Ampk alpha2 T172 activation dictates exercise performance and energy transduction in skeletal muscle

AMPK (5′-AMP-activated protein kinase) is an energetic sensor for metabolic regulation and integration. Here, we employed CRISPR/Cas9 to generate non-activatable Ampkα knock-in (KI) mice with a mutation of the threonine 172 phosphorylation site to alanine, circumventing the limitations of previous genetic interventions that disrupt the protein stoichiometry. KI mice of Ampkα2, but not Ampkα1, demonstrated phenotypic changes with increased fat-to-lean mass, impaired endurance exercise capacity, and diminished mitochondrial maximal respiration and conductance in skeletal muscle. Integrated temporal multi-omic analysis (proteomics/ phosphoproteomics/ metabolomics) in skeletal muscle at rest and during exercise establishes a pleiotropic yet imperative role of Ampkα2 T172 activation for glycolytic and oxidative metabolism, mitochondrial respiration, and contractile function. Importantly, there is a significant overlap of skeletal muscle proteomic changes in Ampkα2 T172A KI mice with those of..., LC-MS/MS Proteomics & Phosphoproteomics
Muscle tissue samples from male and female WT and KI mice were collected before exercise (i.e., sedentary control, SED), after 10-min running (10 min), and after exhaustion (Exh.) and stored at -80ºC until undergoing processing. Sample processing for global proteomics and phosphoproteomics analysis was conducted as previously described (71) with several modifications. Briefly, frozen muscle tissue samples were minced into small pieces on a pre-chilled aluminum tray with dry ice. ~ 25 mg of tissue were collected and homogenized in cold lysis buffer (50 mM Tris, 8 M urea, 75 mM NaCl, 1 mM EDTA, 2 µg/mL aprotinin, 10 µg/mL leupeptin, 1 mM PMSF, 10 mM NaF, 1% phosphatase inhibitor cocktail 2, 1% phosphatase inhibitor cocktail 3, pH 8.0) on a pre-chilled bead beater using 2-min cycle. Samples were then centrifuged at 13,000xg at 4 °C for 10 min to remove debris. The extracted protein was quantified by the CA assay. The same quantity of protein was ..., # Data from: Ampk alpha2 T172 activation dictates exercise performance and energy transduction in skeletal muscle

Dataset DOI: [10.5061/dryad.f1vhhmh96](https://doi.org/10.5061/dryad.f1vhhmh96)

## Description of the data and file structure

Muscle tissues from male and female WT and KI mice were collected at rest (SED), after 10 min of running, and at exhaustion, snap-frozen, and stored at −80 °C. Samples were processed for global proteomics and phosphoproteomics with minor modifications to established methods, including tissue homogenization, protein extraction, digestion, TMT18 labeling, high-pH fractionation, IMAC phosphoenrichment, and LC–MS/MS analysis on an Orbitrap Fusion Lumos. Untargeted metabolomics was performed using chloroform–methanol extraction, AcquireX-enabled LC–MS/MS on an Orbitrap IDX Tribrid, and rigorous QC procedures. AMPK consensus motifs were identified from phosphopeptides using strict, relaxed, and minimal sequence criteria based on known substrate preferenc...,