title: Identification and characterization of a novel protein inhibitor of type 1 protein phosphatase.
abstract: We have isolated human cDNA for a novel type 1 protein phosphatase (PP1) inhibitory protein, named inhibitor-4 (I-4), from a cDNA library of germ cell tumors. I-4, composed of 202 amino acids, is 44% identical to a PP1 inhibitor, inhibitor-2 (I-2). I-4 conserves functionally important structure of I-2 and exhibited similar biochemical properties. I-4 inhibited activity of the catalytic subunit of PP1 (PP1C), specifically with an IC(50) of 0.2 nM, more potently than I-2 with an IC(50) of 2 nM. I-4 weakly inhibited the activity of myosin-associated phosphates (PP1M). However, the level of inhibition of PP1M was increased during preincubation of PP1M with I-4, suggesting that the inhibition is caused by interaction of I-4 with PP1C in such a manner that it competes with the M subunit of PP1M. Gel overlay experiments showed that I-4 binds PP1C directly. Three I-4 peptides containing the N-terminal residues 1-123, 1-131, and 1-142 all showed strong binding ability to PP1C but did not show PP1 inhibitory activity, whereas an I-2 peptide (residues 1-134), lacking the corresponding C-terminal residues, potently inhibited PP1C activity as previously reported. Removal of the 18 N-terminal amino acid residues from I-4 dramatically reduced the PP1 binding activity with a correlated loss of inhibitory activity, whereas removal of the 10 N-terminal residues had only a little effect. The two peptides GST-I-4(19-131) and GST-I-4(132-202) showed ability to bind to PP1C, albeit very weakly. These results strongly suggest a multiple-point interaction between I-4 and PP1C, which is thought to cause the inhibition of I-4 which is stronger than the inhibition of I-2.
authors.affiliation: Division of Biochemical Oncology and Immunology, Institute for Genetic Medicine, Hokkaido University, Kita-15, Nishi-7, Kita-ku, Sapporo 060-0815, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Shirato
authors.forename: H
authors.initials: H
authors.lastname: Shima
authors.forename: G
authors.initials: G
authors.lastname: Sakashita
authors.forename: T
authors.initials: T
authors.lastname: Nakano
authors.forename: M
authors.initials: M
authors.lastname: Ito
authors.forename: E Y
authors.initials: EY
authors.lastname: Lee
authors.forename: K
authors.initials: K
authors.lastname: Kikuchi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D003001:Cloning, Molecular; D004331:Drosophila melanogaster; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000302:isolation & purification* / Q000494:pharmacology; D005982:Glutathione Transferase / Q000235:genetics; D020035:Holoenzymes / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics; D008969:Molecular Sequence Data; D043263:Myosin-Light-Chain Phosphatase; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D054645:Protein Phosphatase 1; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000494:pharmacology; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein; D017384:Sequence Deletion
chemical_list: D004791:Enzyme Inhibitors; D020035:Holoenzymes; D007527:Isoenzymes; D011506:Proteins; D011993:Recombinant Fusion Proteins; C417224:protein phosphatase inhibitor-4; D005982:Glutathione Transferase; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D043263:Myosin-Light-Chain Phosphatase