Proximity labeling of DAF-16 FOXO highlights aging regulatory proteins Artan, Murat ; https://orcid.org/0000-0001-8945-6992 Schön, Hanna De Bono, Mario ; https://orcid.org/0000-0001-8347-0443 ddc:570 Insulin/insulin-like growth factor signaling inhibits FOXO transcription factors to control development, homeostasis, and aging. Here, we use proximity labeling to identify proteins interacting with the C. elegans FOXO DAF-16. We show that in well-fed, unstressed animals harboring active insulin signaling, DAF-16 forms a complex with the PAR-1/MARK serine/threonine kinase, a key regulator of cell polarity. PAR-1 inhibits DAF-16 accumulation and promotes DAF-16 phosphorylation at S249, at a conserved motif that PAR-1/human MARK2 phosphorylates in vitro. DAF-2 insulin-like receptor signaling stimulates DAF-16 S249 phosphorylation, suggesting DAF-2 activates PAR-1. DAF-2 also promotes PAR-1 expression by inhibiting DAF-16. PAR-1 knockdown, or DAF-16 S249A, prolong lifespan, whereas phosphomimetic DAF-16 S249D suppresses the longevity of daf-2 mutants. At low insulin signaling, DAF-16 proximity labeling highlights transcription factors, chromatin regulators, and DNA repair proteins. One interactor, the zinc finger/homeobox protein ZFH-2/ZFHX3, forms a complex with DAF-16 and prolongs lifespan. Our work provides entry points for hypothesis-driven studies of FOXO function and longevity. Springer Nature 2025 info:eu-repo/semantics/article doc-type:Article Article http://purl.org/coar/resource_type/c_2df8fbb1 https://research-explorer.ista.ac.at/record/20929 https://research-explorer.ista.ac.at/download/20929/20941 Artan M, Schön H, de Bono M. Proximity labeling of DAF-16 FOXO highlights aging regulatory proteins. <i>Nature Communications</i>. 2025;16. doi:<a href="https://doi.org/10.1038/s41467-025-66409-0">10.1038/s41467-025-66409-0</a> eng info:eu-repo/semantics/altIdentifier/doi/10.1038/s41467-025-66409-0 info:eu-repo/semantics/altIdentifier/e-issn/2041-1723 info:eu-repo/semantics/altIdentifier/pmid/41381452 info:eu-repo/semantics/openAccess