Proteome-wide base editor screens to assess phosphorylation site functionality in high-throughput
Kennedy, P. H.; Deh Sheikh, A. A.; Balakar, M.; Jones, A. C.; Olive, M. E.; Hedge, M.; Matias, M. I.; Pirete, N.; Burt, R.; Levy, J.; Little, T.; Hogan, P. G.; Liu, D. R.; Doench, J. G.; Newton, A. C.; Gottschalk, R. A.; de Boer, C.; Alarcon, S.; Newby, G.; Myers, S. A.
Show abstract
Signaling pathways that drive gene expression are typically depicted as having a dozen or so landmark phosphorylation and transcriptional events. In reality, thousands of dynamic post-translational modifications (PTMs) orchestrate nearly every cellular function, and we lack technologies to find causal links between these vast biochemical pathways and genetic circuits at scale. Here, we describe "signaling-to-transcription network" mapping through the development of PTM-centric base editing coupled to phenotypic screens, directed by temporally-resolved phosphoproteomics. Using T cell activation as a model, we observe hundreds of unstudied phosphorylation sites that modulate NFAT transcriptional activity. We identify the phosphorylation-mediated nuclear localization of the phosphatase PHLPP1 which promotes NFAT but inhibits NF{kappa}B activity. We also find that specific phosphosite mutants can alter gene expression in subtle yet distinct patterns, demonstrating the potential for fine-tuning transcriptional responses. Overall, base editor screening of PTM sites provides a powerful platform to dissect PTM function within signaling pathways.
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