Committed skeletal muscle myoblasts undergo terminal differentiation when shifted from a high-
mitogen medium to a low-
mitogen medium. However, expression of the
myogenic regulatory factor MyoD seems to be similar in proliferating and differentiating cells, suggesting that its function is attenuated in proliferating myoblasts. To further understand the potential mechanisms that may attenuate MyoD function, we have examined the effect of posttranslational modification. By analogy with
myogenin, we have examined the role of phosphorylation in regulating the function of MyoD. MyoD contains two putative
protein kinase C (PKC) phosphorylation sites (Thr115 and Ser200). The former site is analogous to Thr85 within the highly conserved basic domain of
myogenin that has been demonstrated to negatively regulate the myogenic differentiation functions of
myogenin. To test whether hyperphosphorylation of the same PKC site in MyoD would attenuate its function, we generated a mutant MyoD with a single amino acid substitution (Thr115-Ala) that disrupts the PKC phosphorylation site (Thr115) within the conserved basic domain. Wild-type and mutant MyoD were introduced into cells using an E1, E3-deleted adenoviral vector. In mouse C3H10T1/2 fibroblasts, both wild-type and mutant MyoD induced terminal myogenic differentiation when
growth factors were withdrawn from the cell culture. Consistent with these results, nuclear extracts from infected cells, but not those from uninfected cells, demonstrated complex formation with an
oligonucleotide containing an E-box consensus sequence. Growth arrest was associated with the up-regulation of p21cip1, cell fusion to multinucleated myotubes, and the expression of a muscle
differentiation marker (
myosin heavy chain). On the other hand, when infected cells were maintained under high mitogenic conditions (in the presence of 10%
fetal bovine serum), the expression of wild-type or mutant MyoD slowed cell growth and induced p21cip1. Only mutant MyoD caused cell fusion,
myosin heavy chain expression, and altered mobility of the E-box
oligonucleotide in gel shift assays. Furthermore, after
infection, MyoD was phosphorylated, and
phosphothreonine was detected in wild-type MyoD immunoprecipitated only from C3H10T1/2 cells grown under high mitogenic conditions. These results suggest that Thr115 may play an important role in the regulation of MyoD function under conditions of high mitogenesis. MyoD was also phosphorylated in malignant
rhabdomyosarcoma (RMS) cells in which MyoD function was attenuated.
Phosphothreonine was also detected in MyoD immunoprecipitates. Rh30 alveolar RMS cells were infected with an adenovirus expressing either wild-type or mutant MyoD. In contrast to the results in fibroblasts, when overexpressed in malignant Rh30 RMS cells, mutant MyoD arrested cell growth without inducing p21cip1 and caused cell fusion. However, no muscle
differentiation markers were detected, indicating that an overexpression of mutant MyoD lacking Thr115 caused Rh30 cells to become quiescent and recapitulate at least some aspects of myogenesis (cell fusion).