Bypass of Activation Loop Phosphorylation by Aspartate 836 in Activation of the Endoribonuclease Activity of Ire1.
Bypass of Activation Loop Phosphorylation by Aspartate 836 in Activation of the Endoribonuclease Activity of Ire1.
复制标题
DOI：
10.1128/mcb.00655-16
复制
发表时间：
2017-08-15
影响因子：
5.3
通讯作者：
Schröder M
中科院分区：
文献类型：
--
作者：
Armstrong MC;Šestak S;Ali AA;Sagini HAM;Brown M;Baty K;Treumann A;Schröder M
当未折叠的蛋白质在内质网中积累时，双功能蛋白激酶-内切核糖核酸酶 Ire1 启动转录因子 Hac1 的 mRNA 剪接。酿酒酵母 Ire1 的激活与其激活环 S840、S841、T844 和 S850 处的自磷酸化同时发生。对大肠杆菌中表达的 Ire1 进行质谱分析，确定 S837 是体内另一个潜在的磷酸化位点。激活环中所有五个潜在磷酸化位点的突变均减少，但并未完全消除 HAC1 mRNA 的剪接、KAR2 和 PDI1 mRNA 的诱导以及 Hac1i 激活的 β-半乳糖苷酶报告基因的表达。磷酸化位点突变体比 IRE1 缺失菌株能更好地承受低水平的内质网应激。 Ire1 的体内聚集和失活不受磷酸化位点突变体的影响。磷酸化位点突变体激活环中的 D836 突变为丙氨酸几乎完全消除了 HAC1 剪接、KAR2、PDI1 和 β-半乳糖苷酶报告基因的诱导以及 ER 应激的存活，但对 Ire1 的聚类没有影响。 D836A 突变本身并不赋予表型。这些数据表明，D836 可以部分替代 Ire1 核糖核酸内切酶结构域激活中的激活环磷酸化。
The bifunctional protein kinase-endoribonuclease Ire1 initiates splicing of the mRNA for the transcription factor Hac1 when unfolded proteins accumulate in the endoplasmic reticulum. Activation of Saccharomyces cerevisiae Ire1 coincides with autophosphorylation of its activation loop at S840, S841, T844, and S850. Mass spectrometric analysis of Ire1 expressed in Escherichia coli identified S837 as another potential phosphorylation site in vivo. Mutation of all five potential phosphorylation sites in the activation loop decreased, but did not completely abolish, splicing of HAC1 mRNA, induction of KAR2 and PDI1 mRNAs, and expression of a β-galactosidase reporter activated by Hac1i. Phosphorylation site mutants survive low levels of endoplasmic reticulum stress better than IRE1 deletions strains. In vivo clustering and inactivation of Ire1 are not affected by phosphorylation site mutants. Mutation of D836 to alanine in the activation loop of phosphorylation site mutants nearly completely abolished HAC1 splicing, induction of KAR2, PDI1, and β-galactosidase reporters, and survival of ER stress, but it had no effect on clustering of Ire1. By itself, the D836A mutation does not confer a phenotype. These data argue that D836 can partially substitute for activation loop phosphorylation in activation of the endoribonuclease domain of Ire1.