title: Fibroblast growth factor inhibits MRF4 activity independently of the phosphorylation status of a conserved threonine residue within the DNA-binding domain.
abstract: MRF4 is a member of the muscle-specific basic helix-loop-helix transcription factor family that also includes MyoD, myogenin, and Myf-5. Each of these proteins, when overexpressed in fibroblasts, converts the cells to differentiated muscle fibers that express several skeletal muscle genes, such as those for alpha-actin, muscle creatine kinase, and troponin I. Despite the fact that MRF4 functions as a positive transcriptional 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 specific posttranslational modifications, we examined whether MRF4 exists in vivo as a phosphoprotein and whether the phosphorylation status of the protein regulates its activity. Our results indicate that MRF4 is phosphorylated predominantly on serine residues, with weak phosphorylation 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 MRF4 activity and thus blocks terminal differentiation. PKC-directed phosphorylation 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 activity, 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 negative regulation of MRF4 imposed by bFGF does not involve a direct modification of the protein at the identified PKA and PKC sites but instead may involve the modification of specific coregulators that interact with this muscle regulatory factor.
authors.affiliation: Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907-1392.
authors.forename: S
authors.initials: S
authors.lastname: Hardy
authors.forename: Y
authors.initials: Y
authors.lastname: Kong
authors.forename: S F
authors.initials: SF
authors.lastname: Konieczny
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D003001:Cloning, Molecular; D017124:Conserved Sequence; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D005346:Fibroblast Growth Factors / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D018006:Myogenic Regulatory Factors / Q000037:antagonists & inhibitors* / Q000378:metabolism; D018008:Myogenin; D010449:Peptide Mapping; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013912:Threonine / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: C503799:MYOG protein, human; C503801:Myog protein, mouse; C503803:Myog protein, rat; D018006:Myogenic Regulatory Factors; D018008:Myogenin; D014157:Transcription Factors; C063403:myogenic factor 6; D013912:Threonine; D005346:Fibroblast Growth Factors; D004247:DNA; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C