Lightning Talk & Poster 28th Annual Lorne Proteomics Symposium 2023

Functional phosphoproteomic analysis of insulin signalling in ageing bone (#18)

Mriga Dutt 1 , Luoping Liao 1 , Hani Jieun KIm 2 3 , Ronnie Blazev 1 , Audrey Chan 1 , Natalie K.Y. Wee 4 , Jeffrey Molendijk 1 , Vanessa R. Haynes 1 , Yaan-KIt Ng 1 , Veronica U. Sokolov 1 , Kelly A. Smith 1 , Pengyi Yang 2 3 5 , Garron Dodd 1 , Gordon Lynch 1 , Natalie Sims 4 , Benjamin Parker 1
  1. Department of Anatomy and Physiology, Faculty of Medicine, School of Biomedical Sciences, University of Melbourne, Parkville, VIC, Australia
  2. Computational Systems Biology Group, Children's Medical Research Institute, Westmead, NSW, 2145, Australia
  3. School of Mathematics and Statistics, University of Sydney, Sydney, NSW, Australia
  4. St. Vincent's Institute of Medical Research and Department of Medicine at St. Vincent's Hospital, University of Melbourne, Parkville, VIC, 3010
  5. Charles Perkin Centre, University of Sydney, Sydney, NSW, Australia

Insulin signalling in bone plays a critical role in development and the regulation of energy metabolism. However, a systems biology analysis to map in vivo signalling has yet to be performed. Furthermore, whether signalling is rewired during ageing and insulin-resistance is unknown. Here we present the first mouse bone phosphoproteome study of 8- and 73-week-old mice following acute in vivo insulin stimulation and identified >16,000 phosphorylation sites mapped to 4528 bone phosphoproteins, of which >4,600 sites are novel. Hundreds of phosphorylation sites were differentially regulated between young and old bone revealing dramatic rewiring and defects in insulin signalling. Kinase:substrate prediction using machine learning coupled to phosphosite evolutionary conservation analysis and integration with human bone mineral density GWAS enabled us to prioritise novel proteins containing differential phosphorylation and highly likely to play important roles in bone function. We next developed a CRISPR/Cas9 loss-of-function screening pipeline in zebrafish to assess the role of these proteins on bone development. This identified several novel insulin-regulated phosphoproteins as causal regulators of bone formation including AFF4, the core scaffold of the transcriptional Super Elongation Complex (SEC). Using targeted phosphoproteomics and affinity-purification coupled to mass spectrometry, we show that AFF4 is a novel substrate of nuclear-localised P70S6K, and phosphorylation regulates SEC formation. Furthermore, we show the activity of the SEC is defective under insulin-resistant conditions and is associated with reduced phosphorylation of AFF4 and an inability to engage efficient transcription of Immediate Early Genes. Taken together, we have defined in vivo defective insulin-signalling events in old mouse bone and identified novel functional phosphorylation events.