Muscle cell development is dependent on the activity of cell type-specific basic-helix-loop-helix transcription factors, MyoD, Myf-5, myogenin, and MRF4 which collaborate with myocyte enhancer factor 2 proteins to activate muscle-specific gene expression. Growth factors and activated Ras prevent differentiation of myoblasts in culture but the downstream signalling pathways are not well understood. Here, we demonstrate that active Raf kinase (Raf-BxB) completely inhibits myogenic conversion of 10T1/2 cells mediated by Myf-5 and differentiation of L6 myoblasts as indicated by the absence of myotubes, lack of myogenin expression, and markedly reduced expression of myosin heavy chain. However, activated Raf inhibits transcriptional activation by Myf-5 only partially suggesting that other potential targets of Ras/Raf signalling may be involved. Significantly, we observed that elevated Raf kinase activity in L6 muscle cells suppresses the accumulation of MEF2 protein in nuclei, while MEF2 transcription appears unaffected. Moreover, forced expression of MEF2A in 10T1/2 cells rescues MyoD dependent myogenic conversion in the presence of constitutively active Raf kinase and partially restores transactivation of a myogenin promoter-dependent reporter gene in L6 muscle cells containing activated Raf kinase. From these observations we conclude that persistent activation of Raf signalling affects nuclear MEF2 functions which may explain why myogenin expression and myoblast differentiation are inhibited.

REFERENCES

Alema
S.
,
Tato
F.
(
1994
).
Oncogenes and muscle differentiation: multiple mechanisms of interference.
Cancer Biol
5
,
147
156
Avruch
J.
,
Zhang
X. F.
,
Kyriakis
J. M.
(
1994
).
Raf meets Ras: completing the framework of a signal transduction pathway.
Trends Biochem. Sci
19
,
279
283
Arnold
H. H.
,
Winter
B.
(
1998
).
Muscle differentiation: more complexity to the network of myogenic regulators.
Curr. Opin. Genet. Dev
8
,
539
544
Bader
D.
,
Masaki
T.
,
Fischman
D. A.
(
1982
).
Immunochemical analysis of myosin heavy chain during avian myogenesis in vivo and in vitro.
J. Cell Biol
95
,
763
770
Bennett
A. M.
,
Tonks
N. K.
(
1997
).
Regulation of distinct stages of skeletal muscle differentiation by mitogen-activated protein kinases.
Science
278
,
1288
1291
Black
B. L.
,
Molkentin
J. D.
,
Olson
E. N.
(
1998
).
Multiple roles for the MyoD basic region in transmission of transcriptional activation signals and the interaction with MEF2.
Mol. Cell. Biol
18
,
69
77
Brennan
T. J.
,
Edmondson
D. G.
,
Li
L.
,
Olson
E. N.
(
1991
).
Transforming growth factor β represses the action of myogenin through a mechanism independent of DNA binding.
Proc. Nat. Acad. Sci. USA
88
,
3822
3826
Buchberger
A.
,
Ragge
K.
,
Arnold
H. H.
(
1994
).
The myogenin gene is activated during myocyte differentiation by pre-existing, not newly synthesized transcription factor MEF-2.
J. Biol. Chem
269
,
17289
17296
Dorman
C. M.
,
Johnson
S. E.
(
1999
).
Activated Raf inhibits avian myogenesis through a MAPK-dependent mechanism.
Oncogene
18
,
5167
5176
Eckner
R.
,
Ewen
M. E.
,
Newsome
D.
,
Gerdes
M.
,
DeCaprio
J. A.
,
Lawrence
J. B.
,
Livingston
D. M.
(
1994
).
Molecular cloning and functional analysis of the adenovirus E1A-associated 300-kD protein (p300) reveals a protein with properties of a transcriptional adaptor.
Genes Dev
3
,
869
884
Gredinger
E.
,
Gerber
A. N.
,
Tamir
Y.
,
Tapscott
S. J.
,
Bengal
E.
(
1998
).
Mitogen-activated protein kinase pathway is involved in the differentiation of muscle cells.
J. Biol. Chem
273
,
10436
10444
Han
J.
,
Jiang
Y.
,
Li
Z.
,
Kravchenko
V. V.
,
Ulevitch
R. J.
(
1997
).
Activation of the transcription factor MEF2C by the MAP kinase p38 in inflammation.
Nature
386
,
296
299
Hasty
P.
,
Bradley
A.
