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Proteome‐Wide Analysis of Phospho‐Regulated PDZ Domain Interactions

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Proteome‐Wide Analysis of Phospho‐Regulated PDZ Domain Interactions Published online: August 20, 2018 Method Proteome-wide analysis of phospho-regulated PDZ domain interactions Gustav N Sundell1, Roland Arnold2,*, Muhammad Ali1 , Piangfan Naksukpaiboon2, Julien Orts3, Peter Güntert3,4, Celestine N Chi5,** & Ylva Ivarsson1,*** Abstract Introduction A key function of reversible protein phosphorylation is to regulate Reversible protein phosphorylation is crucial for regulation of cellu- protein–protein interactions, many of which involve short linear lar processes and primarily occurs on Ser, Thr, and Tyr residues in motifs (3–12 amino acids). Motif-based interactions are difficult to eukaryotes (Seet et al, 2006). Phosphorylation may have different capture because of their often low-to-moderate affinities. Here, functional effects on the target protein, such as inducing conforma- we describe phosphomimetic proteomic peptide-phage display, tional changes, altering cellular localization, or enabling or disabling a powerful method for simultaneously finding motif-based interaction sites. Hundreds of thousands of such phosphosites have interaction and pinpointing phosphorylation switches. We compu- been identified in different cell lines and under different conditions tationally designed an oligonucleotide library encoding human (Olsen et al, 2006; Hornbeck et al, 2015). An unresolved question is C-terminal peptides containing known or predicted Ser/Thr phos- which of these phosphosites are of functional relevance and not phosites and phosphomimetic variants thereof. We incorporated background noise caused by the off-target activity of kinases these oligonucleotides into a phage library and screened the PDZ revealed by the high sensitivity in the mass spectrometry analysis. (PSD-95/Dlg/ZO-1) domains of Scribble and DLG1 for interactions So far, only a minor fraction of identified phosphosites has been potentially enabled or disabled by ligand phosphorylation. We linked to functional effects, and only 35% of phosphosites are identified known and novel binders and characterized selected evolutionarily conserved, which would support that they play non- interactions through microscale thermophoresis, isothermal titra- redundant functional roles (Landry et al, 2009). Sifting functional tion calorimetry, and NMR. We uncover site-specific phospho-regu- from non-functional phosphosites by experimental approaches is lation of PDZ domain interactions, provide a structural framework thus of fundamental importance in order to better understand the for how PDZ domains accomplish phosphopeptide binding, and function of the phosphoproteome. discuss ligand phosphorylation as a switching mechanism of PDZ Phosphosites are often located in the intrinsically disordered domain interactions. The approach is readily scalable and can be regions of proteins, which in eukaryotes are estimated to cover 30– used to explore the potential phospho-regulation of motif-based 40% of the populated protein sequence space (Ward et al, 2004). interactions on a large scale. These regions are enriched in short linear motifs (SLiMs, 3–12 amino acids) that are recognized by modular domains (Van Roey Keywords PDZ domain; phage display; phosphorylation; protein–protein et al, 2014). Discrete phosphorylation events may regulate and tune interaction; Scribble the strength of interactions. Phosphopeptide binding domains, such Subject Categories Methods & Resources; Post-translational Modifications, as the 14-3-3 proteins, serve as readers of this phosphorylation code Proteolysis & Proteomics (Yaffe et al, 1997; Reinhardt & Yaffe, 2013). Deciphering the phos- DOI 10.15252/msb.20178129 | Received 29 November 2017 | Revised 24 July phorylation code by linking the reader domains to their preferred 2018 | Accepted 24 July 2018 binding phosphosites is a major challenge. Adding to the complex- Mol Syst Biol. (2018) 14:e8129 ity, phosphorylation of a SLiM may have a switch-like effect, making the interaction stronger (enabling) with a given domain, while weakening interactions (disabling) with other domains (Van Roey et al, 2013), in other worlds regulating interactions. Such a switch-like mechanism was recently found to regulate interactions 1 Department of Chemistry – BMC, Uppsala University, Uppsala, Sweden 2 Institute of Cancer and Genomic Sciences, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, UK 3 Laboratory of Physical Chemistry, ETH Zürich, Zürich, Switzerland 4 Institute of Biophysical Chemistry, Goethe University, Frankfurt am Main, Germany 5 Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden *Corresponding author. Tel: +44 7936624996; E-mail: [email protected] **Corresponding