Abstract
Inhibitor-1 (I-1) is a selective inhibitor of protein phosphatase-1 (PP1) and regulates several PP1-dependent signaling pathways, including cardiac contractility and regulation of learning and memory. The human I-1 gene has been spliced to generate two alternative mRNAs, termed I-1alpha and I-1beta, encoding polypeptides that differ from I-1 in their C-terminal sequences. Reverse transcription-PCR established that I-1alpha and I-1beta mRNAs are expressed in a developmental and tissue-specific manner. Functional analysis of I-1 in a Saccharomyces cerevisiae strain dependent on human I-1 for viability established that a novel domain encompassing amino acids 77-110 is necessary for PP1 inhibition in yeast. Expression of human I-1 in S. cerevisiae with a partial loss-of-function eukaryotic initiation factor-2alpha (eIF2alpha) kinase (Gcn2p) mutation permitted growth during amino acid starvation, consistent with the inhibition of Glc7p/PP1, the yeast eIF2alpha phosphatase. In contrast, human I-1alpha, which lacks amino acids 83-134, and I-1 with C-terminal deletions were significantly less effective in promoting yeast growth under starvation conditions. These data suggest that C-terminal sequences of I-1 enhance regulation of the eukaryotic eIF2alpha phosphatase. In vitro studies established that C-terminal sequences, deleted in both I-1alpha and I-1beta, enhance PP1 binding and inhibition. Expression of full-length and C-terminally truncated I-1 in HEK293T cells established the importance of the I-1 C terminus in transducing cAMP signals that promote eIF2alpha phosphorylation. This study demonstrates that multiple domains in I-1 target cellular PP1 complexes and establishes I-1 as a cellular regulator of eIF2alpha phosphorylation.
Publication types
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Research Support, U.S. Gov't, Non-P.H.S.
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Alternative Splicing
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Amino Acids / chemistry
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Animals
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Cell Line
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Cell Proliferation
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Cell Survival
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Cyclic AMP / metabolism
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Cyclic AMP-Dependent Protein Kinases / metabolism
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DNA, Complementary / metabolism
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Dose-Response Relationship, Drug
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Electrophoresis, Polyacrylamide Gel
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Eukaryotic Initiation Factor-2 / metabolism*
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Exons
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Gene Deletion
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Glutathione Transferase / metabolism
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Hormones / metabolism*
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Humans
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Immunoblotting
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Intracellular Signaling Peptides and Proteins
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Mice
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Mice, Inbred C57BL
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Mutation
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Nuclear Proteins
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Peptides / chemistry
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Phosphoprotein Phosphatases / antagonists & inhibitors
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Phosphoprotein Phosphatases / metabolism*
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Phosphorylase Phosphatase / metabolism
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Phosphorylation
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Plasmids / metabolism
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Protein Isoforms
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Protein Kinases / chemistry
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Protein Kinases / genetics
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Protein Phosphatase 1
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Protein Serine-Threonine Kinases
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Protein Structure, Tertiary
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Proteins / chemistry*
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Proteins / physiology
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RNA, Messenger / metabolism
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RNA-Binding Proteins
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Recombinant Proteins / chemistry
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Reverse Transcriptase Polymerase Chain Reaction
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Saccharomyces cerevisiae / metabolism
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Saccharomyces cerevisiae Proteins
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Signal Transduction
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Structure-Activity Relationship
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Time Factors
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Tissue Distribution
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eIF-2 Kinase / metabolism
Substances
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ANP32A protein, human
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Amino Acids
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DNA, Complementary
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Eukaryotic Initiation Factor-2
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Hormones
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Intracellular Signaling Peptides and Proteins
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Nuclear Proteins
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Peptides
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Protein Isoforms
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Proteins
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RNA, Messenger
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RNA-Binding Proteins
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Recombinant Proteins
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Saccharomyces cerevisiae Proteins
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Cyclic AMP
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Glutathione Transferase
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Protein Kinases
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Eif2ak4 protein, mouse
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GCN2 protein, S cerevisiae
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Protein Serine-Threonine Kinases
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eIF-2 Kinase
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Cyclic AMP-Dependent Protein Kinases
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Phosphoprotein Phosphatases
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Protein Phosphatase 1
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Phosphorylase Phosphatase