Coupling of Stress in the ER to Activation of JNK Protein Kinases by Transmembrane Protein Kinase IRE1

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The targeted mutation leads to deletion of the exon encoding the transmembrane domain of murine IRE1α. The 5′ homology arm of the targeting vector is the 6-kb Kpn I–Mun I fragment ending 5′ of the transmembrane domain and the 3′ homology arm is the 4-kb Eco RI–Pst I fragment starting 3′ of the transmembrane domain. Gene targeting was achieved in W4 ES cells and germ line transmission was obtained from two independently derived mutant ES lines with identical phenotypes. Heterozygous mice were intercrossed and embryonic fibroblasts were procured and genotyped at day 9.5 of gestation. Pools of cells, immortalized with simian virus 40 large tumor antigen derived from individual sibling embryos of wild-type and IRE1α^−/− genotypes were evaluated.

Antiserum to IRE1α was raised in rabbits against a bacterially expressed fusion protein consisting of the COOH-terminal 542 residues of mouse IRE1α that encode for the entire kinase-endonuclease domain of the protein fused to a polyhistidine tag.

The COOH-terminal IRE1β bait consisted of a fusion between the GAL4 DNA-binding domain and the 460 COOH-terminal residues encompassing the entire kinase and endonuclease domain of IRE1β in the pAS2-1 vector (Clontech). The larger of the two in-frame TRAF2 clones obtained by screening the library made in the pACT2 vector were modified to delete the COOH-terminal region (233 to 501) of TRAF2 (ΔTRAF domain) or to create an in-frame deletion of the first 199 residues (ΔN-term). Interactions were studied in the Matchmaker two-hybrid system 2 (Clontech) according to the manufacturer's instructions.

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Supported by NIH grants ES08681 and DK47119. F.U., H.P.H., and A.B. were supported by fellowships from the Japan Society for the Promotion of Science, NIH, and the European Molecular Biology Organisation, respectively. We thank E. Skolnik and E. Becker (New York University) for advice, R. Kaufman (University of Michigan) for the IRE1α expression plasmids, D. Goeddel (Tularik Corp., San Francisco) for the anti-TRAF2 antiserum, and T. Fujino (Keio University, Tokyo) for the fibroblast cDNA library in pACT2 plasmid.