Degradation of Myogenic Transcription Factor MyoD by the Ubiquitin Pathway In Vivo and In Vitro: Regulation by Specific DNA Binding

Abstract

MyoD is a tissue-specific transcriptional activator that acts as a master switch for skeletal muscle differentiation. Its activity is induced during the transition from proliferating, nondifferentiated myoblasts to resting, well-differentiated myotubes. Like many other transcriptional regulators, it is a short-lived protein; however, the targeting proteolytic pathway and the underlying regulatory mechanisms involved in the process have remained obscure. It has recently been shown that many short-lived regulatory proteins are degraded by the ubiquitin system. Degradation of a protein by the ubiquitin system proceeds via two distinct and successive steps, conjugation of multiple molecules of ubiquitin to the target protein and degradation of the tagged substrate by the 26S proteasome. Here we show that MyoD is degraded by the ubiquitin system both in vivo and in vitro. In intact cells, the degradation is inhibited by lactacystin, a specific inhibitor of the 26S proteasome. Inhibition is accompanied by accumulation of high-molecular-mass MyoD-ubiquitin conjugates. In a cell-free system, the proteolytic process requires both ATP and ubiquitin and, like the in vivo process, is preceded by formation of ubiquitin conjugates of the transcription factor. Interestingly, the process is inhibited by the specific DNA sequence to which MyoD binds: conjugation and degradation of a MyoD mutant protein which lacks the DNA-binding domain are not inhibited. The inhibitory effect of the DNA requires the formation of a complex between the DNA and the MyoD protein. Id1, which inhibits the binding of MyoD complexes to DNA, abrogates the effect of DNA on stabilization of the protein.MyoD is a tissue-specific transcriptional activator that acts as a master switch for skeletal muscle development. Following binding to specific upstream DNA regulatory elements, it leads to activation of a wide array of muscle-specific genes and consequently to conversion of proliferating myoblasts to terminally differentiated mature myotubes (10,30,43). MyoD belongs to the family of muscle-specific basic helix-loop-helix (bHLH) proteins, which also includes Myf5, myogenin, and MRF4 (42,43). These myogenic regulators have 80% homology within a segment of about 70 amino acid residues that encompasses the basic and helix-loop-helix motifs. These motifs mediate DNA binding and dimerization, respectively (41). MyoD binds to DNA as a homodimer; however, a more stable complex is generated when MyoD heterodimerizes with other ubiquitously expressed bHLH proteins, such as E2A, E12, and E47 (28). The activity of MyoD is negatively regulated by members of the Id (inhibitors of differentiation) family of proteins. These proteins can heterodimerize with E12/E47 or MyoD, but since they lack the basic region, the complexes cannot bind to DNA and are therefore inactive (2, 24, 37).Like many other transcriptional factors, MyoD is an extremely short-lived protein, with a half-life of ϳ30 min (38). However, the proteolytic system(s) that targets the protein, as well as the ...

Ubiquitin-Proteasome-mediated Degradation, Intracellular Localization, and Protein Synthesis of MyoD and Id1 during Muscle Differentiation

“…Because MyoD engages in a network of transcriptional activities in C2C12 cells as well as in other cells of the muscle lineage, one potential explanation for the inaccessibility of the hypophosphorylated MyoD species to phosphorylation and subsequent ubiquitin-proteasome-mediated degradation is that it is actively engaged in the transcription process and is possibly associated with DNA or other binding partners. Consistent with this notion, it has been shown that the formation of MyoD-ubiquitin conjugates is inhibited by the specific DNA sequence to which MyoD binds; conjugation and degradation of a MyoD mutant protein that lacks the DNA-binding domain are not inhibited (19). Moreover, substitution of serine 200 in MyoD to a nonphosphorylatable alanine abolished the slower migrating hyperphosphorylated form of MyoD; this mutant also significantly enhances both the muscle gene-specific transcriptional activity of MyoD and the ability of MyoD to induce myogenic conversion of nonmuscle cells (32).…”

“…Studies in a cell culture model that has a phenotype similar to that observed in myoblast cultures derived from myotonic dystrophy 1 patient muscle suggest that C2C12 myogenic differentiation is disrupted by mutant myotonic dystrophy protein kinase 3Ј-untranslated region transcripts via posttranscriptional reduction of MyoD protein levels (27). Previous studies in vitro or in non-muscle cells also show that MyoD degradation is regulated by phosphorylation, DNA binding, and protein-protein interactions (19,28). Phosphorylation of MyoD is required for its rapid degradation.…”

“…MyoD and Id1 have both been shown to be degraded by the ubiquitin-proteasome system (13)(14)(15)(16)(17)(18)(19). This pathway involves the activation of ubiquitin by the ubiquitin-activating enzyme, E1, followed by transfer of ubiquitin to E2, a ubiquitin-conjugating enzyme.…”

“…Sequestering the ubiquitous E proteins allows Id1 to control the transcriptional activity of muscle-specific MyoD. In cultured myoblasts, Id1 over-expression via a "dominant-negative" effect inhibits the transactivation by MyoD, thereby inhibiting the synthesis of proteins participating in muscle differentiation and consequently the fusion of myoblasts to myotubes (12).MyoD and Id1 have both been shown to be degraded by the ubiquitin-proteasome system (13)(14)(15)(16)(17)(18)(19). This pathway involves the activation of ubiquitin by the ubiquitin-activating enzyme, E1, followed by transfer of ubiquitin to E2, a ubiquitin-conjugating enzyme.…”

Abduction and asylum in the lives of transcription factors

“…Additionally, some studies show that certain transcription factors with mutations in the DNA binding domain become resistant to degradation (18). On the other hand, some transcription factors are resistant to degradation when bound, such as p53 (19) and MyoD (20). Because situations exhibiting both binding vulnerability and binding protection exist, we will investigate both scenarios here in the context of an autoregulated gene surrounded by a variable number of nonfunctional transcription factor binding sites.…”