Preferential MyoD homodimer formation demonstrated by a general method of dominant negative mutation employing fusion with a lysosomal protease.

Li, F Q; Coonrod, Archie; Horwitz, Marshall S.

doi:10.1083/jcb.135.4.1043

Cited by 17 publications

(22 citation statements)

References 35 publications

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“…In our model, the early MRF, MyoD, acts predominantly as a homodimer in muscle cells to regulate the transcription of specific sets of contractile genes. MyoD homodimers are the predominant multiplex in C2C12 myoblasts (28,47). Moreover, the presence of MyoD-MyoD transcriptional units is further supported by the inability of MEKK1⌬ to disrupt MyoD-directed transcription from a minimal muscle E-box reporter (Fig.…”

Section: Fig 9 Mekk1⌬ Inhibition Of E47 Does Not Alter Tad Functionmentioning

confidence: 88%

MEKK1 Signaling through p38 Leads to Transcriptional Inactivation of E47 and Repression of Skeletal Myogenesis

Page

Wang

Sordillo

et al. 2004

Journal of Biological Chemistry

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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 MEKindependent signaling in response to elevated Raf activity. Myogenic cells overexpressing activated Raf and kinasedefective 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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Section: Fig 9 Mekk1⌬ Inhibition Of E47 Does Not Alter Tad Functionmentioning

confidence: 88%

MEKK1 Signaling through p38 Leads to Transcriptional Inactivation of E47 and Repression of Skeletal Myogenesis

Page

Wang

Sordillo

et al. 2004

Journal of Biological Chemistry

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“…10 6 transfected cells were washed in phosphate-buffered saline (PBS), and the cell pellet was resuspended in 150 l of 250 mM Tris-Cl, pH 7.4. 30 l was aliquoted for ␤-galactosidase assay, performed as described previously (24). The concentration was adjusted to 100 mM Tris-Cl, pH 7.4, 1 M MgCl 2 , 0.1% Triton X-100 in a 300-l volume.…”

Section: Cdna Mutagenesis and Construction Of Cell Expressionmentioning

confidence: 99%

Characterization of Mutant Neutrophil Elastase in Severe Congenital Neutropenia

Horwitz

2001

Journal of Biological Chemistry

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Severe congenital neutropenia is a heritable human disorder characterized by neutropenia and acute myelogenous leukemia. We recently determined that the majority of cases result from de novo or autosomal dominantly inherited heterozygous mutations in ELA2, encoding neutrophil elastase. Neutrophil elastase is a chymotryptic serine protease localized in granules of neutrophils and monocytes and is the major target of inhibition of the serpin ␣ 1 -antitrypsin. The mutations causing severe congenital neutropenia consist of amino acid missense substitutions, in-frame deletion, splice donor mutation producing a deletion, splice acceptor mutation causing insertion of novel residues, and protein truncating mutations of the carboxyl terminus resulting from nonsense substitutions and deletions leading to frameshifts. We have expressed 14 mutant forms of neutrophil elastase in vitro and have characterized their biochemical properties. The mutations have variable effects on proteolytic activity, eliminating the possibility that the disease results from haploinsufficiency. There is no evidence that the mutant enzymes are cytotoxic. The mutant enzymes retain vulnerability to inhibition by ␣ 1 -antitrypsin, but demonstrate variable avidity for interaction with this serpin. Somewhat surprisingly, the mutant enzymes inhibit the wild type enzyme when both are coexpressed within the same cell, suggesting the potential to interfere with normal subcellular trafficking or post-translational processing.

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“…CaPO 4 -DNA precipitate remained on the cells for about 17 h before feeding with fresh GM. Cell extracts and ␤-galactosidase assay were performed as before (15). To establish stable transfectants, C2C12 cells were transfected with 20 g of plasmid DNA containing a neo gene; 17 h later, transfectants were selected by placing cells in GM containing G418 (500 g/ml; Gibco).…”

