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Principal Investigator: Anne Lyons
Organization: UNIVERSITY OF CALIFORNIA, SAN DIEGO
Fiscal Year: 2024
Award: $41,755
Funding agency: National Heart Lung and Blood Institute
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.
Terms: <2'-AMP><2'-adenosine monophosphate><2'-adenylic acid><21+ years old><5'-Adenylic acid><ACE2><ATP-protein phosphotransferase><Adenosine Monophosphate><Adenylic Acid><Adult><Adult Human><Affect><Angiotensins><Antibodies><Assay><Autoregulation><BP homeostasis><BP regulation><Bioassay><Bioavailability><Biochemical><Biological Assay><Biological Availability><Biosensor><Blood Pressure><Blood Vessels><Ca(2+)-Calmodulin Dependent Protein Kinase><CaMK><Calcium/calmodulin-dependent protein kinase><Calmodulin-Dependent Protein Kinases><Calmodulin-Kinase><Cardiovascular><Cardiovascular Agents><Cardiovascular Body System><Cardiovascular Diseases><Cardiovascular Drugs><Cardiovascular Organ System><Cardiovascular system><Cell Body><Cell Communication and Signaling><Cell Signaling><Cell membrane><Cells><Cellular Assay><Chaperone><Chemical Fractionation><Clinical><Closure by Ligation><Cytoplasmic Membrane><Data><Disease><Disorder><ELISA><Electron Microscopy><Endogenous Nitrate Vasodilator><Endoplasmic Reticulum><Endothelial Cells><Endothelium><Endothelium-Derived Nitric Oxide><Enzyme-Linked Immunosorbent Assay><Ergastoplasm><FRACN><Fluorescence Light Microscopy><Fluorescence Microscopy><Fortification><Fractionation><Fractionation Radiotherapy><Goals><Health><Heart Vascular><Homeostasis><Hypertension><Image><Integral Membrane Protein><Intracellular Communication and Signaling><Intrinsic Membrane Protein><Kinase Family Gene><Kinases><L-Serine><Ligation><Light><Link><Location><Maintenance><Mediating><Membrane><Metabolic><Molecular><Molecular Chaperones><Monitor><Mononitrogen Monoxide><Nitric Oxide><Nitrogen Monoxide><Nitrogen Protoxide><Pathway interactions><Peptide Signal Sequences><Peptides><Phosphorylation><Phosphotransferase Gene><Phosphotransferases><Photoradiation><Physiologic Availability><Physiological Homeostasis><Plasma Enhancement><Plasma Membrane><Protein Kinase><Protein Phosphorylation><Proteins><Receptor Protein><Regulation><Reporter><Research><Role><Serine><Signal Peptide><Signal Sequences><Signal Transduction><Signal Transduction Systems><Signaling><Signaling Molecule><Site><Specificity><Testing><Therapeutic><Transmembrane Protein><Transmembrane Protein Gene><Transphosphorylases><Vascular Hypertensive Disease><Vascular Hypertensive Disorder><Work><adulthood><angiotensin converting enzyme 2><angiotensin converting enzyme II><biological sensor><biological signal transduction><blood pressure homeostasis><blood pressure regulation><calcium-calmodulin-dependent PK><calcium-calmodulin-dependent PK type II><calmodulin dependent protein kinase><cardiovascular disorder><cardiovascular health><cell assay><circulatory system><drug development><drug efficacy><endothelial cell derived relaxing factor><enzyme linked immunoassay><experiment><experimental research><experimental study><experiments><fluorophore><glycogen synthase a kinase><high blood pressure><hydroxyalkyl protein kinase><hyperpiesia><hyperpiesis><hypertensive disease><hypertensive disorder><imaging><improved><knock-down><knockdown><membrane structure><microtubule associated protein 2 kinase><novel><pathway><phosphorylase b kinase kinase><plasmalemma><protein kinase II><protein signal sequence><ratiometric><receptor><regulate BP><regulate blood pressure><social role><spatiotemporal><sub-cellular targeting><subcellular targeting><tool><trafficking><upstream kinase><vascular>