MOLECULAR MECHANISM OF THE SYNERGISTIC PHOSPHORYLATION OF PHOSPHATASEINHIBITOR-2 - CLONING, EXPRESSION, AND SITE-DIRECTED MUTAGENESIS OF INHIBITOR-2

Inhibitor-2 (I-2) is the regulatory subunit of the ATP-Mg-dependent ph osphatase, a cytosolic form of type 1 protein phosphatase. Phosphoryla tion of 1-2 at Thr-72 by the protein kinase glycogen synthase kinase-3 (GSK-3) leads to activation of the enzyme. Casein kinase II action wa s shown to synergistically enhance phosphorylation and activation by G SK-3 (DePaoli-Roach, A. A. (1984) J. Biol. Chem. 259, 12144-12152). Ra bbit skeletal muscle and liver I-2 cDNA clones have been isolated. Rab bit skeletal muscle cDNAs could be placed in two subtypes, differing i n the length of the 3'-untranslated region. The coding sequence of 612 nucleotides was identical in the two skeletal muscle and the liver cD NAs and predicted a protein of 204 amino acids, consistent with analys is of the purified protein. Northern hybridization analysis indicated that the two mRNAs of 1.7 and 2.7 kilobase pairs were present in all r abbit tissues examined, except in liver, where only the larger transcr ipt was detected, and in testis, where additional transcripts were pre sent. Expression in Escherichia coli of wild-type and phosphorylation site mutants resulted in the production of I-2 polypeptides with appar ent M(r) values of approximately 31,000 on sodium dodecyl sulfate-poly acrylamide gel electrophoresis. The inhibitory activity of the recombi nant proteins was similar to that of native rabbit skeletal muscle I-2 and was unaffected by the substitution of alanine for the GSK-3 site (Thr-72) and for the casein kinase II sites (Ser-86 and Ser-120/121) o r by substitution of glutamic acid and aspartic acid for Thr-72 and Se r-86. Recombinant wild-type I-2 and the Ala-120/121 mutant were phosph orylated synergistically by GSK-3 and casein kinase II. The Thr-72 and Ser-86 mutants, however, did not undergo this synergistic phosphoryla tion. Our studies indicate that Thr-72 is the only GSK-3 site and that Ser-86 is the casein kinase II site required for the potentiation of GSK-3 action. Furthermore, acidic residues cannot substitute for the p hosphate group either in enhancing GSK-3 phosphorylation or in activat ing the phosphatase.