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 (proteinfragment 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.
