Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.12.2 (MEK)
 18,161 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Rhabdomyosarcoma (RMS) has deregulated proliferation and is blocked in the differentiation program despite Myf-5, MyoD and myogenin expression. Here we show that ectopic expression of MRF4, which is not subject to an autoregulatory pathway but regulated by the other MRFs protein family, induces growth arrest and terminal differentiation in RD cells. Deletion mapping identified a positive-acting C-terminal domain in MRF4 as the mediator of transcriptional activity, revealing a conserved motif with helix III in MyoD previously found to initiate expression of endogenous skeletal muscle genes. By using chimeric MyoD/MRF4 proteins, we observe that the C-terminal motif of MRF4 rescues MyoD activity in RD cells. Moreover, comparative induction of muscle-specific genes following activation of MyoD, through the expression of a constitutively activated MKK6 either in the absence or presence of MRF4, shows that MyoD and MRF4 can differently regulate muscle genes expression. Together, these results demonstrate that the MRF4 C-terminus functions as specification as well as activation domain in tumor cells. They provide a basis to identify gene products necessary for b-HLH-mediated differentiation versus tumor progression.
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PMID:Muscle regulatory factor MRF4 activates differentiation in rhabdomyosarcoma RD cells through a positive-acting C-terminal protein domain. 1294 14

Activation of the Raf kinase signal transduction pathway in skeletal myoblasts causes a complete cessation of myofiber formation and muscle gene expression. The negative impacts of the signaling pathway are realized through downstream activation of mitogen and extracellular kinase (MEK) phosphorylation-dependent events and MEK-independent signal transmission. MEKK1, a kinase that can physically associate with Raf, may contribute to the MEK-independent signaling in response to elevated Raf activity. Myogenic cells overexpressing activated Raf and kinase-defective MEKK1 remain differentiation-defective, suggesting that MEKK1 does not contribute to the inhibitory actions of Raf. However, constitutive activation of MEKK1 dramatically inhibits biochemical and morphological measures of muscle formation. MEKK1 inhibits MyoD-directed transcriptional activity without altering the ability of the protein to form heterodimers with E2A proteins or bind DNA. By contrast, the transcriptional activity of E47, the preferred dimer partner of the myogenic regulatory factors, is severely compromised by MEKK1-initiated signaling. Inhibition of MEK1/2 and JNK1/2 function did not reinstate E47-directed transcription, indicating that these two downstream kinases likely are not involved in the MEKK1-controlled transcriptional block. Inhibition of p38 signaling overcame the negative effects exerted by MEKK1 on the amino terminus of E47. Closer examination indicates that E47 is phosphorylated in vitro by p38, and deletion analysis predicts that the critical amino acid(s) phosphorylated by p38 lie outside of the minimal transcriptional activation domains. Thus, modification of E47 by p38 likely disrupts higher order protein complex formation that is necessary for muscle gene transcription.
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PMID:MEKK1 signaling through p38 leads to transcriptional inactivation of E47 and repression of skeletal myogenesis. 1515 7

Lysosomal sialidase, encoded by neu1, is required for the removal of terminal sialic acid residues from a variety of sialoglycoconjugates. In humans, deficiency of this enzyme results in the inborn error of metabolism sialidosis, characterized by the accumulation of sialoglycoconjugates within the nervous system and in peripheral organs. A subset of sialidosis patients present with symptoms of profound muscle dysfunction, including progressive muscular atrophy. We have previously shown that the 5' regulatory region of murine neu1 is typical of skeletal muscle-specific genes due to the presence of several E-boxes and its responsiveness to stimulation by muscle regulatory factors (MRFs) such as MyoD. Here, we report that sialidase activity is increased 6-fold during the first 24 h of differentiation of C2C12 myoblasts followed by an attenuation to pre-differentiation levels by 48 h. We demonstrate that the lysosomal sialidase promoter is highly upregulated by MyoD through a mechanism that is dependent on the MyoD chromatin remodeling domain. We also show that the sialidase promoter is repressed by activated MEK. Inappropriate overexpression of sialidase 48 h after the onset of differentiation results in downregulation of myogenin as well as myosin heavy chain expression and in a halt of the differentiation cascade. This study indicates that lysosomal sialidase is a potent regulator of the early stages of myogenesis.
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PMID:Overexpression of MyoD-inducible lysosomal sialidase (neu1) inhibits myogenesis in C2C12 cells. 1621 42

