Rewiring of RSK-PDZ interactions by linear motif phosphorylation

Abstract

Protein phosphorylation is a key regulator of protein-protein interactions. How does the interactome of a protein change during extracellular stimulations? While many individual examples of phosphorylation-regulated interactions were described previously, studies addressing the interactome changes induced by a particular phosphorylation event remain scarce. Here, we try to answer this question, by focusing on interactions between a phosphorylable PDZ-binding linear motif and the entire complement of human PDZ domains. Using a combination of in vitro quantitative techniques and cell-based approaches, we demonstrate that the activation of the mitotic effector kinase RSK1 causes dramatic changes in its connectivity with PDZ domain containing proteins. These changes consist of modulations of the binding affinity of numerous interactions, rather than on/off switching of a few interactions. Our results highlight the previously unappreciated role of phosphorylation in the complex and subtle rewiring of large numbers of protein-protein interactions.

Quantitative fragmentomics allow affinity mapping of interactomes

“…Upon phosphorylation at p-3, RPS6KA1 gains a negative charge at this position, thereby getting closer to SYNJ2BP's logo. Accordingly, the p-3 phosphorylated variant RPS6KA1 gains affinity to SYNJ2BP (pK d = 4.48; ΔppK d = 0.66; Figure 3D), as previously published (Gógl et al, 2019). The structure of p-3 phosphorylated RPS6KA1 bound to SYNJ2BP explains this enhanced binding by revealing a network of specific phosphoryl-PDZ contacts (Figure 3E).…”

“…Most LYSC data originate from previous articles (Vincentelli et al, 2015) (Gógl et al, 2019) (Gogl et al, 2020) (Jané et al, 2020) (Genera et al, 2021) (Giraud et al, 2021. Additional LYSC holdup experiments were carried out as previously described, using only the original layout.…”

“…To explore the PDZ-PBM interactome, we expressed a recombinant PDZome library covering all the 266 known human PDZs (Duhoo, Girault et al, 2019), and we synthetized a 10-mer peptide library of 24 viral and 424 human PBMs, of which 323, 63, 51, and 11 belong to class 1, 2, 3, and to atypical or non-C-terminal subgroups, respectively (Table S1). 8 PBMs harboured post-translational modifications (phosphorylation or acetylation) which may modulate binding specificities (Gógl et al, 2019) (Gogl et al, 2020) (Jané et al, 2020). The viral PBMs included notably 12 PBMs from oncoproteins HPV E6 (11 distinct types) and HTLV1 TAX1.…”

“…PBMs are mostly (though not exclusively) C-terminal, with their COOimplicated in binding, and are classified based on position -2, being respectively Ser/Thr, hydrophobic or acidic in classes 1, 2, and 3 (Stiffler et al, 2007) (Tonikian et al, 2008). PDZ-PBM interactions are rather transient and promiscuous (Vincentelli et al, 2015) (Gógl et al, 2019) (Gogl et al, 2020) (Jané et al, 2020).…”