Abstract
Phosphorylation is a ubiquitous post-translation modification that regulates protein function by promoting, inhibiting or modulating protein–protein interactions. Hundreds of thousands of phosphosites have been identified but the vast majority have not been functionally characterised and it remains a challenge to decipher phosphorylation events modulating interactions. We generated a phosphomimetic proteomic peptide-phage display library to screen for phosphosites that modulate short linear motif-based interactions. The peptidome covers ~13,500 phospho-serine/threonine sites found in the intrinsically disordered regions of the human proteome. Each phosphosite is represented as wild-type and phosphomimetic variant. We screened 71 protein domains to identify 248 phosphosites that modulate motif-mediated interactions. Affinity measurements confirmed the phospho-modulation of 14 out of 18 tested interactions. We performed a detailed follow-up on a phospho-dependent interaction between clathrin and the mitotic spindle protein hepatoma-upregulated protein (HURP), demonstrating the essentiality of the phospho-dependency to the mitotic function of HURP. Structural characterisation of the clathrin-HURP complex elucidated the molecular basis for the phospho-dependency. Our work showcases the power of phosphomimetic ProP-PD to discover novel phospho-modulated interactions required for cellular function.
Originalsprog | Engelsk |
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Artikelnummer | e11164 |
Tidsskrift | Molecular Systems Biology |
Vol/bind | 19 |
Udgave nummer | 7 |
Antal sider | 21 |
ISSN | 1744-4292 |
DOI | |
Status | Udgivet - 2023 |
Bibliografisk note
Funding Information:This work was supported by the grants from the Swedish Research Council (YI: 2020‐03380), the Ollie and Elof Ericsson foundation (YI), the Carl Trygger foundation (DD) and the Cancer Research UK (CRUK) (NED: Senior Cancer Research Fellowship C68484/A28159). Sequencing was performed by the SNP&SEQ Technology Platform in Stockholm. The facility is part of the National Genomic Infrastructure (NGI) Sweden and Science for Life Laboratory and is also supported by the Swedish Research Council and the Knut and Alice Wallenberg. Work at the Novo Nordisk Foundation Center for Protein Research is supported by grant NNF14CC0001. We thank Dr. Pedro Beltrao and Dr. David Ochoa for useful discussions on the library design. We thank Dr. Sachdev Sidhu for providing the phagemid and several of the expression constructs.
Publisher Copyright:
© 2023 The Authors. Published under the terms of the CC BY 4.0 license.