.
Over-expression of homodimer of MEF2 proteins inhibited c-jun transcription
induced by TNF-alpha, while over-expression of heterodimer MEF2A/MEF2D enhanced
c-jun transcription induced by TNF-alpha. Phosphorylation of MEF2A and MEF2D by
p38 and BMK1 respectively appeared very important in TNF-alpha induced
MEF2A/MEF2D heterodimer formation to enhance c-jun gene expression.

PMID: 12075451 [PubMed - as supplied by publisher]


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5: Arch Biochem Biophys. 2002 Apr 15;400(2):199-207. 

Vascular smooth muscle cell proliferation requires both p38 and BMK1 MAP
kinases.

Zhao M, Liu Y, Bao M, Kato Y, Han J, Eaton JW.

Division of Pathology II, Faculty of Health Sciences, Linkoping University,
Linkoping SE-581 85, Sweden. ming.zhao@inr.liu.se

Vascular smooth muscle cell (VSMC) proliferation is a key event in the
progression of atherosclerosis. Induction of both c-fos (through the
transcription factor Elk-1) and c-jun, both immediate early genes, is important
for the stimulation of VSMC proliferation and migration. It was earlier found
that p38 mitogen-activated protein (MAP) kinase upregulates c-jun gene
transcription through phosphorylation of two myocyte enhancer factor 2 (MEF2)
family transcription factors, MEF2A and MEF2C, while big MAP kinase 1 (BMK1) may
upregulate c-jun gene transcription through MEF2A, MEF2C, and also MEF2D. Here,
we report that inhibition of BMK1 by a dominant negative form of MEK5 or
pharmacologic inhibition of p38 by SB 203580 additively suppress serum-induced
VSMC proliferation. This additive effect of p38 and BMK1 inhibition implies that
these two kinases coordinately regulate MEF2 transcription factors. The
exclusive activation of MEF2D by BMK1 appears required for this cooperative
upregulation of c-jun in VSMC, and coactivation of p38 and BMK1 also has
additive effects on the activation of a reporter gene linked to the c-jun
promoter in our experimental system. Thus, coordinate activity of both the p38
and BMK1 pathways appears necessary for optimal transcription of c-jun and, pari
pasu, VSMC proliferation. These results may have implications for the future
design of pharmacologic agents for inhibition of VSMC growth.

PMID: 12054430 [PubMed - indexed for MEDLINE]


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6: Circ Res. 2002 May 17;90(9):1004-11. 

Angiotensin II induces myocyte enhancer factor 2- and calcineurin/nuclear factor
of activated T cell-dependent transcriptional activation in vascular myocytes.

Suzuki E, Nishimatsu H, Satonaka H, Walsh K, Goto A, Omata M, Fujita T, Nagai R,
Hirata Y.

Department of Internal Medicine, Faculty of Medicine, University of Tokyo,
Tokyo, Japan. suzuki-2im@h.u-tokyo.ac.jp

It is well known that angiotensin II (Ang II) is implicated in the phenotypic
modulation and hypertrophy of vascular smooth muscle cells (VSMCs). To study the
mechanisms by which Ang II contributes to the pathological changes of VSMCs, we
examined whether Ang II stimulated myocyte enhancer factor 2 (MEF2)- and
calcineurin/nuclear factor of activated T cell (NFAT)-dependent transcriptional
activation of genes in VSMCs. Ang II increased the DNA binding activity of MEF2A
and its expression at the protein level. Ang II induced c-jun promoter activity,
and this increase was inhibited by dominant-negative mutants of MEF2A and
mitogen-activated protein kinase kinase 6 but not by calcineurin inhibitors. Ang
II stimulated NFAT DNA binding activity and NFAT-dependent gene transcription,
and these effects of Ang II were inhibited by calcineurin inhibitors.
Furthermore, Ang II induced the promoter activity of the nonmuscle-type myosin
heavy chain B gene, which we used as a marker of the dedifferentiated state of
VSMCs, and this increase was inhibited by calcineurin inhibitors but not by the
dominant-negative mutants of MEF2A or mitogen-activated protein kinase kinase 6.
Finally, Ang II increased protein synthesis, and this increase was inhibited by
infection with an adenovirus construct that expresses the dominant-negative
mutant of MEF2A but not by calcineurin inhibitors. These results suggest that
Ang II stimulates the MEF2- and calcineurin/NFAT-dependent pathways and that
these pathways have distinct roles in VSMCs.

