AKAP1/PKA-mediated GRP75 phosphorylation at mitochondria-associated endoplasmic reticulum membranes protects cancer cells against ferroptosis

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.