Dynamic Control of Signaling by Phosphorylation of PDZ Binding Motifs.

The dynamic regulation of protein-protein interactions (PPIs) involves phosphorylation of short liner motifs in disordered protein regions modulating binding affinities. The ribosomal-S6-kinase 1 is capable of binding to scaffold proteins containing PDZ domains through a PDZ-binding motif (PBM) located at the disordered C-terminus of the kinase. Phosphorylation of the PBM dramatically changes the interactome of RSK1 with PDZ domains exerting a fine-tuning mechanism to regulate PPIs. Here we present in detail highly effective biophysical (fluorescence polarization, isothermal calorimetry) and cellular (protein-fragment complementation) methods to study the effect of phosphorylation on RSK1-PDZ interactions that can be also applied to investigate phosphoregulation of other PPIs in signaling pathways.