PMID: 12016267 [PubMed - indexed for MEDLINE]


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7: J Biol Chem. 2002 May 10;277(19):16426-32. Epub 2002 Mar 4. 

Composition and function of AP-1 transcription complexes during muscle cell
differentiation.

Andreucci JJ, Grant D, Cox DM, Tomc LK, Prywes R, Goldhamer DJ, Rodrigues N,
Bedard PA, McDermott JC.

Department of Biology, York University, Toronto, Ontario M3J 1P3, Canada.

The role of activating protein-1 (AP-1) in muscle cells is currently equivocal.
While some studies propose that AP-1 is inhibitory for myogenesis, others
implicate a positive role in this process. We tested whether this variation may
be due to different properties of the AP-1 subunit composition in
differentiating cells. Using Western analysis we show that c-Jun, Fra-2, and
JunD are expressed throughout the time course of differentiation. Phosphatase
assays indicate that JunD and Fra-2 are phosphorylated in muscle cells and that
at least two isoforms of each are expressed in muscle cells. Electrophoretic
mobility shift assays combined with antibody supershifts indicate the appearance
of Fra-2 as a major component of the AP-1 DNA binding complex in differentiating
cells. In this context it appears that Fra-2 heterodimerizes with c-Jun and
JunD. Studying the c-jun enhancer in reporter gene assays we observed that the
muscle transcription factors MEF2A and MyoD can contribute to robust
transcriptional activation of the c-jun enhancer. In differentiating muscle
cells mutation of the MEF2 site reduces transactivation of the c-jun enhancer
and MEF2A is the predominant MEF2 isoform binding to this cis element.
Transcriptional activation of an AP-1 site containing reporter gene (TRE-Luc) is
enhanced under differentiation conditions compared with growth conditions in
C2C12 muscle cells. Further studies indicate that Fra-2 containing AP-1
complexes can transactivate the MyoD enhancer/promoter. Thus, an AP-1 complex
containing Fra-2 and c-Jun or JunD is consistent with muscle differentiation,
indicating that AP-1 function during myogenesis is dependent on its subunit
composition.

PMID: 11877423 [PubMed - indexed for MEDLINE]


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8: Tsitologiia. 2001;43(10):961-8. 

[Constitutive activity of MAP kinase cascades in REF cells transformed by E1A
and cHa-ras oncogenes]

[Article in Russian]

Svetlikova SB, Abramova MV, Kukushkin AN, Darieva ZA, Pospelova TV, Pospelov VA.

Institute of Cytology, RAS, St. Petersburg.

Proteins of Ras family play an important role in regulation of cell growth and
proliferation, and their mutations can lead to growth factor-independent
proliferation due to constitutive activity of various signal transduction
cascades. In the present work, we studied the activity of ERK, JNK and p38
MAP-kinase cascades in rat embryo fibroblast cells transformed with oncogenes
E1A and cHa-ras. These transformed cells are characterized by a high and
non-regulated activity of transcription factor AP-1 involved in the regulation
of cell proliferation. Since phosphorylation of AP-1 depends on the activity of
relevant MAP-kinase cascades (ERK, JNK and p38), we analysed the expression of
non-phosphorylated forms of the kinases and their phosphorylated state in E1A +
cHa-ras cells using antibodies specific to non-phosphorylated and phosphorylated
proteins. It has been established that transformed cells contain higher amounts
of non-phosphorylated ERK, JNK and p38 kinases, thus implying a reduced
degradation of these and other proteins in the transformants. The content of
phosphorylated (active) forms studied in Western blot-analysis with
phosphoantibodies was shown to be also higher in exponentially growing E1A +
cHa-ras cells. But serum stimulation of the starved cells gave insignificant
rise to an increase of ERK, JNK and p38 phosphorylation. Nevertheless, an in
vitro kinase assay performed with the kinases, either immunoprecipitated by
antibody or bound to GST-fusion substrates, enabled us to show a certain level
of stimulation of c-Jun-associated (JNK) and MEF2A-associated (p38) kinase
activity in serum stimulated E1A + cHa-ras cells. Thus, the obtained results
show that transformation of fibroblasts with E1A and ras oncogenes may
contribute to constitutive activation of ERK, JNK and p38 kinase cascades
responsible for a high and non-regulated activity of MAP-kinase-dependent
transcription factors, in particular AP-1.

