Figure 1

Phosphorylation enhances activity of IRE1α in vitro . (A) Schematic of the truncated G547 and H499 IRE1α construct compared to the full-length protein. (B) Deconvoluted mass spectra of lambda phosphatase-treated G547 IRE1α produced in insect cells (grey) and after incubation with Mg/ATP in vitro (black) show the addition of 3 phosphates due to autophosphorylation. (C) Schematic of the in silico designed stembulge RNA containing the XBP-1 splice site labelled 5’ with fluorescein (FAM) and 3’ with Black-Hole Quencher 1 (BHQ1) whose fluorescence quenching is alleviated upon cleavage. (D) 90 nM RNA in C was incubated with increasing concentrations of dephosphorylated IRE1α (open squares, EC₅₀ = 369 nM ) or phosphorylated IRE1α (filled squares, EC₅₀ = 114 nM) for 30 minutes at 30˚C. Error bars S.E.M of 3 independent experiments. (E) Linker regions of human and yeast IRE1. The linker domain is defined by the first residue after the transmembrane domain and the last residue before the kinase domain (human P465-S570, yeast Q556-L673). Human IRE1α linker domain is more Ser/Thr-rich 26/106aa (24.5%) than yeast Ire1 16/118aa (13.6%) linker domain. The lysine-rich region of the yeast linker domain is boxed. Full-length human IRE1α and yeast IRE1 sequences were aligned using EMBOSS stretcher [http://www.ebi.ac.uk/Tools/psa/emboss_stretcher/]. (F) Deconvoluted mass spectra of lambda phosphatase-treated H499 IRE1α produced in insect cells (grey) and after incubation with Mg/ATP in vitro (black) show the addition of multiple phosphates (8–11) due to autophosphorylation. (G) As in D, dephosphorylated H499 IRE1α (open squares, EC₅₀ = 440 nM), autophosphorylated H499 IRE1α (filled squares, EC₅₀ = 77 nM).