Open AccessThe catalytic subunits of protein phosphatase‐1 and protein phosphatase 2A are distinct gene products
Author(s) -
TUNG H. Y. Lim,
RESINK Therese J.,
HEMMINGS Brian A.,
SHENOLIKAR Shirish,
COHEN Philip
Publication year - 1984
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1984.tb07962.x
Subject(s) - phosphatase , phosphorylase kinase , biochemistry , microbiology and biotechnology , protein subunit , gel electrophoresis , glycogen phosphorylase , biology , enzyme , polyacrylamide gel electrophoresis , chemistry , protein phosphatase 2 , gene
Three forms of protein phosphatase‐1 were isolated from rabbit skeletal muscle that had M r values of 37000, 34000 and 33000 determined by sodium dodecyl sulphate (SDS) gel electrophoresis. Each species dephosphorylated the β‐subunit of phosphorylase kinase very much faster than the α‐subunit, was inhibited by inhibitors 1 and 2 with equal potency, and was converted to a form dependent on glycogen synthase kinase‐3 and Mg‐ATP for activity by incubation with inhibitor‐2. Digestion with cyanogen bromide or Staphylococcus aureus proteinase followed by SDS gel electrophoresis showed a very similar pattern of cleavage products for all three forms. The M r ‐37000 and M r ‐34000 species were converted to the M r ‐33000 form by incubation with chymotrypsin. It is concluded that the M r ‐33000 and M r ‐34000 forms are derived from the M r ‐37000 component by limited proteolysis. Conversion of the M r ‐37000 to the M r ‐33000 form was accompanied by a two‐fold increase in activity, indicating that an M r ‐4000 fragment at one end of the polypeptide is an inhibitory domain that decreases enzyme activity. The catalytic subunit of protein phosphatase 2A from rabbit skeletal muscle had an M r of 36000 determined by SDS gel electrophoresis and its specific activity (3 kU/mg) was much lower than that of the M r ‐37000 (15–20 kU/mg) or M r ‐33/34000 (40–50 kU/mg) forms of protein phosphatase‐1. It dephosphorylated the α‐subunit of phosphorylase kinase 4–5‐fold faster than the β‐subunit, was unaffected by inhibitor‐1 or inhibitor‐2, and preincubation with the latter protein did not result in the production of a glycogen synthase kinase‐3 and Mg‐ATP‐dependent form of the enzyme. Digestion with chymotrypsin did not alter the electrophoretic mobility of protein phosphatase 2A under conditions that caused quantitative conversion of the M r ‐37000 form of protein phosphatase‐1 to the M r ‐33000 species. Digestion with cyanogen bromide or S. aureus proteinase, followed by SDS gel electrophoresis, showed a quite different pattern of cleavage products to those observerd with protein phosphatase 1. Antibody to protein phosphatase‐2A raised in sheep did not cross‐react with any of the forms of protein phosphatase‐1, as judged by immunoelectrophoretic and immunotitration experiments. It is concluded that protein phosphatase‐1 and protein phosphatase‐2A are distinct gene products.