Synergistic phosphorylation and activation of ATP-Mg-dependent phosphoprotein phosphatase by F(A)/GSK-3 and casein kinase II (PC0.7)

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Abstract

The ATP-Mg-dependent phosphoprotein phosphatase is believed to consist of a catalytic subunit and a regulatory component identified as phosphatase inhibitor-2. It was found in this study that isolated inhibitor-2 was phosphorylated in serine residues by casein kinase II to at least 3 mol of phosphate per mol of inhibitor-2 while another protein kinase, F(A)/GSK-3, introduced no more than 0.3 mol of phosphate per mol exclusively in threonine residues. Analysis of tryptic digests by high performance liquid chromatography indicated that casein kinase II action resulted in two major (peaks 1 and 2) and two minor phosphopeptides whereas F(A)/GSK-3 action generated only peak 2. Combined action of the two protein kinases introduced an additional 0.4-0.6 mol of phosphate per mol over that predicted for simple additive behavior. This synergistic phosphorylation was associated with increased phosphate in peak 2 and correlated with unchanged phosphoserine but increased phosphothreonine, to a level approaching 1 mol/mol. ATP-Mg-dependent protein phosphatase was either reconstituted from purified inhibitor-2 and low molecular weight type 1 phosphatase or isolated as an inactive complex (F(c)). Both phosphatase complexes were activated by F(A)/GSK-3 which caused a transient phosphorylation of the inhibitor-2 component. Casein kinase II alone phosphorylated the inhibitor-2 in both phosphatase complexes without affecting the enzyme activity. Exposure to the combination of F(A)/GSK-3 and casein kinase II resulted in a synergistic phosphorylation. Furthermore, the combined action of the two protein kinases caused a synergistic activation of the phosphatase at submaximal F(A)/GSK-3 levels. The results suggest that interactions between phosphorylation sites may play a role in the activation of the ATP-Mg-dependent phosphatase, in particular that phosphorylation by casein kinase II at serine can potentiate the phosphorylation of threonine by F(A)/GSK-3 with subsequent influence on phosphatase activation.

Original languageEnglish
Pages (from-to)12144-12152
Number of pages9
JournalJournal of Biological Chemistry
Volume259
Issue number19
StatePublished - 1984

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Glycogen Synthase Kinase 3
Casein Kinase II
Phosphorylation
Phosphoprotein Phosphatases
Phosphoric Monoester Hydrolases
Adenosine Triphosphate
Chemical activation
Phosphates
Protein Kinases
Threonine
Serine
Phosphothreonine
Phosphoserine
Phosphopeptides
High performance liquid chromatography
Enzyme activity
Catalytic Domain
Molecular Weight
Molecular weight
High Pressure Liquid Chromatography

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "Synergistic phosphorylation and activation of ATP-Mg-dependent phosphoprotein phosphatase by F(A)/GSK-3 and casein kinase II (PC0.7)",
abstract = "The ATP-Mg-dependent phosphoprotein phosphatase is believed to consist of a catalytic subunit and a regulatory component identified as phosphatase inhibitor-2. It was found in this study that isolated inhibitor-2 was phosphorylated in serine residues by casein kinase II to at least 3 mol of phosphate per mol of inhibitor-2 while another protein kinase, F(A)/GSK-3, introduced no more than 0.3 mol of phosphate per mol exclusively in threonine residues. Analysis of tryptic digests by high performance liquid chromatography indicated that casein kinase II action resulted in two major (peaks 1 and 2) and two minor phosphopeptides whereas F(A)/GSK-3 action generated only peak 2. Combined action of the two protein kinases introduced an additional 0.4-0.6 mol of phosphate per mol over that predicted for simple additive behavior. This synergistic phosphorylation was associated with increased phosphate in peak 2 and correlated with unchanged phosphoserine but increased phosphothreonine, to a level approaching 1 mol/mol. ATP-Mg-dependent protein phosphatase was either reconstituted from purified inhibitor-2 and low molecular weight type 1 phosphatase or isolated as an inactive complex (F(c)). Both phosphatase complexes were activated by F(A)/GSK-3 which caused a transient phosphorylation of the inhibitor-2 component. Casein kinase II alone phosphorylated the inhibitor-2 in both phosphatase complexes without affecting the enzyme activity. Exposure to the combination of F(A)/GSK-3 and casein kinase II resulted in a synergistic phosphorylation. Furthermore, the combined action of the two protein kinases caused a synergistic activation of the phosphatase at submaximal F(A)/GSK-3 levels. The results suggest that interactions between phosphorylation sites may play a role in the activation of the ATP-Mg-dependent phosphatase, in particular that phosphorylation by casein kinase II at serine can potentiate the phosphorylation of threonine by F(A)/GSK-3 with subsequent influence on phosphatase activation.",
author = "{De Paoli-Roach}, Anna",
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T1 - Synergistic phosphorylation and activation of ATP-Mg-dependent phosphoprotein phosphatase by F(A)/GSK-3 and casein kinase II (PC0.7)