PMID: 11769129 [PubMed - indexed for MEDLINE]


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9: Genes Dev. 2001 Mar 1;15(5):535-53. 

Regulation of gene expression by the small GTPase Rho through the ERK6 (p38
gamma) MAP kinase pathway.

Marinissen MJ, Chiariello M, Gutkind JS.

Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial
Research, National Institutes of Health, Bethesda, Maryland 20892, USA.

Small GTP-binding proteins of the Rho-family, Rho, Rac, and Cdc42, have been
traditionally linked to the regulation of the cellular actin-based cytoskeleton.
Rac and Cdc42 can also control the activity of JNK, thus acting in a molecular
pathway transmitting extracellular signals to the nucleus. Interestingly, Rho
can also regulate gene expression, albeit by a not fully understood mechanism.
Here, we found that activated RhoA can stimulate c-jun expression and the
activity of the c-jun promoter. As the complexity of the signaling pathways
controlling the expression of c-jun has begun to be unraveled, this finding
provided a unique opportunity to elucidate the biochemical routes whereby RhoA
regulates nuclear events. We found that RhoA can initiate a linear kinase
cascade leading to the activation of ERK6 (p38 gamma), a recently identified
member of the p38 family of MAPKs. Furthermore, we present evidence that RhoA,
PKN, MKK3/MKK6, and ERK6 (p38 gamma) are components of a novel signal
transduction pathway involved in the regulation of gene expression and cellular
transformation.

PMID: 11238375 [PubMed - indexed for MEDLINE]


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10: J Biol Chem. 2000 Jun 16;275(24):18534-40. 

Big mitogen-activated kinase regulates multiple members of the MEF2 protein
family.

Kato Y, Zhao M, Morikawa A, Sugiyama T, Chakravortty D, Koide N, Yoshida T,
Tapping RI, Yang Y, Yokochi T, Lee JD.

Department of Microbiology and Immunology, Aichi Medical University, Nagakute,
Aichi 480-1195, Japan.

Big mitogen-activated protein (MAP) kinase (BMK1), a member of the mammalian MAP
kinase family, is activated by growth factors. The activation of BMK1 is
required for growth factor-induced cell proliferation and cell cycle
progression. We have previously shown that BMK1 regulates c-jun gene expression
through direct phosphorylation and activation of transcription factor MEF2C.
MEF2C belongs to the myocyte enhancer factor 2 (MEF2) protein family, a
four-membered family of transcription factors denoted MEF2A, -2B, -2C, and -2D.
Here, we demonstrate that, in addition to MEF2C, BMK1 phosphorylates and
activates MEF2A and MEF2D but not MEF2B. The blocking of BMK1 signaling inhibits
the epidermal growth factor-dependent activation of these three MEF2
transcription factors. The sites phosphorylated by activated BMK1 were mapped to
Ser-355, Thr-312, and Thr-319 of MEF2A and Ser-179 of MEF2D both in vitro and in
vivo. Site-directed mutagenesis reveals that the phosphorylation of these sites
in MEF2A and MEF2D are necessary for the induction of MEF2A and 2D
transactivating activity by either BMK1 or by epidermal growth factor. Taken
together, these data demonstrate that, upon growth factor induction, BMK1
directly phosphorylates and activates three members of the MEF2 family of
transcription factors thereby inducing MEF2-dependent gene expression.

PMID: 10849446 [PubMed - indexed for MEDLINE]


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11: Mol Cell Biol. 2000 Jun;20(11):3951-64. 

p38 and extracellular signal-regulated kinases regulate the myogenic program at
multiple steps.

Wu Z, Woodring PJ, Bhakta KS, Tamura K, Wen F, Feramisco JR, Karin M, Wang JY,
Puri PL.

Laboratory of Gene Regulation and Signal Transduction, University of California,
San Diego, La Jolla, California 92093-0322, USA.

