Domains of phosphatase inhibitor-2 involved in the control of the ATP-Mg-dependent protein phosphatase.
Inhibitor-2 (I-2) inhibits the free catalytic subunit of type 1 phosphatase (CS1) and controls the cyclic inactivation/activation of CS1 in the ATP-Mg-dependent protein phosphatase complex. We report here the effect of mutations on these two properties of I-2. Substitution of Thr-72 with Ala, Asp, or Glu generated complexes with CS1 that could not be activated. Mutation of Ser-86 did not affect activation by glycogen synthase kinase-3 (GSK-3) alone but impaired synergistic activation by casein kinase II and GSK-3. Mutations in the region between Thr-72 and Ser-86 did not alter the inhibitory potency of I-2 but prevented complete inactivation of CS1. A mutant without the 35 NH2-terminal residues exhibited an IC50 for CS1 200-fold higher than that of wild-type I-2. However, it formed an inactive phosphatase complex with CS1, which was activated by GSK-3. A mutant with the 59 COOH-terminal residues deleted retained full inhibitory activity and formed an inactive complex that could not be activated by GSK-3. We conclude that the NH2-terminal region of I-2 is involved in inhibition, that the sequence between Thr-72 and Ser-86 is necessary for the conversion of CS1 from an active to an inactive conformation, and that the COOH terminus is required for activation by GSK-3. Thus, different functional domains of I-2 may interact with distinct regions of CS1.
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis 46202-5122.
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Park
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DePaoli-Roach
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D047390:Casein Kinase II; D038362:Glycogen Synthase Kinase 3; D038341:Glycogen Synthase Kinases; D008969:Molecular Sequence Data; D009154:Mutation; D009838:Oligodeoxyribonucleotides; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011392:Proline / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D011817:Rabbits; D017384:Sequence Deletion; D000097847:Protein Phosphatase Inhibitory Proteins
D000255:Adenosine Triphosphate; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D047390:Casein Kinase II; D038362:Glycogen Synthase Kinase 3; D038341:Glycogen Synthase Kinases; D009838:Oligodeoxyribonucleotides; D010749:Phosphoprotein Phosphatases; D011392:Proline; D017346:Protein Serine-Threonine Kinases; D011506:Proteins; C024833:protein phosphatase inhibitor-2; D000097847:Protein Phosphatase Inhibitory Proteins