Cyclin E–Cdk2 Phosphorylation Promotes Late G1-Phase Degradation of MyoD in Muscle Cells (original) (raw)
Related papers
Molecular and cellular biology, 1999
We have examined the role of protein phosphorylation in the modulation of the key muscle-specific transcription factor MyoD. We show that MyoD is highly phosphorylated in growing myoblasts and undergoes substantial dephosphorylation during differentiation. MyoD can be efficiently phosphorylated in vitro by either purified cdk1-cyclin B or cdk1 and cdk2 immunoprecipitated from proliferative myoblasts. Comparative two-dimensional tryptic phosphopeptide mapping combined with site-directed mutagenesis revealed that cdk1 and cdk2 phosphorylate MyoD on serine 200 in proliferative myoblasts. In addition, when the seven proline-directed sites in MyoD were individually mutated, only substitution of serine 200 to a nonphosphorylatable alanine (MyoD-Ala200) abolished the slower-migrating hyperphosphorylated form of MyoD, seen either in vitro after phosphorylation by cdk1-cyclin B or in vivo following overexpression in 10T1/2 cells. The MyoD-Ala200 mutant displayed activity threefold higher tha...
Phosphorylation of nuclear MyoD is required for its rapid degradation
Molecular and cellular biology, 1998
MyoD is a basic helix-loop-helix transcription factor involved in the activation of genes encoding skeletal muscle-specific proteins. Independent of its ability to transactivate muscle-specific genes, MyoD can also act as a cell cycle inhibitor. MyoD activity is regulated by transcriptional and posttranscriptional mechanisms. While MyoD can be found phosphorylated, the functional significance of this posttranslation modification has not been established. MyoD contains several consensus cyclin-dependent kinase (CDK) phosphorylation sites. In these studies, we examined whether a link could be established between MyoD activity and phosphorylation at putative CDK sites. Site-directed mutagenesis of potential CDK phosphorylation sites in MyoD revealed that S200 is required for MyoD hyperphosphorylation as well as the normally short half-life of the MyoD protein. Additionally, we determined that turnover of the MyoD protein requires the proteasome and Cdc34 ubiquitin-conjugating enzyme ac...
MyoD induces growth arrest independent of differentiation in normal and transformed cells
Proceedings of The National Academy of Sciences, 1990
MyoD is a gene involved in the control of muscle differentiation. We show that MyoD causes growth arrest when expressed in cell lines derived from tumors or transformed by different oncogenes. MyoD-induced growth inhibition was demonstrated by reduction in the efficiency of colony formation and at the single-cell level. We further show that MyoD growth inhibition can occur in cells that are not induced to activate muscle differentiation markers. The inhibitory activity of MyoD was mapped to the same 68-amino acid segment necessary and sufficient for induction of muscle differentiation, the basic-helix-oop-helix motif. Mutants with alterations in the basic region of MyoD that fail to bind or do not activate a muscle-specific enhancer inhibited growth; mutants with deletions in the helix-oop-helix region failed to inhibit growth. Thus, inhibition of cell growth by MyoD seems to occur by means of a parallel pathway to the one that leads to myogenesis. We conclude that MyoD is a prototypic gene capable of functionally activating intracellular growth inhibitory pathways.