The extracellular signals which regulate the myogenic program are transduced to
the nucleus by mitogen-activated protein kinases (MAPKs). We have investigated
the role of two MAPKs, p38 and extracellular signal-regulated kinase (ERK),
whose activities undergo significant changes during muscle differentiation. p38
is rapidly activated in myocytes induced to differentiate. This activation
differs from those triggered by stress and cytokines, because it is not linked
to Jun-N-terminal kinase stimulation and is maintained during the whole process
of myotube formation. Moreover, p38 activation is independent of a parallel
promyogenic pathway stimulated by insulin-like growth factor 1. Inhibition of
p38 prevents the differentiation program in myogenic cell lines and human
primary myocytes. Conversely, deliberate activation of endogenous p38 stimulates
muscle differentiation even in the presence of antimyogenic cues. Much evidence
indicates that p38 is an activator of MyoD: (i) p38 kinase activity is required
for the expression of MyoD-responsive genes, (ii) enforced induction of p38
stimulates the transcriptional activity of a Gal4-MyoD fusion protein and allows
efficient activation of chromatin-integrated reporters by MyoD, and (iii)
MyoD-dependent myogenic conversion is reduced in mouse embryonic fibroblasts
derived from p38alpha(-/-) embryos. Activation of p38 also enhances the
transcriptional activities of myocyte enhancer binding factor 2A (MEF2A) and
MEF2C by direct phosphorylation. With MEF2C, selective phosphorylation of one
residue (Thr293) is a tissue-specific activating signal in differentiating
myocytes. Finally, ERK shows a biphasic activation profile, with peaks of
activity in undifferentiated myoblasts and postmitotic myotubes. Importantly,
activation of ERK is inhibitory toward myogenic transcription in myoblasts but
contributes to the activation of myogenic transcription and regulates
postmitotic responses (i.e., hypertrophic growth) in myotubes.

PMID: 10805738 [PubMed - indexed for MEDLINE]


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12: J Biol Chem. 2000 Jul 14;275(28):21730-6. 

Signaling from G protein-coupled receptors to ERK5/Big MAPK 1 involves Galpha q
and Galpha 12/13 families of heterotrimeric G proteins. Evidence for the
existence of a novel Ras AND Rho-independent pathway.

Fukuhara S, Marinissen MJ, Chiariello M, Gutkind JS.

Oral and Pharyngeal Cancer Branch, NIDCR, National Institutes of Health,
Bethesda, Maryland 20892-4330, USA.

The regulation of gene expression by cell surface receptors often involves the
stimulation of signaling pathways including one or more members of the MAPK
superfamily of serine-threonine kinases. Upon their activation in the cytosol,
MAPKs can translocate to the nucleus and affect the activity of a variety of
transcription factors. Recently, it has been observed that a novel member of the
MAPK superfamily, ERK5, can be potently activated by transforming G
protein-coupled receptors (GPCRs) and that ERK5 participates in the regulation
of c-jun expression through the activation of MEF2 transcription factors. How
cell surface receptors, including GPCRs, stimulate ERK5 is still poorly
understood. In this study, we have used transiently transfected COS-7 cells to
begin delineating the biochemical route linking GPCRs to ERK5. We show that
receptors that can couple to the G(q) and G(12/13) families of heterotrimeric G
proteins, m1 and thrombin receptors, respectively, but not those coupled to
G(i), such as m2 receptors, are able to regulate the activity of ERK5. To
investigate which heterotrimeric G proteins signal to ERK5, we used a chimeric
system by which Galpha(q)- and Galpha(13)-mediated signaling pathways can be
conditionally activated upon ligand stimulation. Using this system, as well as
the expression of activated forms of G protein subunits, we show that the
Galpha(q) and Galpha(12/13) families of heterotrimeric G proteins, but not the
Galpha(i), Galpha(s), and betagamma subunits, are able to regulate ERK5.
Furthermore, we provide evidence that the stimulation of ERK5 by GPCRs involves
a novel signaling pathway, which is distinct from those regulated by Ras and Rho
GTPases.

PMID: 10781600 [PubMed - indexed for MEDLINE]


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13: J Mol Biol. 2000 Mar 24;297(2):437-49. 

Crystal structure of MEF2A core bound to DNA at 1.5 A resolution.

Santelli E, Richmond TJ.

Institut fur Molekularbiologie und Biophysik, ETH Zurich, Zurich, CH,
Switzerland.

