Bypassing a Kinase Activity with an ATP-Competitive Drug

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 RNAto 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.

[1]  J. Sambrook,et al.  Protein folding in the cell , 1992, Nature.

[2]  P. Walter,et al.  Basis for regulated RNA cleavage by functional analysis of RNase L and Ire1p. , 2001, RNA.

[3]  P. Walter,et al.  tRNA Ligase Is Required for Regulated mRNA Splicing in the Unfolded Protein Response , 1996, Cell.

[4]  Peter Walter,et al.  The Transmembrane Kinase Ire1p Is a Site-Specific Endonuclease That Initiates mRNA Splicing in the Unfolded Protein Response , 1997, Cell.

[5]  Peter G. Schultz,et al.  A chemical switch for inhibitor-sensitive alleles of any protein kinase , 2000, Nature.

[6]  宁北芳,et al.  疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A , 2005 .

[7]  P. Walter,et al.  Intracellular signaling from the endoplasmic reticulum to the nucleus: the unfolded protein response in yeast and mammals. , 2001, Current opinion in cell biology.

[8]  Xiaohua Shen,et al.  The unfolded protein response in nutrient sensing and differentiation , 2002, Nature Reviews Molecular Cell Biology.

[9]  P. Walter,et al.  Oligomerization and phosphorylation of the Ire1p kinase during intracellular signaling from the endoplasmic reticulum to the nucleus. , 1996, The EMBO journal.

[10]  B. Dong,et al.  Alternative function of a protein kinase homology domain in 2', 5'-oligoadenylate dependent RNase L. , 1999, Nucleic acids research.

[11]  Arthur Weiss,et al.  Switching Signals On or Off by Receptor Dimerization , 1998, Cell.

[12]  Peter Walter,et al.  Transcriptional induction of genes encoding endoplasmic reticulum resident proteins requires a transmembrane protein kinase , 1993, Cell.

[13]  Yi Liu,et al.  Design of allele-specific inhibitors to probe protein kinase signaling , 1998, Current Biology.

[14]  P. Walter,et al.  Block of HAC1 mRNA Translation by Long-Range Base Pairing Is Released by Cytoplasmic Splicing upon Induction of the Unfolded Protein Response , 2001, Cell.

[15]  J. Sambrook,et al.  A transmembrane protein with a cdc2+ CDC28 -related kinase activity is required for signaling from the ER to the nucleus , 1993, Cell.

[16]  Kevan M. Shokat,et al.  Chemical genetic analysis of the budding-yeast p21-activated kinase Cla4p , 2000, Nature Cell Biology.

[17]  S. Hubbard,et al.  Autoregulatory Mechanisms in Protein-tyrosine Kinases* , 1998, The Journal of Biological Chemistry.

[18]  F. Stevens,et al.  Protein folding in the ER. , 1999, Seminars in cell & developmental biology.

[19]  Peter Walter,et al.  Functional and Genomic Analyses Reveal an Essential Coordination between the Unfolded Protein Response and ER-Associated Degradation , 2000, Cell.

[20]  R. Steele,et al.  Deceiving appearances: signaling by “dead” and “fractured” receptor protein‐tyrosine kinases , 2000, BioEssays : news and reviews in molecular, cellular and developmental biology.

[21]  J. Kuriyan,et al.  Crystal structure of Hck in complex with a Src family-selective tyrosine kinase inhibitor. , 1999, Molecular cell.

[22]  J. Paranjape,et al.  RNase L dimerization in a mammalian two-hybrid system in response to 2',5'-oligoadenylates. , 1998, Nucleic acids research.

[23]  P. Walter,et al.  A Novel Mechanism for Regulating Activity of a Transcription Factor That Controls the Unfolded Protein Response , 1996, Cell.

[24]  J. Kuriyan,et al.  The Conformational Plasticity of Protein Kinases , 2002, Cell.

[25]  F. Urano,et al.  IRE1 and efferent signaling from the endoplasmic reticulum. , 2000, Journal of cell science.

[26]  D. Pipeleers,et al.  Cellular Origin of Hexokinase in Pancreatic Islets* , 1999, The Journal of Biological Chemistry.

[27]  K. Shokat,et al.  Engineering unnatural nucleotide specificity for Rous sarcoma virus tyrosine kinase to uniquely label its direct substrates. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[28]  J. Kuriyan,et al.  Reciprocal regulation of Hck activity by phosphorylation of Tyr(527) and Tyr(416). Effect of introducing a high affinity intramolecular SH2 ligand. , 2000, The Journal of biological chemistry.

[29]  P. Brown,et al.  Exploring the metabolic and genetic control of gene expression on a genomic scale. , 1997, Science.

[30]  Anthony C. Bishop,et al.  Chemical inhibition of the Pho85 cyclin-dependent kinase reveals a role in the environmental stress response , 2001, Proceedings of the National Academy of Sciences of the United States of America.