Papa Feroz R, Zhang Chao, Shokat Kevan, Walter Peter
Department of Medicine, University of California, San Francisco, CA 94143-2200, USA.
Science. 2003 Nov 28;302(5650):1533-7. doi: 10.1126/science.1090031. Epub 2003 Oct 16.
Unfolded proteins in the endoplasmic reticulum cause trans-autophosphorylation of the bifunctional transmembrane kinase Ire1, which induces its endoribonuclease activity. The endoribonuclease initiates nonconventional splicing of HAC1 messenger RNA to trigger the unfolded-protein response (UPR). We explored the role of Ire1's kinase domain by sensitizing it through site-directed mutagenesis to the ATP-competitive inhibitor 1NM-PP1. Paradoxically, rather than being inhibited by 1NM-PP1, drug-sensitized Ire1 mutants required 1NM-PP1 as a cofactor for activation. In the presence of 1NM-PP1, drug-sensitized Ire1 bypassed mutations that inactivate its kinase activity and induced a full UPR. Thus, rather than through phosphorylation per se, a conformational change in the kinase domain triggered by occupancy of the active site with a ligand leads to activation of all known downstream functions.
内质网中未折叠的蛋白质会导致双功能跨膜激酶Ire1发生反式自磷酸化，从而诱导其核糖核酸内切酶活性。该核糖核酸内切酶启动HAC1信使核糖核酸的非常规剪接，以触发未折叠蛋白反应（UPR）。我们通过定点诱变使Ire1的激酶结构域对ATP竞争性抑制剂1NM-PP1敏感，从而探究其作用。矛盾的是，药物敏感的Ire1突变体非但没有被1NM-PP1抑制，反而需要1NM-PP1作为激活的辅助因子。在1NM-PP1存在的情况下，药物敏感的Ire1绕过了使其激酶活性失活的突变，并诱导了完全的UPR。因此，激酶结构域的构象变化并非通过磷酸化本身，而是由配体占据活性位点引发，进而导致所有已知下游功能的激活。
