Abstract
Insulin action on the skeleton is essential for bone development and whole-body energy metabolism, however a global view of signaling in this tissue is lacking. Furthermore, whether there are signaling differences that drive the gene-specific activation under insulin-resistant (IR) or ageing conditions is unknown. Here, we perform a phosphoproteomic analysis of insulin signaling in the bones of young, lean, insulin-sensitive versus old, obese, IR mice revealing a rewiring of phosphorylation. We target dysregulated phosphoproteins in a zebrafish functional genomic screen of bone development and mineralization revealing candidates important for skeletal formation. One of these is ALF Transcription Elongation Factor 4 (AFF4), the core scaffold of the Super Elongation Complex and we show that phosphorylation of S831 on AFF4 is an insulin-dependent substrate of P70S6K and attenuated in aged, IR bone. Phosphorylation of S831 is defective in IR osteoblasts and associated with reduced transcriptional elongation at discrete locations in the genome. Mechanistically, we show phosphorylation of S831 increases recruitment of chromatin remodelers, ENL/AF9 to crotonylated histone via the YEATS domain, and promotes gene-specific activation. Our analysis identifies regulators of insulin action on the skeleton, further uncovering a mechanism of IR via locus-specific changes in transcriptional elongation and gene activation.
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Data availability
The phosphoproteomics and proteomics data generated in this study are deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org/cgi/GetDataset) via the PRIDE113 and can be accessed through the links provided below: PRIDE: PXD054205 (Phosphoproteomic and proteomics of insulin signaling in aged mouse bone). PXD054212 (Zebrafish caudal fin phosphoproteomic of Rps6kb1a/b knockdown). PXD054247 (Analysis of insulin-regulated phosphorylation of AFF4 with Akti or S6Ki and S6K in vitro kinase assay). PXD054250 Affinity purification – mass spectrometry of AFF4 WT or S829/S831/3/5/8 A mutant). PXD054479(Proteomic and secretomic analysis of Kusa 4B10 osteoblasts). The targeted proteomic data can be accessed via Panorama Web Repository114 through the accession code Panorama Web: U of Melbourne – Parker Lab: PRM of mouse AFF4 S831 phosphorylation (Targeted phosphoproteomics of mouse S831 AFF4 phosphorylation in control or insulin resistant osteoblasts). The transcriptomic dataset generated in this study is deposited to NCBI and can be accessed through the accession code below: PRJNA1146056 (Transcriptomics of HEK293T cells expressing AFF4-wild type or AFF4-T829/S831/S833/S834/S835A mutant treated with or without insulin). Full list of Deposited Data is also provided in Supplementary Table 1. Source data are provided with this paper.
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Acknowledgements
We thank Nicholas Williamson, Ching-Seng Ang, Shuai Nie, Swati Varshney and Michael Leeming for instrument support in the Bio21 Mass Spectrometry and Proteomics Facility. We thank Mr. Cameron Mackey and Mr. Bryan Ko for providing technical support for zebrafish maintenance. This research was supported by access to the Melbourne Mouse Metabolic Phenotyping Platform at The University of Melbourne. This work was funded by a University of Melbourne Driving Research Momentum Grant, an NHMRC Emerging Leader Investigator Grant APP2009642 and Australian Research Council Discovery Grant DP250100201 to B.L.P. G.T.D is funded by NHMRC Grants 2022/GNT2021126, 2020/GNT2002427, 2018/GNT1160043, Australian Research Council Grant DP220102910, Diabetes Australia Grants Y23G-DodG, Y20G-DodG and The University of Melbourne Deans Innovation Award. S.J.V. is supported by a CSL Centenary Fellowship and SNOW Medical Fellowship. We thank Prof Benjamin M. Hogan and Mr. Scott Paterson from the Peter MacCallum Cancer Center, Australia for assistance with the zebrafish functional screen. We thank Dr Marco Tarasco from The University of Algarve, Portugal, for designing the ZFBONE brightfield ImageJ macro used in the zebrafish bone mineralization analysis.
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B.L.P. conceptualized the study. M.D., L.L., R.B., A.C., A.B.-G., L.D., I.S.R., N.K.Y.W., V.R.H., V.U., J.P.H.W., B.L.P. performed the experiments. M.D., L.L., H.J.K., R.B., A.C., H.K., A.B.-G., J.M., P.Y., B.L.P. analyzed the data. G.S.L., K.A.S., M.K.M., M.J.W., G.T.D., S.J.V., N.A.S., B.L.P provided resources, guided experimental design, supervised and funded the research.
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Dutt, M., Liao, L., Kim, H.J. et al. Phosphoproteomics of aged insulin-resistant bone identifies P70S6K phosphorylation of AFF4 as a gene-specific transcriptional regulator. Nat Commun (2025). https://doi.org/10.1038/s41467-025-68106-4
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DOI: https://doi.org/10.1038/s41467-025-68106-4
