Functional analysis of human protein phosphorylation sites
December 15 @ 16:00 – 17:00 CET
Matthias Selbach
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.
Selected publications
- Vieira-Vieira, CH, Dauksaite, V., Sporbert, A., Gotthardt, M. and Selbach M. (2022) Proteome-wide quantitative RNA-interactome capture identifies phosphorylation sites with regulatory potential in RBM20. Mol Cell 82, 2069-2083.
- Meyer, K., Kirchner, M., Uyar, B., Cheng, J.Y., Russo, G., …, and Selbach, M. (2018). Mutations in Disordered Regions Can Cause Disease by Creating Dileucine Motifs. Cell 175, 239-253.
- Imami, K., Milek, M., Bogdanow, B., Yasuda, T., Kastelic, N., …, and Selbach, M. (2018). Mol Cell. Phosphorylation of the Ribosomal Protein RPL12/uL11 Affects Translation during Mitosis. Mol Cell 72,
- Zauber, H., Kirchner, M., and Selbach, M. (2018). Picky: a simple online PRM and SRM method designer for targeted proteomics. Nat Methods 15, 156-157.
- McShane, E., Sin, C., Zauber, H, Wells JN, Donnelly N, …, and Selbach M. (2016). Kinetic analysis of protein stability reveals age-dependent degradation. Cell 167, 803-815.
- Schwanhausser, B., Busse, D., Li, N., Dittmar, G., Schuchhardt, J., Wolf, J., Chen, W., and Selbach, M. (2011). Global quantification of mammalian gene expression control. Nature 473, 337-342.
janne.lehtio@scilifelab.se
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