Abstract
Unfolded proteins in the endoplasmic reticulum cause trans-autophosphorylation of the bifunctional transmembrane kinase Ire1, which induces its endoribonuclease activity. The endoribonuclease initiates nonconventional splicing of HAC1 messenger RNA to trigger the unfolded-protein response (UPR). We explored the role of Ire1's kinase domain by sensitizing it through site-directed mutagenesis to the ATP-competitive inhibitor 1NM-PP1. Paradoxically, rather than being inhibited by 1NM-PP1, drug-sensitized Ire1 mutants required 1NM-PP1 as a cofactor for activation. In the presence of 1NM-PP1, drug-sensitized Ire1 bypassed mutations that inactivate its kinase activity and induced a full UPR. Thus, rather than through phosphorylation per se, a conformational change in the kinase domain triggered by occupancy of the active site with a ligand leads to activation of all known downstream functions.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Adenosine Diphosphate / pharmacology
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Adenosine Triphosphate / analogs & derivatives
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Adenosine Triphosphate / chemistry
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Adenosine Triphosphate / metabolism*
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Adenosine Triphosphate / pharmacology
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Basic-Leucine Zipper Transcription Factors
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Binding Sites
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Binding, Competitive
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Cytosol / metabolism
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Dithiothreitol / pharmacology
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Endoplasmic Reticulum / metabolism*
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Endoribonucleases / metabolism
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Enzyme Activation
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Ligands
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Membrane Glycoproteins / antagonists & inhibitors
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Membrane Glycoproteins / chemistry*
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Membrane Glycoproteins / genetics
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Membrane Glycoproteins / metabolism*
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Models, Biological
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Mutagenesis, Site-Directed
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Phosphorylation
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Protein Conformation
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Protein Folding*
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Protein Serine-Threonine Kinases / antagonists & inhibitors
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Protein Serine-Threonine Kinases / chemistry*
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Protein Serine-Threonine Kinases / genetics
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Protein Serine-Threonine Kinases / metabolism*
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Protein Structure, Tertiary
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Pyrazoles / chemistry
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Pyrazoles / metabolism*
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Pyrazoles / pharmacology*
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Pyrimidines / chemistry
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Pyrimidines / metabolism*
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Pyrimidines / pharmacology*
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RNA Splicing
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RNA, Messenger / genetics
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RNA, Messenger / metabolism
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Repressor Proteins / genetics
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Repressor Proteins / metabolism
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Saccharomyces cerevisiae Proteins / antagonists & inhibitors
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Saccharomyces cerevisiae Proteins / chemistry*
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Saccharomyces cerevisiae Proteins / genetics
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Saccharomyces cerevisiae Proteins / metabolism*
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Signal Transduction
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Structure-Activity Relationship
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Substrate Specificity
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Transcription Factors / genetics
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Transcription Factors / metabolism
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Up-Regulation
Substances
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1-tert-butyl-3-naphthalen-1-ylmethyl-1H-pyrazolo(3,4-d)pyrimidin-4-ylemine
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Basic-Leucine Zipper Transcription Factors
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HAC1 protein, S cerevisiae
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Ligands
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Membrane Glycoproteins
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Pyrazoles
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Pyrimidines
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RNA, Messenger
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Repressor Proteins
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Saccharomyces cerevisiae Proteins
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Transcription Factors
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Adenosine Diphosphate
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Adenosine Triphosphate
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IRE1 protein, S cerevisiae
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Protein Serine-Threonine Kinases
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Endoribonucleases
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Dithiothreitol