Members of the myocyte enhancer factor-2 (MEF2) family of transcription factors
bind to and activate transcription through A+T-rich DNA sequences found
primarily, but not exclusively, in the promoters of muscle-specific genes. Their
importance has been established for myogenic development and in activation of
the immediate-early gene, c-jun, and recently further functional roles in the
immune system have emerged. The MEF2 factors belong to the MADS-box superfamily,
sharing homology in a 58 amino acid domain that mediates DNA binding and
dimerization. The structures of two MADS-box proteins, SRF and MCM1, bound to
their cognate DNA have been previously reported and shown to share extensive
similarity in their mode of DNA binding. We have solved the structure of MEF2A
2-78 bound to its DNA consensus sequence at 1.5 A resolution. It reveals how the
absence of amino acids N-terminal to the MADS-box contributes to the DNA binding
properties of MEF2 proteins and shows that the MEF domain C-terminal to the
MADS-box adopts a conformation considerably different from the same region in
SRF and MCM1. Copyright 2000 Academic Press.

PMID: 10715212 [PubMed - indexed for MEDLINE]


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14: Mol Cell Biol. 1999 Nov;19(11):7816-27. 

HDAC4, a human histone deacetylase related to yeast HDA1, is a transcriptional
corepressor.

Wang AH, Bertos NR, Vezmar M, Pelletier N, Crosato M, Heng HH, Th'ng J, Han J,
Yang XJ.

Molecular Oncology Group, Department of Medicine, McGill University, Montreal,
Quebec, Canada.

Histone acetylation plays an important role in regulating chromatin structure
and thus gene expression. Here we describe the functional characterization of
HDAC4, a human histone deacetylase whose C-terminal part displays significant
sequence similarity to the deacetylase domain of yeast HDA1. HDAC4 is expressed
in various adult human tissues, and its gene is located at chromosome band 2q37.
HDAC4 possesses histone deacetylase activity intrinsic to its C-terminal domain.
When tethered to a promoter, HDAC4 represses transcription through two
independent repression domains, with repression domain 1 consisting of the
N-terminal 208 residues and repression domain 2 containing the deacetylase
domain. Through a small region located at its N-terminal domain, HDAC4 interacts
with the MADS-box transcription factor MEF2C. Furthermore, HDAC4 and MEF2C
individually upregulate but together downmodulate c-jun promoter activity. These
results suggest that HDAC4 interacts with transcription factors such as MEF2C to
negatively regulate gene expression.

PMID: 10523670 [PubMed - indexed for MEDLINE]


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15: Mol Immunol. 1999 Feb;36(3):197-203. 

CD28-mediated regulation of the c-jun promoter involves the MEF2 transcription
factor in Jurkat T cells.

Shin HM, Han TH.

Department of Microbiology and Immunology, Sungkyunkwan University School of
Medicine, Suwon, South Korea.

Within a few minutes of T-cell activation, transcription of a set of genes
including c-fos and c-jun is activated. For maximal induction of c-jun, at least
two major signal pathways are required. One can be triggered by T-cell receptor
engagement or phorbol esters and the other by anti-CD28 engagement. The c-jun
promoter region between -117 and -50 contains binding sites for the
transcription factors Spl, CTF, ATF/CREB, and MEF2. In this study, we sought to
map the sequences in the c-jun promoter responsible for CD28-mediated induction
in activated Jurkat T cell by point mutational analysis. We found that mutation
of the c-jun MEF2 site strongly reduces CD28 induction of the promoter in Jurkat
T cells and that MEF2D is the major binding molecule to the c-jun MEF2 site in
Jurkat T cells. Mutation of the c-jun ATF site also partially reduced CD28
induction of the promoter. In addition, pretreatment with an endolysomotropic
agent NH4Cl, an acidic sphingomyelinase inhibitor, completely inhibited the
activation of the c-jun promoter by anti-CD28 antibody treatment, whereas
pretreatment with wortmannin, a PI3-kinase inhibitor, did not affect the
induction of the c-jun promoter. These results suggest that CD28 signaling
leading to the c-jun promoter involves acidic sphingomyelinase, but not
PI3-kinase, to activate factors binding to the MEF2 and ATF sites.

PMID: 10403485 [PubMed - indexed for MEDLINE]


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16: Mol Cell Biol. 1999 Jun;19(6):4028-38. 

