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Kirschstein NRSA Individual Predoctoral Fellowship under Award Number","award":["F31HL178305"],"award-info":[{"award-number":["F31HL178305"]}]}],"content-domain":{"domain":["www.science.org"],"crossmark-restriction":true},"short-container-title":["Sci. Adv."],"published-print":{"date-parts":[[2026,9,4]]},"abstract":"<jats:p>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\u2032-monophosphate\u2013activated 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)\u2013mediated phosphorylation or allosteric activator PF-739 both triggering extensive autophosphorylation with \u03b11-Ser496 (S496) showing the highest kinetic priority. Proteoform-resolved analysis uncovers channeled \u03b21-Ser24\/25 (S24\/25)\u00a0+\u00a0Ser108 (S108) cophosphorylation linking subcellular localization with allosteric responsiveness. Site-directed mutagenesis demonstrates CaMKK2 targets only \u03b11-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\u2019s temporal kinetics and proteoform architecture during activation enables a proteoform-centric understanding on kinase regulation.<\/jats:p>","DOI":"10.1126\/sciadv.aeg5294","type":"journal-article","created":{"date-parts":[[2026,9,4]],"date-time":"2026-09-04T17:58:46Z","timestamp":1788544726000},"update-policy":"https:\/\/doi.org\/10.34133\/aaas_crossmark","source":"Crossref","is-referenced-by-count":1,"title":["AMPK phosphorylation proceeds through hierarchical proteoform cascades revealed by integrated mass spectrometry"],"prefix":"10.1126","volume":"12","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9667-0719","authenticated-orcid":true,"given":"Boris","family":"Krichel","sequence":"first","affiliation":[{"name":"Department of Cell and Regenerative Biology, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI 53705, USA."},{"name":"Human Proteomics Program, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI 53705, USA."},{"name":"Faculty V: School of Life Sciences, University of Siegen, 57076 Siegen, Germany."},{"name":"CSSB Centre for Structural Systems Biology, Deutsches Elektronen Synchroton DESY, Leibniz Institute of Virology and University of L\u00fcbeck, Notkestra\u00dfe 85, 22607 Hamburg, Germany."},{"name":"Institute of Chemistry and Metabolomics, University of L\u00fcbeck, 23562 L\u00fcbeck, Germany."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4488-5053","authenticated-orcid":true,"given":"Hsin-Ju","family":"Chan","sequence":"additional","affiliation":[{"name":"Department of Chemistry, University of Wisconsin-Madison, Madison, WI 53706, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0008-9587-7273","authenticated-orcid":true,"given":"Liam","family":"Bandura","sequence":"additional","affiliation":[{"name":"Department of Chemistry, University of Wisconsin-Madison, Madison, WI 53706, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-0677-9653","authenticated-orcid":true,"given":"Zhan","family":"Gao","sequence":"additional","affiliation":[{"name":"Department of Chemistry, University of Wisconsin-Madison, Madison, WI 53706, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-2464-839X","authenticated-orcid":true,"given":"Man-Di","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Chemistry, University of Wisconsin-Madison, Madison, WI 53706, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0870-0127","authenticated-orcid":true,"given":"Holden T.","family":"Rogers","sequence":"additional","affiliation":[{"name":"Department of Chemistry, University of Wisconsin-Madison, Madison, WI 53706, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3820-8400","authenticated-orcid":true,"given":"Sean J.","family":"Mcilwain","sequence":"additional","affiliation":[{"name":"Department of Biostatistics and Medical Informatics, University of Wisconsin-Madison, Madison, WI 53705, USA."},{"name":"University of Wisconsin Carbone Cancer Center, University of Wisconsin-Madison, Madison, WI 53705, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1991-7922","authenticated-orcid":true,"given":"Charlotte","family":"Uetrecht","sequence":"additional","affiliation":[{"name":"CSSB Centre for Structural Systems Biology, Deutsches Elektronen Synchroton DESY, Leibniz Institute of Virology and University of L\u00fcbeck, Notkestra\u00dfe 85, 22607 Hamburg, Germany."},{"name":"Institute of Chemistry and Metabolomics, University of L\u00fcbeck, 23562 L\u00fcbeck, Germany."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5211-6812","authenticated-orcid":true,"given":"Ying","family":"Ge","sequence":"additional","affiliation":[{"name":"Department of Cell and Regenerative Biology, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI 53705, USA."},{"name":"Human Proteomics Program, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI 53705, USA."},{"name":"Department of Chemistry, University of Wisconsin-Madison, Madison, WI 53706, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"221","reference":[{"key":"e_1_3_2_2_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2021.03.015"},{"key":"e_1_3_2_3_2","unstructured":"C. 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