Constitutive instability of muscle regulatory factor Myf5 is distinct fromits mitosis-specific disappearance, which requires a D-box-like motif overlapping the basic domain

Transcription factors Myf5 and MyoD play critical roles in controlling myob last identity and differentiation. In the myogenic cell line C2, we have fo und that Myf5 expression, unlike that of MyoD, is restricted to cycling cel ls and regulated by proteolysis at mitosis, In the present study, we have e xamined Myf5 proteolysis through stable transfection of myogenically conver tible U20S cells with Myf5 derivatives under the control of a tetracycline- sensitive promoter. A motif within the basic helix-loop-helix domain of Myf 5 (R93 to Q101) resembles the "destruction box" characteristic of substrate s of mitotic proteolysis and thought to be recognized by the anaphase-promo ting complex or cyclosome (APC), Mutation of this motif in Myf5 stabilizes the protein at mitosis but does not affect its constitutive turnover. Conve rsely, mutation of a serine residue (S158) stabilizes Myf5 in nonsynchroniz ed cultures but not at mitosis, Thus, at least two proteolytic pathways con trol Myf5 levels in cycling cells, The mitotic proteolysis of Myf5 is unlik e that which has been described for other destruction box-dependent substra tes: do,vn-regulation of Myf5 at mitosis appears to precede that of known t argets of the APC and is not affected by a dominant-negative version of the ubiquitin carrier protein UbcH10, implicated in the APC mediated pathway. Finally, we find that induction of Myf5 perturbs the passage of cells throu gh mitosis, suggesting that regulation of Myf5 levels at mitosis may influe nce cell cycle progression of Myf5-expressing muscle precursor cells.