,
Morris
J. H.
,
Edmondson
D. G.
,
Venuti
J. M.
,
Olson
E. N.
(
1993
).
Muscle deficiency and neonatal death in mice with a targeted mutation in the myogenin gene.
Nature
364
,
501
506
Herkowitz
I.
(
1995
).
MAP kinase pathway in yeast: for mating and more.
Cell
80
,
187
197
Hill
C. S.
,
Treisman
R.
(
1995
).
Transcriptional regulation by extracellular signals: mechanisms and specificity.
Cell
80
,
199
211
Kanda
H.
,
Lee
G. H.
,
Nomura
K.
,
Ohtake
K.
,
Kitagawa
T.
(
1993
).
Malignant transformation of the mouse liver epithel cell line by transfection of an activated c-H-ras gene with a point mutation at codon 12.
Carcinogenesis
14
,
1061
1063
Kato
Y.
,
Kravchenko
V. V.
,
Tapping
R. I.
,
Han
J.
,
Ulevitch
R. J.
,
Lee
J. D.
(
1997
).
BMK1/ERK5 regulates serum-induced early geneexpression through transcription factor MEF2C.
EMBO J
16
,
7054
7066
Kerkhoff
E.
,
Rapp
U. R.
(
1997
).
Induction of cell proliferation in quiescent NIH 3T3 cells by oncogenic c-Raf-1.
Mol. Cell. Biol
17
,
2576
2586
Kong
Y.
,
Johnson
S. E.
,
Taparowsky
E. J.
,
Konieczny
S. F.
(
1995
).
Ras p21Val inhibits myogenesis without altering the DNA binding or transcriptional activities of the myogenic basic helix-loop-helix factors.
Mol. Cell Biol
15
,
5205
5213
Konieczny
S. F.
,
Drobes
B. L.
,
Menke
S. L.
,
Taparowsky
E. J.
(
1989
).
Inhibition of myogenic differentiation by the H-ras oncogene is associated with the down regulation of the MyoD1 gene.
Oncogene
4
,
473
481
Lassar
A. B.
,
Thayer
M. J.
,
Overell
R. W.
,
Weintraub
H.
(
1989
).
Transformation by activated ras or fos prevents myogenesis by inhibiting expression of MyoD1.
Cell
58
,
659
667
Lassar
A. B.
,
Munsterberg
A. E.
(
1996
).
The role of positive and negative signals in somite patterning.
Curr. Opin. Neurobiol
1
,
57
63
Li
L.
,
Zhou
J.
,
James
G.
,
Heller-Harrison
R.
,
Czech
M. P.
,
Olson
E. N.
(
1992
).
FGF inactivates myogenic helix-loop-helix proteins through phosphorylation of a conserved protein kinase C site in their DNA-binding domains.
Cell
71
,
1181
1194
Marshall
C. J.
(
1995
).
Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation.
Cell
80
,
179
185
Molkentin
J. D.
,
Black
B. L.
,
Martin
J. F.
,
Olson
E. N.
(
1995
).
Cooperative activation of muscle gene expression by MEF2 and myogenic bHLH proteins.
Cell
83
,
1125
1136
Molkentin
J. D.
,
Olson
E. N.
(
1996
).
Defining the regulatory networks for muscle development.
Curr. Opin. Genet. Dev
4
,
445
453
Olson
E. N.
,
Perry
M.
,
Schulz
R. A.
(
1995
).
Regulation of muscle differentiation by the MEF2 family of MADS box transcription factors.
Dev. Biol
172
,
2
14
Ornatsky
O. I.
,
Andreucci
J. J.
,
McDermott
J. C.
(
1997
).
A MEF2 dominant negative inhibits myogenesis.
J. Biol. Chem
272
,
33271
33278
Ornatsky
O. I.
,
McDermott
J. C.
(
1996
).
MEF2 protein expression, DNA binding specificity and complex composition, and transcriptional activity in muscle and non-muscle cells.
J. Biol. Chem
271
,
24927
24933
Ornatsky
O. I.
,
Cox
D. M.
,
Tangirala
P.
,
Andreucci
J. J.
,
Quinn
Z. A.
,
Wrana
J. L.
,
Prywes
R.
,
Yu
Y.-T.
,
McDermott
J. C.
(
1999
).
Post-translational control of the MEF2A transcriptional regulatory protein.
Nucl. Acids Res
27
,
2646
2654
Puri
P. L.
,
Wu
Z.