Regulation of MyoD function in the dividing myoblast
Febs Letters, 2001
Proliferating myoblasts express MyoD, yet no phenotypic markers are activated as long as mitogen levels are sufficient to keep the cells dividing. Depending upon mitogen levels, a decision is made in G1 that commits the myoblast to either continue to divide or to exit from the cell cycle and activate terminal differentiation. Ectopic expression of MyoD under the control of the RSV or CMV promoters causes 10T1/2 cells to rapidly exit the cell cycle and differentiate as single myocytes, even in growth medium, whereas expression of MyoD under the weaker SV40 promoter is compatible with proliferation. Coexpression of MyoD and cyclin D1, but not cyclins A, B, E or D3, blocks transactivation of a MyoD responsive reporter. Similarly, transfection of myoblasts with the cyclin-dependent kinase (cdk) inhibitors p16 and p21 supports some musclespecific gene expression even in growth medium. Taken altogether, these results suggest cell cycle progression negatively regulates myocyte differentiation, possibly through a mechanism involving the D1 responsive cdks. We review evidence coupling growth status, the cell cycle and myogenesis. We describe a novel mitogen-sensitive mechanism that involves the cyclin D1dependent direct interaction between the G1 cdks and MyoD in the dividing myoblast, which regulates MyoD function in a mitogen-sensitive manner. ß
Crosstalk between cell cycle regulators and the myogenic factor MyoD in skeletal myoblasts
Cellular and Molecular Life Sciences, 2001
During the early process of skeletal muscle differentiation, myogenic factors are not only involved in muscle-specific gene induction but also in regulating the transition from the proliferative stage, when MyoD and Myf5 are already expressed, to the orderly exit from the cell division cycle. This key step in skeletal muscle differentiation involves the down-regulation of cell cycle activators such as cyclins and cdks, and up-regulation of cell cycle inhibitors such as Rb, p21, p27, and p57. In particular, Rb and p21 have been shown to play an important role in the growth arrest of differentiating myoblasts. Their level and/or activity, while being negatively controlled by growth factors, appear to be positively linked with the myogenic factor MyoD, which plays a cooperative role in the induction of growth arrest. MyoD can block proliferation independently of its transcriptional activity. Therefore, the interplay between G1 cyclins and cdk inhibitors, on the one hand, and MyoD and its co-Among positive regulators are the cyclin-dependent kinases (cdks) and their cyclins [1, 2], whereas the negatively acting regulators comprise the cdk inhibitors (CKIs) and pocket protein family: the product of the retinoblastoma susceptibility gene (Rb protein) and the two related Rb family proteins p107 and p130 . The biological activity of cell cycle phase-specific cyclin/cdk complexes allows progression into successive phases of the cell cycle. Cdk1, the first characterized cdk, forms complexes with cyclins A and B, which are crucial
A KAP1 phosphorylation switch controls MyoD function during skeletal muscle differentiation
Genes & Development, 2015
The discovery that fibroblasts could be induced to undergo myogenic differentiation by the forced expression of the DNA-binding basic helix-loop-helix (bHLH) MyoD protein was the first experimental evidence of genetically programmed transdifferentiation (Davis et al. 1987). MyoD was subsequently demonstrated to act as a master regulator of skeletal myogenesis driving myoblasts into a gene expression cascade that leads to their differentiation into multinucleated myotubes (Tapscott 2005; Aziz et al. 2010). It also became apparent that MyoD orchestrates this program by cooperating with a number of other modulators of gene expression, be they transcription factors or chromatin modifiers. How these multiple interactions are regulated and how MyoD-dependent gene targets are defined are still incompletely understood. In muscle precursor cells, MyoD indeed binds as a heterodimer with ubiquitously expressed E proteins to thousands of genomic sites containing a so-called E-box sequence (CAGSTG) (Cao et al. 2010; Soleimani et al. 2012). However, only a fraction of the genes situated nearby are activated during myotube differentiation (Cao et al. 2010). Interestingly, this subset of MyoD-recruiting genes is also enriched in Mef2 (myocyte enhancer factor 2) (Cao et al. 2010), and, more generally, MyoD-and Mef2-binding elements are overrepresented in cis-regulatory modules (CRMs) active during muscle differentiation (Kwon et al. 2011). MyoD and Mef2 also cooperate during muscle regeneration, where MyoD levels first increase in satellite cells, contributing to the expansion of these progenitors (Zhang et al. 2010; Singh and Dilworth 2013) before initiating with Mef2 the myogenic gene expression program that leads to cell cycle exit and formation of functional multinucleated myotubes (Penn et al. 2004; Liu et al. 2012; Singh and Dilworth 2013). This is consistent with the recruitment by the two factors of a combination of coregulatory molecules necessary for activating the muscle gene expression pro
Regulation of MyoD Activity and Muscle Cell Differentiation by MDM2, pRb, and Sp1
Journal of Biological Chemistry, 2003
Muscle cell differentiation is controlled by a complex set of interactions between tissue restricted transcription factors, ubiquitously expressed transcription factors, and cell cycle regulatory proteins. We previously found that amplification of MDM2 in rhabdomyosarcoma cells interferes with MyoD activity and consequently inhibits overt muscle cell differentiation (1). Recently, we found that MDM2 interacts with Sp1 and inhibits Sp1-dependent transcription and that pRb can restore Sp1 activity by displacing MDM2 from Sp1 (2). In this report, we show that forced expression of Sp1 can restore MyoD activity and restore overt muscle cell differentiation in cells with amplified MDM2. Furthermore, we show that pRb can also restore MyoD activity and muscle cell differentiation in cells with amplified MDM2. Surprisingly, we found that the MyoD-interacting domain of pRb is dispensable for this activity. We show that the C-terminal, MDM2-interacting domain of pRb is both necessary and sufficient to restore muscle cell differentiation in cells with amplified MDM2. We also show that the C-terminal MDM2-interacting domain of pRb can promote premature differentiation of proliferating myoblast cells. Our data support a model in which the pRb-MDM2 interaction modulates Sp1 activity during normal muscle cell differentiation.
