Figure 5. The hierarchy of IRE1 activation by flavonols supports a model of ligand binding in the syn orientation of the prime ring.

A. Chemical structures of flavonol-based analogs of quercetin. Quercetin and other relevant flavonols are shown with the 3- and 3’-moieties in the syn orientation; except morin, which is shown in the anti conformation due to clash between the 3- and 2’-hydroxyls.

B. Timecourse of RNase activity of IRE1 (0.5 µM) incubated in the presence of various flavonols (25 µM). The activity of IRE1 in the absence of flavonol is indicated by the x symbols.

C. Timecourse of RNase activity of IRE1 (0.5 µM) incubated in the presence of both ADP (2 mM) and flavonols (25 µM). The activity of IRE1 in the absence of flavonol is indicated by the x symbols.

D. Autoradiograph of IRE1 (0.5 µM) incubated with [³²P]γ-ATP in the presence of the known kinase inhibitor staurosporine (25 µM, a positive control for inhibition) or the flavonols (25 µM), following either 10 or 20 min reaction, as indicated. A Coomassie stain of the 20 min gel is shown below. The asterisk indicates the presence of an activity in the sample containing myrecetin that reproducibly degrades IRE1.

E. RT-PCR analysis of XBP1 mRNA purified from wild-type MEF (+/+) or IRE1 knockout cells transduced with hyIRE1^WT following exposure to thapsigargin (Tg; 0.5 µM for 2 h) or flavonols (450 µM for 2 h), as indicated.

F. RT-PCR analysis of XBP1 mRNA purified from wild-type MEF (+/+) or IRE1 knockout cells transduced with hyIRE1^WT, hyIRE1^S984, hyIRE1^K985A or empty vector (Mock) following exposure to thapsigargin (Tg, 0.5 µM for 2h) or Luteolin (450 µM for 2 h), as indicated.

(also see Supplemental Movie 1).