Abstract
We have investigated the mechanism by which the simian virus 40 large T antigen (SVLT) interferes with the differentiation of C2 myoblasts. SVLT mutants, defective either in the Rb binding site, near the N-terminal end, in a region that affects binding to p53, or in the nuclear transport signal, were also employed to determine whether the interference was especially dependent on these functional domains. It was found that wild-type (wt) SVLT strongly inhibited the terminal differentiation of mouse C2 myoblasts, but this arrest occurred only after the synthesis of myogenin, an initial step in biochemical differentiation. Neither the synthesis nor some basic activities of MyoD appeared to be affected by wt SVLT. In these transformants, mitogen depletion elicited an increase in the Rb level comparable to that in normal C2 cells; wt SVLT, however, promoted the phosphorylation of a large part of the induced Rb. Mutations affecting nuclear transport were far more critical for the ability to interfere with myogenic differentiation than were those affecting the transforming potential; cytoplasmic SVLT expression was fully compatible with the terminal differentiation of C2 cells, despite enabling them to grow in semisolid medium, thus showing that the myogenesis-inhibiting property can be dissociated from transforming competence. The remaining SVLT mutants presented different degrees of ability to inhibit differentiation (as shown by the expression of tissue-specific markers in transformants). The inhibiting mutants, including the Rb binding site mutant, were able to promote a higher state of Rb phosphorylation than that observed in either normal cells or cytoplasmic-SVLT transformants.