Illuminating ER Lumen AMPK Dynamics for Maintaining Vascular Homeostasis via ACE2

University Of California, San Diego, La Jolla CA

Investigators

Abstract

Project Summary Adenosine monophosphate-activated kinase (AMPK) is a master metabolic regulator critical for sensing low cellular energy states and initiating catabolic activities while also inhibiting anabolic activities.1 It has become increasingly clear that spatiotemporal regulation of AMPK is key to achieving specificity in AMPK signaling and fluorescent biosensors have served as powerful tools for monitoring spatiotemporal AMPK activity.2 AMPK helps maintain vascular homeostasis by phosphorylating different substrates such as angiotensin converting enzyme 2 (ACE2), a transmembrane receptor critical for maintaining blood pressure homeostasis, which is phosphorylated by AMPK to maintain stability, though where and how this AMPK/ACE2 interaction occurs is unclear.3 Due to the topology of ACE2 as a type I transmembrane protein, the AMPK/ACE2 interaction would require AMPK to enter the secretory pathway despite having no secretory signal peptides. In this proposal, we aim to (1) identify molecular mechanisms underlying the regulation of AMPK in ER lumen and (2) examine the functional role of ER lumen AMPK activity in ACE2 signaling. In preliminary studies, using an ER lumen-targeted single-fluorophore excitation ratiometric AMPK activity reporter (ExRai AMPKAR), I found AMPK is active within the ER lumen and that such activity is dependent upon the upstream kinase CAMKK2. I also showed that endogenous AMPK is associated with an ER lumen-specific protein, Grp94, using the proximity ligation assay (PLA). I hypothesize that AMPK is phosphorylated by CAMKK2 prior to its Grp94-mediated translocation into the ER lumen and will combine biochemical and live-cell assays to test this hypothesis. As part of our second aim, I found endogenous ACE2 is localized to the ER and that AMPK-phosphomimic ACE2 had enhanced presence at the plasma membrane versus nonphosphorylatable ACE2. I hypothesize that AMPK phosphorylates ACE2 in the ER lumen to facilitate its trafficking to the plasma membrane where it functions to maintain vascular homeostasis. We are now developing an ER lumen AMPK inhibitory peptide to use in ACE2 trafficking and endothelial function assays. The proposed studies will illuminate a novel AMPK activity site not mechanistically described and will fortify our understanding of spatial AMPK signaling to inform cardiovascular drug development and therapeutic strategies, especially for hypertension.