Cellular mechanisms regulating protein phosphatase-1. A key functional interaction between inhibitor-2 and the type 1 protein phosphatase catalytic subunit

John H. Connor, Deborah Frederick, Hsien Bin Huang, Jie Yang, Nicholas R. Helps, Patricia T W Cohen, Angus C. Nairn, Anna De Paoli-Roach, Kelly Tatchell, Shirish Shenolikar

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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.

Original languageEnglish
Pages (from-to)18670-18675
Number of pages6
JournalJournal of Biological Chemistry
Volume275
Issue number25
DOIs
StatePublished - Jun 23 2000

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Protein Phosphatase 1
Catalytic Domain
Enzyme inhibition
Amino Acids
Enzymes
Mutagenesis
Coprecipitation
Site-Directed Mutagenesis
Alanine
Yeast
Saccharomyces cerevisiae
Assays
Substitution reactions
Peptides
protein phosphatase inhibitor-2
protein phosphatase inhibitor-1

ASJC Scopus subject areas

  • Biochemistry

Cite this

Cellular mechanisms regulating protein phosphatase-1. A key functional interaction between inhibitor-2 and the type 1 protein phosphatase catalytic subunit. / Connor, John H.; Frederick, Deborah; Huang, Hsien Bin; Yang, Jie; Helps, Nicholas R.; Cohen, Patricia T W; Nairn, Angus C.; De Paoli-Roach, Anna; Tatchell, Kelly; Shenolikar, Shirish.

In: Journal of Biological Chemistry, Vol. 275, No. 25, 23.06.2000, p. 18670-18675.

Research output: Contribution to journalArticle

Connor, John H. ; Frederick, Deborah ; Huang, Hsien Bin ; Yang, Jie ; Helps, Nicholas R. ; Cohen, Patricia T W ; Nairn, Angus C. ; De Paoli-Roach, Anna ; Tatchell, Kelly ; Shenolikar, Shirish. / Cellular mechanisms regulating protein phosphatase-1. A key functional interaction between inhibitor-2 and the type 1 protein phosphatase catalytic subunit. In: Journal of Biological Chemistry. 2000 ; Vol. 275, No. 25. pp. 18670-18675.
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