Molecular mechanism of the synergistic phosphorylation of phosphatase inhibitor-2: Cloning, expression, and site-directed mutagenesis of inhibitor-2

In Kyung Park, Paris Roach, Jeffry Bondor, Stephen P. Fox, Anna De Paoli-Roach

Research output: Contribution to journalArticle

71 Citations (Scopus)

Abstract

Inhibitor-2 (I-2) is the regulatory subunit of the ATP-Mg-dependent phosphatase, a cytosolic form of type 1 protein phosphatase. Phosphorylation 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 was shown to synergistically enhance phosphorylation and activation by GSK-3 (DePaoli-Roach, A. A. (1984) J. Biol. Chem. 259, 12144-12152). Rabbit skeletal muscle and liver 1-2 cDNA clones have been isolated. Rabbit skeletal muscle cDNAs could be placed in two subtypes, differing in the length of the 3′-untranslated region. The coding sequence of 612 nucleotides was identical in the two skeletal muscle and the liver cDNAs and predicted a protein of 204 amino acids, consistent with analysis of the purified protein. Northern hybridization analysis indicated that the two mRNAs of 1.7 and 2.7 kilobase pairs were present in all rabbit tissues examined, except in liver, where only the larger transcript was detected, and in testis, where additional transcripts were present. Expression in Escherichia coli of wild-type and phosphorylation site mutants resulted in the production of I-2 polypeptides with apparent Mr. values of ∼31,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The inhibitory activity of the recombinant 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) or by substitution of glutamic acid and aspartic acid for Thr-72 and Ser-86. Recombinant wild-type 1-2 and the Ala-120/121 mutant were phosphorylated synergistically by GSK-3 and casein kinase II. The Thr-72 and Ser-86 mutants, however, did not undergo this synergistic phosphorylation. 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 phosphate group either in enhancing GSK-3 phosphorylation or in activating the phosphatase.

Original languageEnglish
Pages (from-to)944-954
Number of pages11
JournalJournal of Biological Chemistry
Volume269
Issue number2
StatePublished - Jan 14 1994

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Glycogen Synthase Kinase 3
Mutagenesis
Phosphorylation
Cloning
Site-Directed Mutagenesis
Organism Cloning
Casein Kinase II
Muscle
Skeletal Muscle
Liver
Rabbits
Complementary DNA
Phosphoric Monoester Hydrolases
Substitution reactions
Chemical activation
Protein Phosphatase 1
Enzyme Activation
3' Untranslated Regions
protein phosphatase inhibitor-2
Electrophoresis

ASJC Scopus subject areas

  • Biochemistry

Cite this

Molecular mechanism of the synergistic phosphorylation of phosphatase inhibitor-2 : Cloning, expression, and site-directed mutagenesis of inhibitor-2. / Park, In Kyung; Roach, Paris; Bondor, Jeffry; Fox, Stephen P.; De Paoli-Roach, Anna.

In: Journal of Biological Chemistry, Vol. 269, No. 2, 14.01.1994, p. 944-954.

Research output: Contribution to journalArticle

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AU - Park, In Kyung

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AU - Bondor, Jeffry

AU - Fox, Stephen P.

AU - De Paoli-Roach, Anna

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N2 - Inhibitor-2 (I-2) is the regulatory subunit of the ATP-Mg-dependent phosphatase, a cytosolic form of type 1 protein phosphatase. Phosphorylation 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 was shown to synergistically enhance phosphorylation and activation by GSK-3 (DePaoli-Roach, A. A. (1984) J. Biol. Chem. 259, 12144-12152). Rabbit skeletal muscle and liver 1-2 cDNA clones have been isolated. Rabbit skeletal muscle cDNAs could be placed in two subtypes, differing in the length of the 3′-untranslated region. The coding sequence of 612 nucleotides was identical in the two skeletal muscle and the liver cDNAs and predicted a protein of 204 amino acids, consistent with analysis of the purified protein. Northern hybridization analysis indicated that the two mRNAs of 1.7 and 2.7 kilobase pairs were present in all rabbit tissues examined, except in liver, where only the larger transcript was detected, and in testis, where additional transcripts were present. Expression in Escherichia coli of wild-type and phosphorylation site mutants resulted in the production of I-2 polypeptides with apparent Mr. values of ∼31,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The inhibitory activity of the recombinant 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) or by substitution of glutamic acid and aspartic acid for Thr-72 and Ser-86. Recombinant wild-type 1-2 and the Ala-120/121 mutant were phosphorylated synergistically by GSK-3 and casein kinase II. The Thr-72 and Ser-86 mutants, however, did not undergo this synergistic phosphorylation. 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 phosphate group either in enhancing GSK-3 phosphorylation or in activating the phosphatase.

AB - Inhibitor-2 (I-2) is the regulatory subunit of the ATP-Mg-dependent phosphatase, a cytosolic form of type 1 protein phosphatase. Phosphorylation 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 was shown to synergistically enhance phosphorylation and activation by GSK-3 (DePaoli-Roach, A. A. (1984) J. Biol. Chem. 259, 12144-12152). Rabbit skeletal muscle and liver 1-2 cDNA clones have been isolated. Rabbit skeletal muscle cDNAs could be placed in two subtypes, differing in the length of the 3′-untranslated region. The coding sequence of 612 nucleotides was identical in the two skeletal muscle and the liver cDNAs and predicted a protein of 204 amino acids, consistent with analysis of the purified protein. Northern hybridization analysis indicated that the two mRNAs of 1.7 and 2.7 kilobase pairs were present in all rabbit tissues examined, except in liver, where only the larger transcript was detected, and in testis, where additional transcripts were present. Expression in Escherichia coli of wild-type and phosphorylation site mutants resulted in the production of I-2 polypeptides with apparent Mr. values of ∼31,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The inhibitory activity of the recombinant 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) or by substitution of glutamic acid and aspartic acid for Thr-72 and Ser-86. Recombinant wild-type 1-2 and the Ala-120/121 mutant were phosphorylated synergistically by GSK-3 and casein kinase II. The Thr-72 and Ser-86 mutants, however, did not undergo this synergistic phosphorylation. 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 phosphate group either in enhancing GSK-3 phosphorylation or in activating the phosphatase.

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