Raf kinase is the upstream activator of MEK1/2 leading to phosphorylation and activation of ERK1/2. Sustained activation of Raf represses skeletal muscle-specific reporter gene transcription and formation of multinucleated myofibers. Inhibition of myogenesis by activated Raf involves downstream ERK1/2 as well as undefined mediators. To identify Raf-interacting proteins that may influence repression of muscle formation, a yeast two-hybrid screen was performed using a MEK1-binding defective Raf (RafBXB-T481A) as bait. Twenty cDNAs coding for Raf-interacting proteins were identified including Ran binding protein 9 (RanBP9), a protein previously reported to interact with receptor tyrosine kinases. Forced expression of RanBP9 in myogenic cells did not alter myogenesis. Co-expression of RanBP9 with constitutively active RafBXB, but not RafBXB-T481A, synergistically inhibited MyoD-directed muscle reporter gene transcription. Knockdown of RanBP9 expression did not restore the differentiation program to Raf-expressing myoblasts. Thus, RanBP9 physically associates with Raf but does not substantially contribute to the inhibitory actions of the kinase.
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PMID:Ran binding protein 9 interacts with Raf kinase but does not contribute to downstream ERK1/2 activation in skeletal myoblasts. 1636 41

During muscle regeneration, the mechanism integrating environmental cues at the chromatin of muscle progenitors is unknown. We show that inflammation-activated MKK6-p38 and insulin growth factor 1 (IGF1)-induced PI3K/AKT pathways converge on the chromatin of muscle genes to target distinct components of the muscle transcriptosome. p38 alpha/beta kinases recruit the SWI/SNF chromatin-remodeling complex; AKT1 and 2 promote the association of MyoD with p300 and PCAF acetyltransferases, via direct phosphorylation of p300. Pharmacological or genetic interference with either pathway led to partial assembly of discrete chromatin-bound complexes, which reflected two reversible and distinct cellular phenotypes. Remarkably, PI3K/AKT blockade was permissive for chromatin recruitment of MEF2-SWI/SNF complex, whose remodeling activity was compromised in the absence of MyoD and acetyltransferases. The functional interdependence between p38 and IGF1/PI3K/AKT pathways was further established by the evidence that blockade of AKT chromatin targets was sufficient to prevent the activation of the myogenic program triggered by deliberate activation of p38 signaling.
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PMID:Functional interdependence at the chromatin level between the MKK6/p38 and IGF1/PI3K/AKT pathways during muscle differentiation. 1796 60

The c-Jun N-terminal kinase (JNK) pathway was reported to be involved in myostatin signaling and MKK4 was suggested as the only upstream kinase for myostatin-induced JNK activation, implying that MKK4 is a suitable target of RNA interference (RNAi) for blocking myostatin activity. The aim of this study was to evaluate the effect of small interfering RNA (siRNA) targeted against MKK4 on myostatin-induced downregulation of differentiation marker gene expression. Real-time quantitative PCR revealed that the level of MKK4 expression was efficiently reduced by MKK4-specific siRNA. Western blot assays showed that knockdown of MKK4 attenuated the myostatin-induced downregulation of MyoD and myogenin expression.
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PMID:Effect of siRNA targeted against MKK4 on myostatin-induced downregulation of differentiation marker gene expression. 1804 64

Mutation R453W in A-type lamins, that are major nuclear envelope proteins, generates Emery-Dreifuss muscular dystrophy. We previously showed that mouse myoblasts expressing R453W-lamin A incompletely exit the cell cycle and differentiate into myocytes with a low level of multinucleation. Here we attempted to improve differentiation by treating these cells with a mixture of PD98059, an extracellular-regulated kinase (ERK) kinase (also known as mitogen-activated kinase, MEK) inhibitor, and insulin-like growth factor-II, an activator of phosphoinositide 3-kinase. We show that mouse myoblasts expressing R453W-lamin A were sensitive to the drug treatment as shown by (i) an increase in multinucleation, (ii) downregulation of proliferation markers (cyclin D1, hyperphosphorylated Rb), (iii) upregulation of myogenin, and (iv) sustained activation of p21 and cyclin D3. However, nuclear matrix anchorage of p21 and cyclin D3 in a complex with hypophosphorylated Rb that is critical to trigger cell cycle arrest and myogenin induction was deficient and incompletely restored by drug treatment. As the turn-over of R453W-lamin A at the nuclear envelope was greatly enhanced, we propose that R453W-lamin A impairs the capacity of the nuclear lamina to serve as scaffold for substrates of the MEK-ERK pathway and for MyoD-induced proteins that play a role in the differentiation process.
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PMID:Differentiation of C2C12 myoblasts expressing lamin A mutated at a site responsible for Emery-Dreifuss muscular dystrophy is improved by inhibition of the MEK-ERK pathway and stimulation of the PI3-kinase pathway. 1829 30

