Possible role of phosphorylation in the function of chicken MyoD1.
Chicken MyoD1 (CMD1), an equivalent to the mouse MyoD1, expressed in chicken skeletal muscle (Lin, Z.-Y., Dechesne, C. A., Eldridge, J., and Paterson, B. M. (1989) Genes & Dev. 3, 986-996), was produced in Spodoptera frugiperda (Sf9) cells by Baculovirus expression vector and purified to almost homogeneity. This CMD1 was directly demonstrated to be a phosphoprotein by a 32P-labeling experiment. Phosphoamino acid analysis revealed that only serine residue was phosphorylated. Phosphoamino acid of CMD1 from chick primary culture of 11-day embryonic breast muscle was also serine. Electrophoretic mobility on SDS-polyacrylamide gel electrophoresis of the phosphorylated CMD1 produced in Sf9 cells and that obtained from primary culture of muscle were indistinguishable. Gel retardation and methylation interference assays showed that purified CMD1 bound specifically to the mouse muscle creatine kinase enhancer in combination with the in vitro translated E12. CMD1 alone had almost no affinity to the target DNA. When purified CMD1 was treated with calf intestinal phosphatase, its affinity to the target DNA in combination with E12 was reduced by approximately 5-fold. The affinity recovered at least partially by possible rephosphorylation of CMD1 by kinase(s) contained in wheat germ extract. These results suggested that phosphorylation of CMD1 could be involved in the regulation of muscle differentiation.
Nakamura
S
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Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D002478:Cells, Cultured; D002642:Chick Embryo; D002645:Chickens; D003402:Creatine Kinase / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D005822:Genetic Vectors; D008715:Methionine / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009036:Moths; D009124:Muscle Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009132:Muscles / Q000378:metabolism*; D017570:MyoD Protein; D009838:Oligodeoxyribonucleotides; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011485:Protein Binding; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D013462:Sulfur Radioisotopes; D014158:Transcription, Genetic; D014162:Transfection
D004268:DNA-Binding Proteins; D009124:Muscle Proteins; D017570:MyoD Protein; D009838:Oligodeoxyribonucleotides; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D012333:RNA, Messenger; D013462:Sulfur Radioisotopes; D008715:Methionine; D003402:Creatine Kinase