FIBROBLAST GROWTH-FACTOR INHIBITS MRF4 ACTIVITY INDEPENDENTLY OF THE PHOSPHORYLATION STATUS OF A CONSERVED THREONINE RESIDUE WITHIN THE DNA-BINDING DOMAIN

MRF4 is a member of the muscle-specific basic helix-loop-helix transcr iption factor family that also includes MyoD, myogenin, and Myf-5. Eac h of these proteins, when overexpressed in fibroblasts, converts the c ells to differentiated muscle fibers that express several skeletal mus cle genes, such as those for alpha-actin, muscle creatine kinase, and troponin I. Despite the fact that MRF4 functions as a positive transcr iptional regulator, the MRF4 protein is subject to negative regulation by a variety of agents, most notably by exposure of cells to purified growth factors, such as basic fibroblast growth factor (bFGF). In an effort to establish whether bFGF inhibits MRF4 activity through specif ic posttranslational modifications, we examined whether MRF4 exists in vivo as a phosphoprotein and whether the phosphorylation status of th e protein regulates its activity. Our results indicate that MRF4 is ph osphorylated predominantly on serine residues, with weak phosphorylati on occurring on threonine residues. Both cyclic AMP-dependent protein kinase (PKA) and protein kinase C (PKC) phosphorylate MRF4 in vitro as well as in vivo, and the overexpression of each kinase inhibits MPF4 activity and thus blocks terminal differentiation. PKC-directed phosph orylation of a conserved threonine residue (T-99) situated within the DNA-binding domain inhibits MRF4 from binding in vitro to specific DNA targets. However, although T-99 itself is essential for myogenic acti vity, our studies demonstrate that the phosphorylation status of T-99 does not play a major role in regulating MRF4 activity in vivo, since PKA, PKC, and bFGF inhibit the activity of MRF4 proteins in which the identified PKA and PKC sites have been mutated. We suggest that the ne gative regulation of MRF4 imposed by bFGF does not involve a direct mo dification of the protein at the identified PKA and PKC sites but inst ead may involve the modification of specific coregulators that interac t with this muscle regulatory factor.