Abstract 1429 Spatial compartmentalization of AMPK in the endoplasmic reticulum: regulation & functions in cancer and cardiovascular disease

AMPK is a master metabolic regulator critical for sensing low cellular energy states and initiating catabolic activities.1 The spatiotemporal regulation of AMPK is key to achieving specificity and efficiency in AMPK signaling with downstream targets of AMPK enriched at distinct metabolic locations throughout the cell. One particularly understudied site of AMPK activity is within the endoplasmic reticulum (ER) lumen, where the regulation of AMPK remains uncharacterized.2-4 Programmed death ligand 1 (PD-L1), a pro-tumorigenic transmembrane protein that suppresses immune recognition of cancer, was previously shown to be phosphorylated by AMPK in the ER lumen in breast cancer cells, which led to decreased stability and trafficking of PD-L1. However, it is unclear if AMPK is active in the ER lumen of normal cells or AMPK ER lumen activity is an aberrant cancer phenotype.2 Angiotensin converting enzyme 2 (ACE2), a transmembrane receptor critical for maintaining blood pressure homeostasis, is known to be phosphorylated by AMPK at serine 680, which is critical for maintaining its stability, though where and how this AMPK/ACE2 interaction occurs is unclear.4 The topology of ACE2 would require AMPK to be active in the secretory pathway to phosphorylate S680. We hypothesize that a subcellular pool of AMPK exists within the ER lumen with distinct regulation that functionally modulates transmembrane protein secretory trafficking to impact their functions. To test this hypothesis, we developed and applied a series of native biochemistry tools including fluorescent biosensors for monitoring spatiotemporal AMPK activity.5 Using an ER lumen-targeted AMPK biosensor ExRai AMPKAR, we showed that AMPK activity is present in ER lumen and has distinct regulation compared with AMPK activity at the outer ER surface. We further developed a fluorescent ER lumen translocation assay to view AMPK presence in ER lumen. Using proximity ligation assay, we showed that endogenous AMPK is associated with ER lumen-restricted chaperone Grp94. Using fluorescently-tagged PD-L1 and ACE2 probes, our data shows that phosphomimetic PD-L1 has increased ER localization whereas phosphomimetic ACE2 has increased plasma membrane localization. This work illuminates a novel AMPK activity site not mechanistically described previously and will fortify our understanding of spatial AMPK signaling to inform cancer and cardiovascular disease and therapeutic development. References 1. Trefts E, Shaw RJ. AMPK: restoring metabolic homeostasis over space and time. Molecular Cell. 2021;81(18):3677–3690. 2. Cha J-H, Yang W-H, Xia W, Wei Y, Chan L-C, Lim S-O, Li C-W, Kim T, Chang S-S, Lee H-H, Hsu JL, Wang H-L, Kuo C-W, Chang W-C, Hadad S, et al. Metformin Promotes Antitumor Immunity via Endoplasmic-Reticulum-Associated Degradation of PD-L1. Molecular Cell. 2018;71(4):606-620.e7. 3. Qi J, Gong J, Zhao T, Zhao J, Lam P, Ye J, Li JZ, Wu J, Zhou H-M, Li P. Downregulation of AMP-activated protein kinase by Cidea-mediated ubiquitination and degradation in brown adipose tissue. The EMBO Journal. 2008;27(11):1537–1548. 4. Zhang J, Dong J, Martin M, He M, Gongol B, Marin TL, Chen L, Shi X, Yin Y, Shang F, Wu Y, Huang H-Y, Zhang J, Zhang Y, Kang J, et al. AMP-activated Protein Kinase Phosphorylation of Angiotensin-Converting Enzyme 2 in Endothelium Mitigates Pulmonary Hypertension. American Journal of Respiratory and Critical Care Medicine. 2018;198(4):509–520. 5. Schmitt DL, Curtis SD, Lyons AC, Zhang J, Chen M, He CY, Mehta S, Shaw RJ, Zhang J. Spatial regulation of AMPK signaling revealed by a sensitive kinase activity reporter. Nature Communications. 2022;13(1):3856. This work was supported by NIH R01 HL162302 to J.Z., NIH T32 Pharmacologic Sciences Training Program to Q.Z., and a National Science Foundation (NSF) Graduate Research Fellowship (DGE-2038238) to A.C.L. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the NSF.