Activation of protein phosphatase-1 isoforms and glycogen synthase kinase-3 beta in muscle from mdx mice

Three Protein Phosphatase-1 (PP1) isoforms (PP1 alpha, PP1 gamma-1 and PP1 delta) are found in skeletal muscle. These are bound to regulatory subunits, such as inhibitor 2 (I2) in the cytosol and G in the glycogen and microsomal fractions. In vitro, the PP1-12 complex is activated by Glycogen Synthase Kinase-3 (GSK-3 or FA). We investigated the activities and protein levels of the three PP1 isoforms and of GSK-3 in muscle of mdx dystrophic mice. PP1 was assayed as phosphorylase phosphatase, in the presence of 5 nM okadaic acid (which inhibits PP2A). Peptide antibodies were produced and used to investigate PP1 alpha, PP1 gamma-1 and PP1 delta. GSK-3 was assayed using a previously described peptide. This was synthesized in a pre-phosphorylated from, which avoids the additional use of Casein Kinase II. Higher PP1 activity was assayed in the cytosol from mdx rather than from control muscles. Immunoprecipitation indicated that only PP1 alpha and PP1 gamma-1 were more active. This was most likely due to enzyme activation, since the immunodetected proteins were unchanged. On the other hand, the immunodetected PP1 delta was lower in the glycogen and microsomal fractions from mdx muscle. GSK-3 was more active in the mdx extract Selective immunoprecipitation of GSK-3 alpha and GSK-3 beta indicated that both isoforms were activated. In the case of GSK-3 beta, the immunodetected protein was also increased. The changes described herein may be related to the pathological events occurring in the mdx muscle. These include increased protein degradation and turnover, and fibre regeneration. In fact, the decreased PP1 delta may be due to protein degradation and the increased GSK-3 may be the consequence of increased protein turnover or regeneration. The apparent correlation between the increased PP1 alpha and PP1 gamma-1 activities and the increased GSK-3 may agree with the hypothesis that GSK-3 activates the newly synthesized PP1.