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 regulator y subunits, such as inhibitor 2 (I2) in the cytosol and G in the glyco gen and microsomal fractions. In vitro, the PP1-IZ complex is activate d by Glycogen Synthase Kinase-3 (GSK-3 or F-A). We investigated the ac tivities and protein levels of the three PP1 isoforms and of GSK-3 in muscle of mdx dystrophic mice. PP1 was assayed as phosphorylase phosph atase, in the presence of 5 nM okadaic acid (which inhibits PP2A). Pep tide antibodies were produced and used to investigate PP1 alpha, PP1 g amma-1 and PP1 delta. GSK-3 was assayed using a previously described p eptide. This was synthesized in a pre-phosphorylated form, which avoid s the additional use of Casein Kinase II. Higher PP1 activity was assa yed in the cytosol from mdx rather than from control muscles. Immunopr ecipitation indicated that only PP1 alpha and PP1 gamma-1 were more ac tive. This was most likely due to enzyme activation, since the immunod etected 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 immunopre cipitation 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 increa sed protein degradation and turnover, and fibre regeneration, In fact, the decreased PP1 delta may be due to protein degradation and the inc reased GSK-3 may be the consequence of increased protein turnover or r egeneration. 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.