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

Abstract

Mammalian skeletal myogenesis results in the differentiation of myoblasts to mature syncytial myotubes, a process regulated by an intricate genetic network of at least three protein families: muscle regulatory factors, E proteins, and Id proteins. MyoD, a key muscle regulatory factor, and its negative regulator Id1 have both been shown to be degraded by the ubiquitin-proteasome system. Using C2C12 cells and confocal fluorescence microscopy, we showed that MyoD and Id1 co-localize within the nucleus in proliferating myoblasts. In mature myotubes, in contrast, they reside in distinctive subcellular compartments, with MyoD within the nucleus and Id1 exclusively in the cytoplasm. Cellular abundance of Id1 was markedly diminished from the very onset of muscle differentiation, whereas MyoD abundance was reduced to a much lesser extent and only at the later stages of differentiation. These reductions in MyoD and Id1 protein levels seem to result from a change in the rate of protein synthesis rather than the rate of degradation. In vivo protein stability studies revealed that the rates of ubiquitin-proteasome-mediated MyoD and Id1 degradation are independent of myogenic differentiation state. Id1 and MyoD were both rapidly degraded, each with a t1 ⁄2 Ӎ 1 h in myoblasts and in myotubes. Furthermore, relative protein synthesis rates for MyoD and Id1 were significantly diminished during myoblast to myotube differentiation. These results provide insight as to the interaction between MyoD and Id1 in the process of muscle differentiation and have implications for the involvement of the ubiquitin-proteasome-mediated protein degradation and protein synthesis in muscle differentiation and metabolism under abnormal and pathological conditions. Skeletal muscle differentiation is characterized by the terminal withdrawal of the myoblast from the cell cycle, activation of muscle-specific gene expression, and cell fusion into multinucleated myotubes. These events are coordinated by a family of four muscle-specific basic helix-loop-helix transcription factors, MyoD, Myf5, myogenin, and Mrf4, termed the muscle regulatory factors (1-4). Mice lacking myogenin appropriately specify the skeletal muscle lineage but fail to terminally differentiate. Mrf4 is required for the maintenance of the differentiated myotubes. Although the specification of the myogenic lineage requires myoD and myf5, as double knock-out of both genes yields mice with no skeletal muscle (5), MyoD is also required for healthy self-renewing proliferation of the adult skeletal muscle satellite cells (6 -8). Muscle regulatory factors form heterodimers with ubiquitous E proteins and activate myogenic differentiation through their subsequent binding to specific sequences, termed E boxes, in the promoter regulatory regions of muscle-restricted target genes (4). The transcriptional activities of muscle regulatory factors are negatively regulated by a family of inhibitors of DNA-binding (Id) proteins. The four Id proteins (Id1, Id2, Id3, and Id4) are helixloop-helix proteins tha...

E2A protein degradation by the ubiquitin-proteasome system is stage-dependent during muscle differentiation

“…The E2A proteins are also stabilized in G0 growth-arrested Hela cells compared to that seen in proliferating cells. These results taken together with our previous study of MyoD and Id1 (Sun et al, 2005) support an important role for the ubiquitinproteasome pathway in E2A protein regulation during muscle development.…”

“…We show here that the E2A proteins are localized in the nucleus in myoblasts (Figure 1a). This supports the notion that E2A proteins can heterodimerize with MyoD or Id1, which are also localized to the nucleus in myoblasts (Sun et al, 2005). In myotubes, however, the nuclear localization of the E2A proteins suggests that in the nucleus they form heterodimers primarily with MyoD, but not with Id1, as only MyoD is localized in the myotube in the nucleus, whereas Id1 is localized exclusively to the cytoplasm (Sun et al, 2005).…”

“…We have previously shown that under the experimental conditions in which E2A protein degradation is abolished, the relative rate of E2A protein accumulation correlates with the relative rate of protein synthesis in vivo (Sun et al, 2005). As shown in Figure 4a, E2A protein accumulated to a greater degree in myoblasts than in myotubes, thus suggesting that the E2A protein synthesis rate is faster in myoblasts than in myotubes.…”

“…For example, during myogenic differentiation of C2C12 cells, the degradation rate of extracellular signal-regulated kinase 3 by the ubiquitin system is decreased in a similar fashion to that of the E2A proteins (Coulombe et al, 2003); whereas the 6-phosphofructo-2-kinase undergoes enhanced protein degradation via the ubiquitin-proteasome pathway (Riera et al, 2003). In addition, our recent study of MyoD and Id1 in differentiating C2C12 cells revealed no alteration in the rates of degradation of either protein (Sun et al, 2005).…”

“…Previously we demonstrated that the abundance of MyoD and Id1 are highly regulated with different mechanisms governing protein synthesis and degradation during muscle differentiation (Sun et al, 2005). Further, in non-muscle cells E12 and E47 modulate cellular localization and proteasome-mediated degradation of MyoD and Id1 (Lingbeck et al, 2005).…”