Creatine kinase, Proteins, Myogenesis, Mice-Muscles, Phosphorylation, Genetic transcription, Gene expression, C2C12 cells, Murine, Protein expression, Transcript
Creatine kinase (CK) is an essential phosphoryl transferase enzyme in cellular energy homeostasis. It catalyzes the reversible transfer of the γ-phosphate group from ATP to creatine to yield ADP and creatine phosphate. Because ATP can be regenerated quickly in a single step, CK is critical for tissues that have sudden great needs of energy, such as skeletal muscle. This study investigated three isoforms of CK: CK-B, the nonmuscle cytoplasmic form; CK-M, the cytoplasmic muscle form; and CK-Mt2, the sarcomeric mitochondrial form. The protein expression and phosphorylation of these three CK isoforms were investigated during three stages of C2C12 cell myogenesis. The C2C12 murine myogenic cell line models the process of muscle development and repair after injury. C2C12 cells were harvested as: myoblasts, day 0; early myotubes, day 5; and late myotubes, day 9. Samples from all stages were analyzed by one-dimensional and two-dimensional SDS-PAGE and immunoblotting. Immunoblotting of onedimensional gels with mono-specific antibodies to each CK isoform enabled the quantitation of the relative expression of the three CK isoforms at each stage of myogenesis. CK-B was found to increase in expression from day 0 to day 5 then decrease from day 5 to day 9, whereas CK-M and CK-Mt2 increased in expression throughout myogenesis. Immunoblotting of two-dimensional gels looked for posttranslational phosphorylation of each CK isoform, indicated by a shift in pI of about 0.05 pH units toward the acidic end of the gel. Phosphorylation has been shown to regulate some CK enzymatic activities. Evidence indicating CK phosphorylation was obtained and future work will focus on further elucidating the phosphorylation states of each CK isoform during myogenesis.
Kratkiewicz, Arcadia June, "Analysis of creatine kinase expression and regulation throughout C2C12 myogenesis" (2016). Theses, Dissertations, and Projects. 1610.