Functional analysis of human protein phosphorylation sites
December 15 @ 16:00 – 17:00 CET
Phosphoproteomics routinely quantifies changes in the levels of thousands of phosphorylation sites, but functional analysis of such data remains a major challenge. I will present three different ways to characterise the function of human phosphorylation sites. First, I will show how data from in vitro kinase assays can be used to predict kinase activity in phosphoproteomic datasets. Second, I will show quantitative affinity purification experiments with synthetic phosphopeptides can help to assess their cellular function. Finally, I will outline how quantitative RNA-interactome capture (qRIC) can quantify the fraction of cellular RNA-binding proteins that are pulled down with polyadenylated mRNAs. Combining qRIC with phosphoproteomics allows us to systematically compare pull-down efficiencies of phosphorylated and non-phosphorylated forms of RBPs. Using qRIC, we identify over hundred phosphorylation sites with regulatory potential, including known regulatory sites. Follow-up experiments on the cardiac splicing regulator RBM20 revealed that multiple phosphorylation sites in the C-terminal disordered region affect nucleo-cytoplasmic localisation, association with cytosolic RNA granules and alternative splicing.
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