Emerging evidence suggests that signaling pathways can be spatially regulated to ensure rapid and efficient responses to dynamically changing local cues. Ferroptosis is a recently defined form of lipid peroxidation-driven cell death. Although the molecular mechanisms underlying ferroptosis are emerging, spatial aspects of its signaling remain largely unexplored. By analyzing a public database, we found that a mitochondrial chaperone protein, glucose-regulated protein 75 (GRP75), may have a previously undefined role in regulating ferroptosis. This was subsequently validated. Interestingly, under ferroptotic conditions, GRP75 translocated from mitochondria to mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) and the cytosol. Further mechanistic studies revealed a highly spatial regulation of GRP75-mediated antiferroptotic signaling. Under ferroptotic conditions, lipid peroxidation predominantly accumulated at the ER, which activated protein kinase A (PKA) in a cAMP-dependent manner. In particular, a signaling microdomain, the outer mitochondrial membrane protein A-kinase anchor protein 1 (AKAP1)-anchored PKA, phosphorylated GRP75 at S148 in MAMs. This caused GRP75 to be sequestered outside the mitochondria, where it competed with Nrf2 for Keap1 binding through a conserved high-affinity RGD-binding motif, ETGE. Nrf2 was then stabilized and activated, leading to the transcriptional activation of a panel of antiferroptotic genes. Blockade of the PKA/GRP75 axis dramatically increased the responses of cancer cells to ferroptosis both in vivo and in vitro. Our identification a localized signaling cascade involved in protecting cancer cells from ferroptosis broadens our understanding of cellular defense mechanisms against ferroptosis and also provides a new target axis (AKAP1/PKA/GRP75) to improve the responses of cancer cells to ferroptosis.

新出现的证据表明，信号通路可以在空间上进行调节，以确保对动态变化的局部线索做出快速有效的反应。铁死亡是最近定义的脂质过氧化驱动的细胞死亡形式。尽管铁死亡的分子机制正在出现，但其信号转导的空间方面在很大程度上仍未得到探索。通过分析公共数据库，我们发现线粒体伴侣蛋白葡萄糖调节蛋白 75 （GRP75） 可能在调节铁死亡中具有以前未明确的作用。这随后得到了验证。有趣的是，在铁死亡条件下，GRP75 从线粒体易位到线粒体相关的内质网 （ARM） 膜 （MAM） 和胞质溶胶。进一步的机制研究揭示了 GRP75 介导的抗铁死亡信号传导的高度空间调节。在铁死亡条件下，脂质过氧化主要在 ER 处积累，其以 cAMP 依赖性方式激活蛋白激酶 A （PKA）。特别是，信号转导微结构域，线粒体外膜蛋白 A 激酶锚定蛋白 1 （AKAP1） 锚定的 PKA，在 MAM 中的 S148 位点磷酸化 GRP75。这导致 GRP75 被隔离在线粒体外，在那里它通过保守的高亲和力 RGD 结合基序 ETGE 与 Nrf2 竞争 Keap1 结合。然后 Nrf2 被稳定和激活，导致一组抗铁死亡基因的转录激活。阻断 PKA/GRP75 轴在体内和体外都显着增加了癌细胞对铁死亡的反应。 我们鉴定了一种参与保护癌细胞免受铁死亡的局部信号级联反应，拓宽了我们对细胞防御机制对铁死亡的理解，还提供了一个新的靶轴 （AKAP1/PKA/GRP75） 来改善癌细胞对铁死亡的反应。