Targeting of p38 mitogen-activated protein kinases to MEF2 transcription
factors.

Yang SH, Galanis A, Sharrocks AD.

Department of Biochemistry and Genetics, The Medical School, University of
Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, United Kingdom.

Mitogen-activated protein (MAP) kinase-mediated signalling to the nucleus is an
important event in the conversion of extracellular signals into a cellular
response. However, the existence of multiple MAP kinases which phosphorylate
similar phosphoacceptor motifs poses a problem in maintaining substrate
specificity and hence the correct biological response. Both the extracellular
signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) subfamilies of
MAP kinases use a second specificity determinant and require docking to their
transcription factor substrates to achieve maximal substrate activation. In this
study, we demonstrate that among the different MAP kinases, the MADS-box
transcription factors MEF2A and MEF2C are preferentially phosphorylated and
activated by the p38 subfamily members p38alpha and p38beta2. The efficiency of
phosphorylation in vitro and transcriptional activation in vivo of MEF2A and
MEF2C by these p38 subtypes requires the presence of a kinase docking domain
(D-domain). Furthermore, the D-domain from MEF2A is sufficient to confer p38
responsiveness on different transcription factors, and reciprocal effects are
observed upon the introduction of alternative D-domains into MEF2A. These
results therefore contribute to our understanding of signalling to MEF2
transcription factors and demonstrate that the requirement for substrate binding
by MAP kinases is an important facet of three different subclasses of MAP
kinases (ERK, JNK, and p38).

PMID: 10330143 [PubMed - indexed for MEDLINE]


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17: Development. 1999 May;126(10):2045-52. 

Transcriptional activity of MEF2 during mouse embryogenesis monitored with a
MEF2-dependent transgene.

Naya FJ, Wu C, Richardson JA, Overbeek P, Olson EN.

Department of Molecular Biology, University of Texas Southwestern Medical Center
at Dallas, 6000 Harry Hines Blvd., Dallas, TX 75235-9148, USA.

The four members of the MEF2 family of MADS-box transcription factors, MEF2-A,
MEF2-B, MEF2-C and MEF2-D, are expressed in overlapping patterns in developing
muscle and neural cell lineages during embryogenesis. However, during late fetal
development and postnatally, MEF2 transcripts are also expressed in a wide range
of cell types. Because MEF2 expression is controlled by translational and
post-translational mechanisms, it has been unclear whether the presence of MEF2
transcripts in the embryo reflects transcriptionally active MEF2 proteins. To
define the temporospatial expression pattern of transcriptionally active MEF2
proteins during mouse embryogenesis, we generated transgenic mice harboring a
lacZ reporter gene controlled by three tandem copies of the MEF2 site and
flanking sequences from the desmin enhancer, which is active in cardiac,
skeletal and smooth muscle cells. Expression of this MEF2-dependent transgene
paralleled expression of MEF2 mRNAs in developing myogenic lineages and regions
of the adult brain. However, it was not expressed in other cell types that
express MEF2 transcripts. Tandem copies of the MEF2 site from the c-jun promoter
directed expression in a similar pattern to the desmin MEF2 site, suggesting
that transgene expression reflects the presence of transcriptionally active MEF2
proteins, rather than other factors specific for DNA sequences flanking the MEF2
site. These results demonstrate the presence of transcriptionally active MEF2
proteins in the early muscle and neural cell lineages during embryogenesis and
argue against the existence of lineage-restricted MEF2 cofactors that
discriminate between MEF2 sites with different immediate flanking sequences. The
discordance between MEF2 mRNA expression and MEF2 transcriptional activity in
nonmuscle cell types of embryos and adults also supports the notion that
post-transcriptional mechanisms regulate the expression of MEF2 proteins.

PMID: 10207130 [PubMed - indexed for MEDLINE]


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18: Cell Growth Differ. 1998 Aug;9(8):677-86. 

v-Jun represses c-jun proto-oncogene expression in vivo through a
12-O-tetradecanoylphorbol-13-acetate-responsive element in the proximal gene
promoter.

Hussain S, Kilbey A, Gillespie DA.

