Rewiring of RSK–PDZ Interactome by Linear Motif Phosphorylation

Abstract

Phosphorylation of short linear peptide motifs is a widespread process for the dynamic regulation of protein-protein interactions. However, the global impact of phosphorylation events on the proteinprotein interactome is rarely addressed. The disordered C-terminal tail of ribosomal S6 kinase 1 (RSK1) binds to PDZ domain-containing scaffold proteins, and it harbors a phosphorylatable PDZ binding motif (PBM) responsive to epidermal growth factor (EGF) stimulation. Here, we examined binding of two versions of the RSK1 PBM, either phosphorylated or unphosphorylated at position-3, to almost all (95%) of the 266 PDZ domains of the human proteome. PBM phosphorylation dramatically altered the PDZ domain-binding landscape of RSK1, by strengthening or weakening numerous interactions to various degrees. The RSK-PDZome interactome analyzed in this study reveals how linear motif-based phospho-switches convey stimulus-dependent changes in the context of related network components.

Interactomic affinity profiling by holdup assay: acetylation and distal residues impact the PDZome-binding specificity of PTEN phosphatase

“…Such affinity variations may result from differences of entropy of the free peptides, from altered interface contacts in the resulting PDZ-PBM complexes, or a combination of both. Accordingly, synthetic or recombinant PBMs employed for PDZ interactions generally include at least 9 to 11 residues [4], [5], [15], [17], [28], [50]. Indeed, the presence of distal sites altering PDZ-PBM binding has already been described [51], even at positions as far as at p-36 [52].…”

“…a list of binding strengths in decreasing order exhibited by a given PBM towards the entire PDZome. The high accuracy and efficiency of the holdup assay has been validated previously [4], [15], [28], [30]. Very recently, a manual version of the holdup assay with purified samples and using widespread benchtop equipment has been implemented and has proven to be reliable [31].…”

High throughput competitive fluorescence polarization assay reveals functional redundancy in the S100 protein family

“…Competitive FP, on the other hand, shares the same properties but without any possible interference from the labeling dye. Moreover, it provides comparative results to other, orthogonal, usually low-throughput, label-free biochemical assays, such as ITC or SPR measurements [33]. In summary, competitive FP assay is robust and HTP, thus it is a valuable tool for screening macromolecular interactions involving linear peptide motifs, RNA/DNA oligonucleotides or fluorescent small molecules [34,35].…”

“…Peptide synthesis. The CapZ (265-276), NCX1 (254-265), SIP (188-202), TRPM4 (129-147) and MDM4(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43) peptides were chemically synthesized using solid phase peptide synthesis (PS3 peptide synthesizer, Protein Technologies) with Fmoc/tBu strategy in the case of (5(6)-carboxyfluorescein) labeled and unlabeled version. Peptides were purified by RP-HPLC using a Jupiter 300 Å C 18 column (Phenomenex).…”

Structural characterization and computational analysis of PDZ domains in Monosiga brevicollis

“…[36][37][38] In addition, a number of elegant studies using directed evolution, or other protein engineering techniques, have successfully identified structural elements that determine PDZ selectivity-often through only a small number of amino acid substitutions or post-translational modifications. [39][40][41][42][43] The elucidation of PDZ binding selectivity has enabled investigators to trace the evolution of PDZ specificity throughout the tree of life, including in bacteria, yeast, and plants. 18,42,44 However, what remains to be determined is whether or not the selectivity determinants in PDZ domains related by evolution are also conserved, despite different signaling pathways, for example, in uni-versus multicellular organisms, or those with and without a nervous system.…”