title: Cellular mechanisms regulating protein phosphatase-1. A key functional interaction between inhibitor-2 and the type 1 protein phosphatase catalytic subunit.
abstract: Inhibitor-1 (I-1) and inhibitor-2 (I-2) selectively inhibit type 1 protein serine/threonine phosphatases (PP1). To define the molecular basis for PP1 inhibition by I-1 and I-2 charged-to-alanine substitutions in the Saccharomyces cerevisiae, PP1 catalytic subunit (GLC7), were analyzed. Two PP1 mutants, E53A/E55A and K165A/E166A/K167A, showed reduced sensitivity to I-2 when compared with wild-type PP1. Both mutants were effectively inhibited by I-1. Two-hybrid analysis and coprecipitation or pull-down assays established that wild-type and mutant PP1 catalytic subunits bound I-2 in an identical manner and suggested a role for the mutated amino acids in enzyme inhibition. Inhibition of wild-type and mutant PP1 enzymes by full-length I-2(1-204), I-2(1-114), and I-2(36-204) indicated that the mutant enzymes were impaired in their interaction with the N-terminal 35 amino acids of I-2. Site-directed mutagenesis of amino acids near the N terminus of I-2 and competition for PP1 binding by a synthetic peptide encompassing an I-2 N-terminal sequence suggested that a PP1 domain composed of amino acids Glu-53, Glu-55, Asp-165, Glu-166, and Lys-167 interacts with the N terminus of I-2. This defined a novel regulatory interaction between I-2 and PP1 that determines I-2 potency and perhaps selectivity as a PP1 inhibitor.
authors.affiliation: Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: J H
authors.initials: JH
authors.lastname: Connor
authors.forename: D
authors.initials: D
authors.lastname: Frederick
authors.forename: H b
authors.initials: Hb
authors.lastname: Huang
authors.forename: J
authors.initials: J
authors.lastname: Yang
authors.forename: N R
authors.initials: NR
authors.lastname: Helps
authors.forename: P T
authors.initials: PT
authors.lastname: Cohen
authors.forename: A C
authors.initials: AC
authors.lastname: Nairn
authors.forename: A
authors.initials: A
authors.lastname: DePaoli-Roach
authors.forename: K
authors.initials: K
authors.lastname: Tatchell
authors.forename: S
authors.initials: S
authors.lastname: Shenolikar
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D020134:Catalytic Domain; D017931:DNA Primers; D004791:Enzyme Inhibitors / Q000378:metabolism*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D054645:Protein Phosphatase 1; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D017386:Sequence Homology, Amino Acid
chemical_list: D017931:DNA Primers; D004791:Enzyme Inhibitors; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1