Section: Methodsmentioning

confidence: 99%

“…pCS2ϩ, pCS2ϩmyc6, pCS2ϩCB-myc6, pCS2ϩmyc6-MyoD, pEMSVscribe, and the expression vector for mouse MyoD cDNA (pEMSV-MyoD) have been described previously (15). pCS2ϩCB-myc6-(neo) and pCS2ϩmyc6-(neo) were made by ligating a BamHI-NheI Klenow filled-in fragment derived from plasmid pCD2 into the StuI sites of pCS2ϩCB-myc6 and pCS2ϩmyc6, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…The strategy (15) is based on the demonstration that the lysosomal localization signal in the protease preprocathepsin B (CB) acts in cis dominance to other subcellular localization signals. By fusing CB to a fragment of a gene encoding a subunit of a multimeric complex, the CB fusion protein can dominantly inhibit the function of associating proteins through diversion of the interacting complex from its usual subcellular localization to the hydrolytic environment of the lysosome.…”

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confidence: 99%

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Selection of a Dominant Negative Retinoblastoma Protein (RB) Inhibiting Satellite Myoblast Differentiation Implies an Indirect Interaction between MyoD and RB

Coonrod

Horwitz

2000

Molecular and Cellular Biology

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Satellite myoblasts serve as stem cells in postnatal skeletal muscle, but the genes responsible for choosing between growth versus differentiation are largely undefined. We have used a novel genetic approach to identify genes encoding proteins whose dominant negative inhibition is capable of interrupting the in vitro differentiation of C2C12 murine satellite myoblasts. The screen is based on fusion of a library of cDNA fragments with the lysosomal protease cathepsin B (CB), such that the fusion protein intracellularly diverts interacting factors to the lysosome. Among other gene fragments selected in this screen, including those of known and novel sequence, is the retinoblastoma protein (RB) pocket domain. This unique dominant negative form of RB allows us to genetically determine if MyoD and RB associate in vivo. The dominant negative CB-RB fusion produces a cellular phenotype indistinguishable from recessive loss of function RB mutations. The fact that the dominant negative RB inhibits myogenic differentiation in the presence of nonlimiting concentrations of either RB or MyoD suggests that these two proteins do not directly interact. We further show that the dominant negative RB inhibits E2F1 but cannot inhibit a forced E2F1-RB dimer. Therefore, E2F1 is a potential mediator of the dominant negative inhibition of MyoD by CB-RB during satellite cell differentiation. We propose this approach to be generally suited to the investigation of gene function, even when little is known about the pathway being studied.Satellite cells are a lineage derived from somites that reside under the basement membrane of the myofiber and are responsible for replenishing skeletal muscle during growth in the postnatal period and in response to exercise and injury in the adult animal (24). As muscles hypertrophy, the satellite cells divide and fuse in order to increase the complement of myonuclei in myofibers. Transplant studies in chick embryos indicate that satellite cells are unable to take part in muscle embryogenesis, suggesting that they serve a specialized function as a presumptive skeletal muscle stem cell (3). Satellite cells are required to both proliferate and terminally differentiate, but invariably the proportion of satellite cells in a muscle remains constant, independent of both the age of the animal and the size of the muscle, and returns to this fixed value at the conclusion of muscle regeneration following injury (23).Understanding the molecular mechanisms responsible for the switch between proliferation and differentiation in satellite cells could be a key to understanding skeletal muscle regeneration in response to disease and trauma. However, relatively little is known about the mechanisms at work to ensure maintenance of the satellite cell population. Most of this knowledge comes from embryological studies of mesoderm differentiation in somites or the developing limb bud that have been extrapolated to tissue culture models of satellite myoblast differentiation. As with embryonic myogenesis, expression of MyoD or t...

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Preferential MyoD homodimer formation demonstrated by a general method of dominant negative mutation employing fusion with a lysosomal protease.

Cited by 17 publications

References 35 publications

MEKK1 Signaling through p38 Leads to Transcriptional Inactivation of E47 and Repression of Skeletal Myogenesis

MEKK1 Signaling through p38 Leads to Transcriptional Inactivation of E47 and Repression of Skeletal Myogenesis

Characterization of Mutant Neutrophil Elastase in Severe Congenital Neutropenia

Selection of a Dominant Negative Retinoblastoma Protein (RB) Inhibiting Satellite Myoblast Differentiation Implies an Indirect Interaction between MyoD and RB

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