AU - De Paoli-Roach, Anna

PY - 1984

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N2 - The ATP-Mg-dependent phosphoprotein phosphatase is believed to consist of a catalytic subunit and a regulatory component identified as phosphatase inhibitor-2. It was found in this study that isolated inhibitor-2 was phosphorylated in serine residues by casein kinase II to at least 3 mol of phosphate per mol of inhibitor-2 while another protein kinase, F(A)/GSK-3, introduced no more than 0.3 mol of phosphate per mol exclusively in threonine residues. Analysis of tryptic digests by high performance liquid chromatography indicated that casein kinase II action resulted in two major (peaks 1 and 2) and two minor phosphopeptides whereas F(A)/GSK-3 action generated only peak 2. Combined action of the two protein kinases introduced an additional 0.4-0.6 mol of phosphate per mol over that predicted for simple additive behavior. This synergistic phosphorylation was associated with increased phosphate in peak 2 and correlated with unchanged phosphoserine but increased phosphothreonine, to a level approaching 1 mol/mol. ATP-Mg-dependent protein phosphatase was either reconstituted from purified inhibitor-2 and low molecular weight type 1 phosphatase or isolated as an inactive complex (F(c)). Both phosphatase complexes were activated by F(A)/GSK-3 which caused a transient phosphorylation of the inhibitor-2 component. Casein kinase II alone phosphorylated the inhibitor-2 in both phosphatase complexes without affecting the enzyme activity. Exposure to the combination of F(A)/GSK-3 and casein kinase II resulted in a synergistic phosphorylation. Furthermore, the combined action of the two protein kinases caused a synergistic activation of the phosphatase at submaximal F(A)/GSK-3 levels. The results suggest that interactions between phosphorylation sites may play a role in the activation of the ATP-Mg-dependent phosphatase, in particular that phosphorylation by casein kinase II at serine can potentiate the phosphorylation of threonine by F(A)/GSK-3 with subsequent influence on phosphatase activation.

AB - The ATP-Mg-dependent phosphoprotein phosphatase is believed to consist of a catalytic subunit and a regulatory component identified as phosphatase inhibitor-2. It was found in this study that isolated inhibitor-2 was phosphorylated in serine residues by casein kinase II to at least 3 mol of phosphate per mol of inhibitor-2 while another protein kinase, F(A)/GSK-3, introduced no more than 0.3 mol of phosphate per mol exclusively in threonine residues. Analysis of tryptic digests by high performance liquid chromatography indicated that casein kinase II action resulted in two major (peaks 1 and 2) and two minor phosphopeptides whereas F(A)/GSK-3 action generated only peak 2. Combined action of the two protein kinases introduced an additional 0.4-0.6 mol of phosphate per mol over that predicted for simple additive behavior. This synergistic phosphorylation was associated with increased phosphate in peak 2 and correlated with unchanged phosphoserine but increased phosphothreonine, to a level approaching 1 mol/mol. ATP-Mg-dependent protein phosphatase was either reconstituted from purified inhibitor-2 and low molecular weight type 1 phosphatase or isolated as an inactive complex (F(c)). Both phosphatase complexes were activated by F(A)/GSK-3 which caused a transient phosphorylation of the inhibitor-2 component. Casein kinase II alone phosphorylated the inhibitor-2 in both phosphatase complexes without affecting the enzyme activity. Exposure to the combination of F(A)/GSK-3 and casein kinase II resulted in a synergistic phosphorylation. Furthermore, the combined action of the two protein kinases caused a synergistic activation of the phosphatase at submaximal F(A)/GSK-3 levels. The results suggest that interactions between phosphorylation sites may play a role in the activation of the ATP-Mg-dependent phosphatase, in particular that phosphorylation by casein kinase II at serine can potentiate the phosphorylation of threonine by F(A)/GSK-3 with subsequent influence on phosphatase activation.

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