Profiling the Dual Enzymatic Activities of the Serine/Threonine Kinase IRE1α

: There is an allosteric relationship between the kinase and RNase domains of the ER stress sensor IRE1α. This relationship has been exploited to develop ATP-competitive inhibitors that are able to divergently modulate the RNase activity of IRE1α through its kinase domain. Here, we describe a series of biochemical methods for profiling the dual enzymatic activities of IRE1α. These methods can be used to ascertain how ATP-competitive inhibitors affect the kinase activity of IRE1α and for determining whether these ligands allosterically activate or inactivate RNase activity.