AMPK phosphorylation proceeds through hierarchical proteoform cascades revealed by integrated mass spectrometry.

Protein kinases integrate cellular signals through complex phosphorylation cascades, yet resolving how chemical perturbations trigger and modulate these cascades in therapeutic targets remains a major challenge. Here, we dissect adenosine 5'-monophosphate-activated protein kinase (AMPK) proteoforms during activation through controlled biochemical reactions with a hybrid mass spectrometry (MS) approach integrating bottom-up MS for site-specific kinetics with top-down proteoform characterization. We reveal that AMPK phosphorylation proceeds through hierarchical cascades rather than binary switching, with dual entry points: canonical calcium- and calmodulin-dependent protein kinase kinase 2 (CaMKK2)-mediated phosphorylation or allosteric activator PF-739 both triggering extensive autophosphorylation with α1-Ser496 (S496) showing the highest kinetic priority. Proteoform-resolved analysis uncovers channeled β1-Ser24/25 (S24/25) + Ser108 (S108) cophosphorylation linking subcellular localization with allosteric responsiveness. Site-directed mutagenesis demonstrates CaMKK2 targets only α1-Thr183 (T183), with all other modifications arising through autophosphorylation. Phosphatase competition reveals asymmetric control where PP1A selectively removes activation-loop phosphorylation while autophosphorylation sites remain protected, establishing persistent regulatory states. Resolving AMPK's temporal kinetics and proteoform architecture during activation enables a proteoform-centric understanding on kinase regulation.