Cell differentiation is usually accompanied by irreversible cell cycle exit, which is a critical step for skeletal muscle differentiation. We therefore hypothesise that PD98059 that blocks the MAP kinase kinase (MEK) pathway (proliferation pathway) when administrated to murine C2 skeletal myoblasts will arrest cell cycle and, consequently, enhances differentiation relative to untreated controls. In this study, we aimed to examine this hypothesis using phenotypic differentiation, biochemical assays, flow cytometry and real-time PCR in C2 cells cultured for 48 h in differentiation media only (untreated) or supplemented with either a single dose of 10 ng/ml IGF-I or 20 muM PD98059 for 48 h. Creatine kinase (CK) activity was increased by 7.5-fold (P<0.05) in the presence of PD98059, whereas untreated and IGF-I-treated cells induced 4.5- and 4-fold increase respectively when compared with baseline controls. Increased CK values in the presence of PD98059 were not only associated with myotube formation but also associated with cell cycle arrest in G1 phase (86+/-3.2%; P<0.05). Moreover, the expression of myogenic-specific transcriptional factor mRNAs (MyoD and myogenin) was significantly higher in PD-treated cells (4.7+/-0.15 and 314+/-10.2 ng/reaction respectively; P<0.05) than untreated (2.0+/-0.2 and 233+/-11 ng/reaction respectively) or IGF-treated cells (3.2+/-0.24 and 296+/-16.2 ng/reaction respectively). Unexpectedly, Id3 mRNA, the potent negative regulator of muscle differentiation, was also expressed at significantly higher levels in PD-treated cells (77+/-0.346 ng/reaction; P<0.05) than untreated (49+/-7.7 ng/reaction) or IGF-I-treated cells (47+/-0.7 ng/reaction). Furthermore, expression of the muscle differentiation-specific genes (IGF-binding protein-5, IGF-II receptor and IGF-II) was also increased significantly in PD-treated cells when compared with untreated cells. Phosflow analysis showed a significant increase in the levels of phosphorylation of p38 mitogen-activated protein kinase (49.0+/-6.7%, P<0.05) in PD-treated cells when compared with DM-treated cells (31.7+/-5.7%). These findings uncover a previously unconsidered positive effect of PD98059 on C2 myoblast differentiation and identify the pathway(s) underlying PD-induced C2 myoblast differentiation.
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PMID:PD98059 enhances C2 myoblast differentiation through p38 MAPK activation: a novel role for PD98059. 1846 80

The transcriptional regulator peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) controls mitochondrial biogenesis and energy homeostasis. Although physical exercise induces PGC-1alpha expression in muscle, the underlying mechanism of this effect has remained incompletely understood. We recently identified a novel muscle-enriched isoform of PGC-1alpha transcript (designated PGC-1alpha-b) that is derived from a previously unidentified first exon. We have now cloned and characterized the human PGC-1alpha-b promoter. The muscle-specific transcription factors MyoD and MRF4 transactivated this promoter through interaction with a proximal E-box motif. Furthermore, either forced expression of Ca(2+)- and calmodulin-dependent protein kinase IV (CaMKIV), calcineurin A, or the p38 mitogen-activated protein kinase (p38 MAPK) kinase MKK6 or the intracellular accumulation of cAMP activated the PGC-1alpha-b promoter in cultured myoblasts through recruitment of cAMP response element (CRE)-binding protein (CREB) to a putative CRE located downstream of the E-box. Our results thus reveal a potential molecular basis for isoform-specific regulation of PGC-1alpha expression in contracting muscle.
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PMID:Identification and characterization of an alternative promoter of the human PGC-1alpha gene. 1923 36

In the "canonical" view of transforming growth factor beta (TGF-beta) signaling, Smad7 plays an inhibitory role. While Smad7 represses Smad3 activation by TGF-beta, it does not reverse the inhibitory effect of TGF-beta on myogenesis, suggesting a different function in myogenic cells. We previously reported a promyogenic role of Smad7 mediated by an interaction with MyoD. Based on this association, we hypothesized a possible nuclear function of Smad7 independent of its role at the level of the receptor. We therefore engineered a chimera of Smad7 with a nuclear localization signal (NLS), which serves to prevent and therefore bypass binding to the TGF-beta receptor while concomitantly constitutively localizing Smad7 to the nucleus. This Smad7-NLS did not repress Smad3 activation by TGF-beta but did retain its ability to enhance myogenic gene activation and phenotypic myogenesis, indicating that the nuclear, receptor-independent function of Smad7 is sufficient to promote myogenesis. Furthermore, Smad7 physically interacts with MyoD and antagonizes the repressive effects of active MEK on MyoD. Reporter and myogenic conversion assays indicate a pivotal regulation of MyoD transcriptional properties by the balance between Smad7 and active MEK. Thus, Smad7 has a nuclear coactivator function that is independent of TGF-beta signaling and necessary to promote myogenic differentiation.
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PMID:Nuclear function of Smad7 promotes myogenesis. 1999 10


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