Beatson Institute for Cancer Research, Cancer Research Campaign Beatson
Laboratories, Bearsden, Glasgow, United Kingdom.

c-jun proto-oncogene expression is extinguished in cells transformed by v-Jun;
however, the mechanistic basis of this phenomenon has not been elucidated. c-jun
mRNA levels are greatly reduced in v-Jun-transformed cells, and we show that
this reduction is associated with a similar decrease in the rate of c-jun
transcription. Transcriptional down-regulation was also evident in functional
assays in which the c-jun gene promoter was approximately 10-fold less active in
v-Jun-transformed cells than it was in normal cells. This reduction was largely
attributable to a conserved 12-O-tetradecanoylphorbol-13-acetate-responsive
element (TRE)-like motif at position -72 (the proximal junTRE) that was
essential for efficient basal expression in normal cells but that conferred
little, if any, detectable transcriptional activity in v-Jun-transformed cells.
DNA-binding analysis showed that this element was recognized by a mixture of
c-Jun/Fra and cyclic AMP-responsive element-binding protein/activating
transcription factor-like complexes in normal cells but that v-Jun/Fra
heterodimers predominated in v-Jun-transformed cells. Furthermore, ectopic
expression of v-Jun repressed c-jun promoter activity in normal cells through
the proximal junTRE. Thus, the deficit in transcription mediated by the junTRE
correlates with and is most likely attributable to binding of v-Jun to this
element in vivo. We also find that the c-jun promoter is refractory to induction
via the stress-activated protein kinase/c-jun NH2-terminal kinase pathway in
v-Jun-transformed cells, suggesting that v-Jun interferes with signal-regulated
gene expression. Therefore, c-jun is an example of a cellular gene, the
transcription of which is regulated negatively by v-Jun in vivo.

PMID: 9716184 [PubMed - indexed for MEDLINE]


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19: Mol Cell Biol. 1998 Feb;18(2):1065-73. 

Epidermal growth factor induction of the c-jun promoter by a Rac pathway.

Clarke N, Arenzana N, Hai T, Minden A, Prywes R.

Department of Biological Sciences, Columbia University, New York, New York
10027, USA.

The c-jun proto-oncogene encodes a transcription factor which is activated by
mitogens both transcriptionally and by phosphorylation by Jun N-terminal kinase
(JNK). We have investigated the cellular signalling pathways involved in
epidermal growth factor (EGF) induction of the c-jun promoter. We find that two
sequence elements, which bind ATF1 and MEF2D transcription factors, are required
in HeLa cells, although they are not sufficient for maximal induction. Activated
forms of Ras, RacI, Cdc42Hs, and MEKK increased expression of the c-jun
promoter, while dominant negative forms of Ras, RacI, and MEK kinase (MEKK)
inhibited EGF induction. These and previously published results suggest that EGF
activates the c-jun promoter by a Ras-to-Rac-to-MEKK pathway. This pathway is
similar to that used for posttranslational activation of c-jun by JNK.

PMID: 9448004 [PubMed - indexed for MEDLINE]


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20: J Biol Chem. 1997 Aug 15;272(33):20691-7. 

Signaling from G protein-coupled receptors to the c-jun promoter involves the
MEF2 transcription factor. Evidence for a novel c-jun amino-terminal
kinase-independent pathway.

Coso OA, Montaner S, Fromm C, Lacal JC, Prywes R, Teramoto H, Gutkind JS.

Oral and Pharyngeal Cancer Branch, NIDR, National Institutes of Health,
Bethesda, Maryland 20892-4330, USA.

