{"doi":"10.1101/2025.10.10.681638","title":"AMPK Phosphorylation Proceeds Through Hierarchical Proteoform Cascades Revealed by Integrated Mass Spectrometry","abstract":"Protein phosphorylation creates functionally distinct proteoforms through complex modification cascades, yet capturing their temporal dynamics and combinatorial patterns remains a major analytical challenge. Here, we introduce a hybrid precision mass spectrometry (MS) strategy that integrates intact mass measurements for temporal tracking, bottom-up MS analysis for site-specific kinetics, and top-down MS sequencing for proteoform characterization to resolve phosphorylation dynamics within intact kinase complexes. Using AMP-activated protein kinase (AMPK) as a model system, we uncover coordinated autophosphorylation cascades exhibiting kinetic hierarchies, with α1-S496 showing the highest kinetic efficiency. Allosteric ADaM-site activation bypasses canonical α1-T183 phosphorylation, enabling autophosphorylation even in activation-deficient mutants. Top-down MS sequencing identifies the predominant β1 proteoform as S24/25+S108 double phosphorylation, a pattern linking extranuclear distribution with allosteric responsiveness. Phosphatase competition shows PP1A selectively removes activation-loop phosphorylation while autophosphorylation sites remain protected. This integrated strategy uncovers the proteoform dynamics underlying AMPK activation and provides a broadly applicable framework for studying phosphorylation-based regulation in kinases.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2025,"id":577618,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9554,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1038960,"name":"Hsin‐Ju Chan","orcid":"0000-0002-4488-5053","position":1,"is_corresponding":false},{"id":1431196,"name":"Liam J. Bandura","orcid":null,"position":2,"is_corresponding":false},{"id":1227555,"name":"Zhan Gao","orcid":"0009-0009-0677-9653","position":3,"is_corresponding":false},{"id":1046357,"name":"Man‐Di Wang","orcid":"0009-0002-2464-839X","position":4,"is_corresponding":false},{"id":1038961,"name":"Holden T. Rogers","orcid":"0000-0003-0870-0127","position":5,"is_corresponding":false},{"id":275525,"name":"Sean J. McIlwain","orcid":"0000-0002-3820-8400","position":6,"is_corresponding":false},{"id":167474,"name":"Charlotte Uetrecht","orcid":"0000-0002-1991-7922","position":7,"is_corresponding":false},{"id":256465,"name":"Ying Ge","orcid":"0000-0001-5211-6812","position":8,"is_corresponding":false},{"id":1038959,"name":"Boris Krichel","orcid":"0000-0001-9667-0719","position":0,"is_corresponding":true}],"reference_count":50,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-19T02:58:16.148027Z","pmid":"41279313","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}