2 PUTATIVE PROTEIN-KINASE CK2 PHOSPHORYLATION SITES ARE IMPORTANT FORMYF-5 ACTIVITY

Myf-5, a member of a family of muscle-specific transcription factors, is important for myogenic cell determination and differentiation. Here , we report that Myf-5 protein constitutes a substrate for phosphoryla tion in vitro by protein kinase CK2. We identified two potential phosp horylation sites at serine(49) and serine(133), both of which seem to be necessary for Myf-5 activity. Mutants which can no longer be phosph orylated fail to transactivate E-box-dependent reporter genes and act as trans-dominant repressors of wildtype Myf-5. Normal activity can be restored by replacing the serine residues with glutamate suggesting t hat a negative charge at these sites is obligatory for Myf-5 activity. Although serine(133) is part of helix 2 which mediates dimerization, we find no evidence for impaired DNA-binding or heterodimerization of the Ser-Ala(133) mutant. Some serine(49) mutations exhibit reduced nuc lear localization and/or protein stability Our data suggest that CK2-m ediated phosphorylation of Myf-5 is required for Myf-5 activity.