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 t 1/2 approximately = 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.
Highlights
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
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 have shown that ubiquitin-proteasome-mediated protein degradation can regulate protein abundance by affecting protein stability during muscle differentiation
Summary
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, Myf, myogenin, and Mrf, termed the muscle regulatory factors [1,2,3,4]. Protein degradation studies following co-transfection of MyoD and Id1 to HeLa cells have shown that MyoD is able to modulate both the localization and the degradation of Id1 [15] It remains unclear, how the morphological and biological changes involved in myogenic differentiation affect the ubiquitinproteasome-mediated degradation of MyoD and Id1 in muscle cells and how their degradation and interaction contribute to their cellular abundance and regulate differentiation. The rate of their degradation appears to be unaffected by the differentiation state, whereas a reduction of MyoD and Id1 synthesis rate was observed during myogenic differentiation
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