{"doi":"10.1016/j.cstres.2024.10.002","title":"Flow cytometry FRET reveals post-translational modifications drive Protein Phosphatase-5 conformational changes in mammalian cells","abstract":"The serine/threonine protein phosphatase 5 (PP5) plays an essential role in regulating hormone and stress-induced signaling networks as well as extrinsic apoptotic pathways in cells. Unlike other protein phosphatases, PP5 possesses both regulatory and catalytic domains, and its function is further modulated through post-translational modifications (PTMs). PP5 contains a tetratricopeptide repeat (TPR) domain, which usually inhibits its phosphatase activity by blocking the active site (closed conformation). Certain activators bind to the PP5-TPR domain, alleviating this inhibition and allowing the catalytic domain to adopt an active (open) conformation. While this mechanism has been proposed based on structural and biophysical studies, PP5 conformational changes and activity has yet to be observed in cells. Here, we designed and developed a flow cytometry-based fluorescence resonance energy transfer (FC-FRET) method, enabling real-time observation of PP5 autoinhibition and activation within live mammalian cells. By quantifying FRET efficiency using sensitized emission, we established a standardized and adaptable data acquisition workflow. Our findings revealed that, in a cellular context, PP5 exists in multiple conformational states, none of which alone fully predict its activity. Additionally, we have demonstrated that PTMs such as phosphorylation and SUMOylation impact PP5 conformational changes, representing a significant advancement in our understanding of its regulatory mechanisms. • Development of a novel flow cytometry (FC)-FRET-method for protein phosphatase-5. • FC-FRET revealed multiple PP5 conformational states in real-time within live mammalian cells. • In a cellular context, PP5 primarily is in a closed state but not completely inactive. • Phosphorylation and SUMOylation affect conformational changes and regulatory function of PP5 in cells.","journal":"Cell Stress and Chaperones","year":2024,"id":466526,"datarank":0.26876392038420827,"base_score":1.791759469228055,"endowment":1.791759469228055,"self_citation_contribution":0.26876392038420827,"citation_network_contribution":0.0,"self_endowment_contribution":0.26876392038420827,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":5,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.962,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2024-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":346995,"name":"Sarah J. Backe","orcid":"0000-0001-9275-267X","position":1,"is_corresponding":false},{"id":901063,"name":"Jennifer Heritz","orcid":"0000-0001-7259-8046","position":2,"is_corresponding":false},{"id":346994,"name":"Mark R. Woodford","orcid":"0000-0002-6737-2832","position":3,"is_corresponding":false},{"id":346998,"name":"Dimitra Bourboulia","orcid":"0000-0002-8567-2775","position":4,"is_corresponding":false},{"id":347006,"name":"Mehdi Mollapour","orcid":"0000-0002-2144-1361","position":5,"is_corresponding":false},{"id":349164,"name":"Rebecca Sager","orcid":null,"position":0,"is_corresponding":true}],"reference_count":29,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-19T02:05:06.743102Z","pmid":"39395782","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":[]}