author. Tel: +46 18 471 4557; E-mail: [email protected] ***Corresponding author. Tel: +46 18 471 40 38; E-mail: [email protected] ª 2018 The Authors. Published under the terms of the CC BY 4.0 license Molecular Systems Biology 14:e8129 | 2018 1 of 22 Published online: August 20, 2018 Molecular Systems Biology Phosphomimetic ProP-PD Gustav N Sundell et al of the C-terminal region of PRTM5 with 14-3-3 proteins and PDZ known or predicted phosphorylation sites, and the phosphomimetic (PSD-95/Discs-large/ZO-1) domains. Phosphorylation of the C-term- mutants of these peptides. Second, we use this library in selections inal tail of PRTM5 enables 14-3-3 interactions, while disabling PDZ against the six PDZ domains of human Scribble and DLG1, well- domain interactions (Espejo et al, 2017). Although less common, characterized PDZ proteins with crucial roles in the postsynaptic phosphorylation switches also operate on the interaction interfaces density of excitatory neuronal synapses and in the establishment of folded proteins, hundreds of which were suggested in recent and maintenance of epithelial cell polarity (Humbert et al, 2008; analysis (Betts et al, 2017). Feng & Zhang, 2009). Third, we successfully identify known and There is a paucity of information linking phosphorylation novel interactions, with potential to be regulated by Ser/Thr phos- enabled/disabled SLiMs to their binding partners, in part due to a phorylation, and uncover that Scribble PDZ interactions are enabled lack of suitable experimental methods. Although affinity purifica- by p-3 phosphorylation. Finally, we provide the structural basis of tion coupled to mass spectrometry (AP-MS) can be used to obtain such phosphopeptide binding, expand the analysis to an additional information on phosphorylation states and interactions dynami- nine domains, and discuss the role of Ser/Thr phosphorylation as a cally, the approach does not provide information with resolution at switching mechanism of PDZ domain interactions. In this proof-of- the level of the binding sites. In addition, interactions that rely on concept study, we thus demonstrate that phosphomimetic ProP-PD SLiMs are often elusive to AP-MS as they typically are of low-to- is a powerful approach to identify SLiM-based interactions in the medium affinities, and exhibit rapid association/dissociation kinet- human proteome that are regulated by Ser/Thr phosphorylation. ics. Phosphopeptide libraries can be used to establish preferred The approach is readily scalable and can be used to explore the binding motifs of phosphopeptide binding domains, but the potential phospho-regulation of human protein–protein interactions number of sequences presented in these experiments is typically on a larger scale. limited (Yaffe & Smerdon, 2004). In addition, Grossman and co- workers recently showed the use of yeast-two-hybrid (Y2H) for capturing phospho-Tyr-dependent interactions (Grossmann et al, Results 2015), but phospho-dependent interactions are otherwise typically not captured by Y2H. Thus, there is an urge for novel large-scale Construction of a phosphomimetic ProP-PD library methods for charting phosphorylation-dependent SLiM-based inter- actions. To design a specific phage display library for phosphomimetic Here, we describe a novel large-scale approach termed phospho- ProP-PD, we scanned the human proteome for C-terminal regions mimetic proteomic peptide-phage display (phosphomimetic ProP- containing known or predicted Ser/Thr phosphorylation sites PD) developed to identify SLiM-based interactions that are regulated based on phosphorylation databases and in silico predictions (see by Ser/Thr phosphorylation. Phosphomimetic ProP-PD is a further Materials and Methods). We identified 4,828 unique C-terminal development of ProP-PD, which is a dedicated method for the large- peptides of human full-length proteins that contain one or multi- scale identification of SLiM-based interactions (Ivarsson et al, 2014; ple potential Ser/Thr phosphosites in their last nine amino acids. Davey et al, 2017; Wu et al, 2017). We demonstrate that phospho- We designed the phosphomimetic ProP-PD library to comprehen- mimetic ProP-PD is a straightforward approach for finding ligands sively contain wild-type sequences and phosphomimetic mutants of SLiM-binding domains and for simultaneously pinpointing Ser/ (Ser/Thr to Glu) thereof (7,626 phosphomimetic sequences, Thr phosphorylation events with potential to enable or disable including combinations of multiple phosphorylation sites in one interactions. We showcase the approach by identifying phospho- C-terminus), in order to allow the detection of interactions that regulated interactions of PDZ (PSD-95/Dlg/ZO-1) domains, which are enabled or disabled by phosphomimetic mutations. The are among the most frequent interaction modules in
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