The c-Jun amino-terminal kinases (JNKs) are a subfamily of mitogen-activated
protein kinases that phosphorylate c-Jun and ATF2, and it has been postulated
that phosphorylated c-Jun enhances its own expression through AP-1 sites on the
c-jun promoter. In this study, we asked whether signals activating JNK regulate
the c-jun promoter. Using NIH 3T3 cells expressing G protein-coupled m1
acetylcholine receptors as an experimental model, we have recently shown that
the cholinergic agonist carbachol, but not platelet-derived growth factor,
potently elevates JNK activity. Consistent with these findings, carbachol, but
not platelet-derived growth factor, increased the activity of a c-jun
promoter-driven reporter gene (for chloramphenicol acetyltransferase). However,
coexpression of JNK kinase kinase (MEKK) effectively increased JNK activity, but
resulted in surprisingly limited induction of the c-jun promoter. This raised
the possibility that pathway(s) distinct from JNK control the c-jun promoter,
and prompted us to explore which of its regulatory elements participate in
transcriptional control. We observed that deletion of the 3' AP-1 site
diminished chloramphenicol acetyltransferase activity in response to carbachol,
but only to a limited extent. In contrast, deletion of a MEF2 site dramatically
reduced expression, and deletion of both the MEF2 and 3' AP-1 sites abolished
induction. Furthermore, cotransfection with MEF2C and MEF2D cDNAs potently
enhanced the activity of the c-jun promoter in response to carbachol, and
stimulation of m1 receptors, but not direct JNK activation, induced expression
of a MEF2-responsive plasmid. Taken together, these data strongly suggest that
MEF2 mediates c-jun promoter expression by G protein-coupled receptors through a
yet to be identified pathway, distinct from that of JNK.

PMID: 9252389 [PubMed - indexed for MEDLINE]


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21: J Biol Chem. 1996 Oct 4;271(40):24927-33. 

MEF2 protein expression, DNA binding specificity and complex composition, and
transcriptional activity in muscle and non-muscle cells.

Ornatsky OI, McDermott JC.

Departments of Kinesiology and Biology, Faculty of Pure and Applied Science,
York University, Toronto, Ontario M3J 1P3, Canada.

Tissue-specific gene expression can be mediated by complex transcriptional
regulatory mechanisms. Based on the dichotomy of the ubiquitous distribution of
the myocyte enhancer factor 2 (MEF2) gene mRNAs compared to their cell
type-restricted activity, we investigated the basis for their tissue
specificity. Electrophoretic mobility shift assays using the muscle creatine
kinase MEF2 DNA binding site as a probe showed that HeLa, Schneider, L6E9
muscle, and C2C12 muscle cells have a functional MEF2 binding activity that is
indistinguishable based on competition analysis. Interestingly, chloramphenicol
acetyltransferase reporter assays showed MEF2 site-dependent trans-activation in
myogenic C2C12 cells but no trans-activation by the endogenous MEF2 proteins in
HeLa cells. By immunofluorescence, we detected abundant nuclear localized MEF2A
and MEF2D protein expression in HeLa cells and C2C12 muscle cells. Using
immuno-gel shift analysis and also co-immunoprecipitation studies, we show that
the predominant MEF2 DNA binding complex bound to MEF2 sites from either the
muscle creatine kinase or c-jun regulatory regions in C2C12 muscle cells is
comprised of a MEF2A homodimer, whereas in HeLa cells, it is a MEF2A:MEF2D
heterodimer. Thus, the presence of MEF2 DNA binding complexes is not necessarily
coupled with trans-activation of target genes. The ability of the MEF2 proteins
to activate transcription in vivo correlates with the specific dimer composition
of the DNA binding complex and the cellular context.

PMID: 8798771 [PubMed - indexed for MEDLINE]


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22: Mol Cell Biol. 1995 Jun;15(6):2907-15. 

Regulatory role of MEF2D in serum induction of the c-jun promoter.

Han TH, Prywes R.

Department of Biological Sciences, Columbia University, New York, New York
10027, USA.

Serum induction of c-jun expression in HeLa cells requires a MEF2 site at -59 in
the c-jun promoter. MEF2 sites, found in many muscle-specific enhancers, are
bound by a family of transcription factors, MEF2A through -D, which are related
to serum response factor in their DNA binding domains. We have found that MEF2D
is the predominant protein in HeLa cells that binds to the c-jun MEF2 site.
Serum induction of a MEF2 reporter gene was not observed in a line of NIH 3T3
cells which contain low MEF2 site binding activity. Transfection of MEF2D into
NIH 3T3 cells reconstituted serum induction, demonstrating that MEF2D is
required for the serum response. Deletion analysis of MEF2D showed that its DNA
binding domain, when fused to a heterologous transcriptional activation domain,
was sufficient for serum induction of a MEF2 reporter gene. This is the domain
homologous to that in the serum response factor which is required for serum
induction of the c-fos serum response element, suggesting that serum regulation
of c-fos and c-jun may share a common mechanism.

PMID: 7760790 [PubMed - indexed for MEDLINE]


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