Regulation of AKT phosphorylation at Ser473 and Thr308 by endoplasmic reticulum stress modulates substrate specificity in a severity dependent manner.
Regulation of AKT phosphorylation at Ser473 and Thr308 by endoplasmic reticulum stress modulates substrate specificity in a severity dependent manner.
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DOI：
10.1371/journal.pone.0017894
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发表时间：
2011-03-21
期刊：
影响因子：
3.7
通讯作者：
Burton GJ
中科院分区：
文献类型：
--
作者：
Yung HW;Charnock-Jones DS;Burton GJ
内质网（ER）应激是多种人类疾病病理生理学中的一个共同因素，这些疾病的特征是细胞行为的差异，从癌症的增殖到神经退行性疾病的凋亡。巧合的是，作为细胞生长、增殖和存活的中心调节因子的AKT/PKB活性的失调通常与相同的疾病有关。在这里，我们证明内质网应激以一种严重依赖的方式调节AKT底物特异性，如针对已知AKT靶点的磷酸化特异性抗体所示。内质网应激也以严重依赖的方式降低总AKT和磷酸化AKT，而不影响上游激酶PDK1的活性。对总AKT的正常化显示，在内质网应激下，Thr308的磷酸化被抑制，而Ser473的磷酸化被增加。内质网应激诱导GRP78， sirna介导的GRP78敲除使Ser473位点的磷酸化增加3.6倍，而Thr308位点的磷酸化没有增加。底物特异性再次改变。原位接近结扎实验显示，内质网应激诱导后，细胞质膜上GRP78和AKT之间存在物理相互作用。细胞核形态正常的细胞染色较弱，细胞核圆形、凝聚的细胞染色较强。GRP78和P-AKT（Ser473）的共免疫沉淀证实了免疫复合物由非磷酸化的AKT（Ser473和Thr308）组成。这种相互作用可能是特异性的，因为AKT不与所有分子伴侣结合，GRP78不与p70 S6激酶结合。这些发现为内质网应激如何通过调节AKT信号传导导致不同细胞命运的人类病理变化提供了一种机制解释。
Endoplasmic reticulum (ER) stress is a common factor in the pathophysiology of diverse human diseases that are characterised by contrasting cellular behaviours, from proliferation in cancer to apoptosis in neurodegenerative disorders. Coincidently, dysregulation of AKT/PKB activity, which is the central regulator of cell growth, proliferation and survival, is often associated with the same diseases. Here, we demonstrate that ER stress modulates AKT substrate specificity in a severity-dependent manner, as shown by phospho-specific antibodies against known AKT targets. ER stress also reduces both total and phosphorylated AKT in a severity-dependent manner, without affecting activity of the upstream kinase PDK1. Normalisation to total AKT revealed that under ER stress phosphorylation of Thr308 is suppressed while that of Ser473 is increased. ER stress induces GRP78, and siRNA-mediated knock-down of GRP78 enhances phosphorylation at Ser473 by 3.6 fold, but not at Thr308. Substrate specificity is again altered. An in-situ proximity ligation assay revealed a physical interaction between GRP78 and AKT at the plasma membrane of cells following induction of ER stress. Staining was weak in cells with normal nuclear morphology but stronger in those displaying rounded, condensed nuclei. Co-immunoprecipitation of GRP78 and P-AKT(Ser473) confirmed the immuno-complex consists of non-phosphorylated AKT (Ser473 and Thr308). The interaction is likely specific as AKT did not bind to all molecular chaperones, and GRP78 did not bind to p70 S6 kinase. These findings provide one mechanistic explanation for how ER stress contributes to human pathologies demonstrating contrasting cell fates via modulation of AKT signalling.
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