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.
Department of Chemistry, University of Washington, Box 351700, Seattle, WA, 98195, USA.|Department of Biochemistry, University of Washington, Box 351700, Seattle, WA, 98195, USA.
Hannah C
HC
Feldman
Department of Chemistry, University of Washington, Box 351700, Seattle, WA, 98195, USA. maly@chem.washington.edu.|Department of Biochemistry, University of Washington, Box 351700, Seattle, WA, 98195, USA. maly@chem.washington.edu.
Dustin J
DJ
Maly
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000494:Allosteric Regulation / Q000187:drug effects; D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D004722:Endoribonucleases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D057075:Enzyme Assays; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D015870:Gene Expression; D006801:Humans; D020128:Inhibitory Concentration 50; D008024:Ligands; D004676:Myelin Basic Protein / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D061987:Sf9 Cells; D018411:Spodoptera
D005452:Fluoresceins; D005456:Fluorescent Dyes; D008024:Ligands; D004676:Myelin Basic Protein; D010761:Phosphorus Radioisotopes; D047428:Protein Kinase Inhibitors; D011994:Recombinant Proteins; C024098:6-carboxyfluorescein; D000255:Adenosine Triphosphate; C528229:ERN1 protein, human; D017346:Protein Serine-Threonine Kinases; D004722:Endoribonucleases
Allostery; Endoribonuclease; IC50; Inhibitor; Protein kinase; RNase