The EMBO Journal, 1999
Proliferating myoblasts express the muscle determination factor, MyoD, throughout the cell cycle in the absence of differentiation. Here we show that a mitogen-sensitive mechanism, involving the direct interaction between MyoD and cdk4, restricts myoblast differentiation to cells that have entered into the G 0 phase of the cell cycle under mitogen withdrawal. Interaction between MyoD and cdk4 disrupts MyoD DNA-binding, muscle-specific gene activation and myogenic conversion of 10T1/2 cells independently of cyclin D1 and the CAK activation of cdk4. Forced induction of cyclin D1 in myotubes results in the cytoplasmic to nuclear translocation of cdk4. The specific MyoD-cdk4 interaction in dividing myoblasts, coupled with the cyclin D1-dependent nuclear targeting of cdk4, suggests a mitogen-sensitive mechanism whereby cyclin D1 can regulate MyoD function and the onset of myogenesis by controlling the cellular location of cdk4 rather than the phosphorylation status of MyoD.
Expression of MyoD1 with terminal differentiation in determined but inducible muscle cells
The EMBO Journal
We have examined the expression of MyoDI, a potential determination factor of myogenic cells, in permissive and inducible C2 myoblasts. These two types of myoblasts exhibit distinct requirements to undergo terminal differentiation. Unlike permissive cells, inducible cells fail to differentiate in the presence of growth medium plus fetal calf serum and require insulin to undergo terminal differentiation. We show that while expression of MyoDI is constitutive in permissive cells, no trace of MyoDI transcripts is found in inducible cells at the myoblast stage. In these cells, however, expression of MyoDI accompanies differentiation. This indicates that MyoDl may not be required for the maintenance of the myoblast phenotype, and could act as an effector of terminal differentiation in already determined muscle cells. Our results provide new evidence that permissive and inducible cells represent two distinct stages of the progression of determined muscle cells toward terminal differentiation.
Journal of Biological Chemistry, 2001
Cell cycle arrest is critical for muscle differentiation, and the two processes are closely coordinated but temporally separable. SWI/SNF complexes are ATP-dependent chromatin-remodeling enzymes that have been shown to be required for muscle differentiation in cell culture and have also been reported to be required for Rb-mediated cell cycle arrest. We therefore looked more closely at how SWI/SNF enzymes affect the events that occur during MyoD-induced myogenesis, namely, cell cycle regulation and muscle-specific gene expression, in cells that inducibly express dominant negative versions of Brahma (BRM) and Brahma-related gene 1 (BRG1), the ATPase subunits of two distinct SWI/SNF complexes. Although dominant negative BRM and BRG1 inhibited expression of every muscle-specific regulator and structural gene assayed, there was no effect on MyoD-induced activation of cell cycle regulatory proteins, and thus, cells arrested normally. In particular, in the presence or absence of dominant negative BRM or BRG1, MyoD was able to activate expression of p21, cyclin D3, and Rb, all of which are critical for cell cycle withdrawal in the G 1 /G 0 phase of the cell cycle. These findings suggest that at least one basis for the distinct mechanisms that regulate cessation of cell proliferation and musclespecific gene expression during muscle differentiation is that SWI/SNF-mediated chromatin-remodeling enzymes are required only for the latter.