,
Zhang
P.
,
Wood
L. D.
,
Bhakta
K. S.
,
Han
J.
,
Feramisco
J. R.
,
Karin
M.
,
Wang
J. Y. J.
(
2000
).
Induction of terminal differentiation by constitutive activation of p38 MAP kinase in human rhabdomyosarcoma cells.
Genes Dev
14
,
574
584
Ramocki
M. B.
,
Johnson
S. E.
,
White
M. A. A. C. L.
,
Konieczny
S. F.
,
Taparowsky
E. J.
(
1997
).
Signaling through mitogen-activated protein kinases and Rac/Rho does not duplicate the effects of activated Ras on skeletal myogenesis.
Mol. Cell. Biol
17
,
3547
3555
Ranganayakulu
G.
,
Zhao
B.
,
Dokidis
A.
,
Molkentin
J.
,
Olson
E.
,
Schulz
R. A.
(
1995
).
A series of mutations in the D-MEF2 transcription factor reveal multiple functions in the larval and adult myogenesis in Drosophila.
Dev. Biol
171
,
169
181
Ridgeway
A. G.
,
Wilton
S.
,
Skerjanc
I. S.
(
2000
).
Myocyte enhancer factor 2C and myogenin up-regulate each other's expression and induce the development of skeletal muscle in PC19 cells.
J. Biol. Chem
275
,
41
46
Rosenthal
N.
,
Berglund
E. B.
,
Wentworth
B. M.
,
Donoghue
M.
,
Winter
B.
,
Bober
E.
,
Braun
T.
,
Arnold
H. H.
(
1990
).
A highly conserved enhancer downstream of the human MLC1/3 locus is a target for multiple myogenic determination factors.
Nucl. Acids Res
18
,
6239
6246
Sternberg
E. A.
,
Spizz
G.
,
Perry
M. E.
,
Olson
E. N.
(
1989
).
A ras-dependent pathway abolishes activity of a muscle-specific enhancer upstream from the muscle creatine kinase gene.
Mol. Cell Biol
9
,
594
601
Sun
H.
,
Tonks
N. K.
,
Bar-Sagi
D.
(
1994
).
Inhibition of Ras-induced DNA synthesis by expression of the phosphatase MKP-1.
Science
266
,
285
288
Takano
H.
,
Komuro
I.
,
Oka
T.
,
Shiojima
I.
,
Hiroi
Y.
,
Mizuno
T.
,
Yazaki
Y.
(
1998
).
The Rho family G proteins play a critical role in muscle differentiation.
Mol. Cell. Biol
18
,
1580
1589
Vaidya
T. B.
,
Weyman
C. M.
,
Teegarden
D.
,
Ashendel
C. L.
,
Taparowsky
E. J.
(
1991
).
Inhibition of myogenesis by the H-ras oncogene: implication of a role for protein kinase C.
J. Cell Biol
4
,
809
820
White
M. A.
,
Nicolette
C.
,
Minden
A.
,
Polverino
A.
,
Van Aelst
A.
,
Karin
M.
,
Wigler
M. H.
(
1995
).
Multiple Ras functions can contribute to mammalian cell transformation.
Cell
80
,
533
541
Winter
B.
,
Braun
T.
,
Arnold
H.-H.
) (
1992
).
Co-operativity of functional domains in the muscle-specific transcription factor Myf-5.
EMBO J
11
,
1843
1855
Winter
B.
,
Kautzner
I.
,
Issinger
O.-G.
,
Arnold
H.-H.
) (
1997
).
Two putative protein kinase CK2 phosphorylation sites are important for Myf-5 activity.
Biol. Chem
378
,
1445
1456
Yee
S. P.
,
Rigby
P. W.
(
1993
).
The regulation of myogenin gene expression during the embryonic development of the mouse.
Genes Dev
7
,
1277
1289
Zetser
A.
,
Gredinger
E.
,
Bengal
E.
(
1999
).
p38 mitogen-activated protein kinase pathway promotes skeletal muscle development.
J. Biol. Chem
274
,
5193
5200
Zhao
M.
,
New
L.
,
Kravchenko
V. V.
,
Kato
Y.
,
Gram
H.
,
DiPadova
F.
,
Olson
E. N.
,
Ulevitch
R. J.
,
Han
J.
(
1999
).
Regulation of the MEF2 family of transcription factors by p38.
Mol. Cell. Biol
19
,
21
30
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