Identification of autophosphorylationsites in recombinant Saccharomyces cerevisiae Ire1

This study reports autophosphorylation sites in the serine/threonine protein kinase Ire1. The cytosolic portion of Ire1 was expressed as an glutathione S transferase fusion protein in Escherichia coli. Phosphroyaltion sites in the recombinant wild type protein and a L745A mutant were mapped by mass spectrometry. Phosphorylation sites map of the activation loop in the protein kinase domain and the alphaEF-alphaF insertion loop. In the activation loop phosphorylation was observed at serine 837, serine 840, serine 844, and threonine 844. Phosphorylaton was also observed at serine 850 in the P+1 loop. In the alphaEF-alphaF insertion loop phosphorylation was observed at threonine 873, serine 877, serine 878, threonine 881, serine 884, serine 885, and serine 887.