Chapter 11
Dynamic Control of Signaling by Phosphorylation of PDZ
Binding Motifs
Ma ´ rton A. Simon and La ´ szlo ´ Nyitray
Abstract
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.
Key words PDZ domain, Fluorescence polarization, Isothermal titration calorimetry, Bimolecular
fragment complementation assay, NanoBiT, Protein–protein interaction
1 Introduction
Dynamic regulation of protein–protein interactions (PPIs) is essential for maintaining cellular physiology [1, 2]. One of the key
mechanisms for such regulation, reversible phosphorylation, frequently occurs in disordered protein regions and can alter interactions with structured domains, controlling the functional network
of the proteome [3–5]. An example of this scenario, the globular
PDZ (PSD95/DLG1/ZO-1) domains interact with disordered
binding regions, called PDZ-binding motifs (PBMs) that are usually located at the disordered C-terminus of their targets [6–8]. The
interactions between PDZ domains and their partners containing
the PBM are affected by phosphorylation events, exerting a
dynamic control of signaling pathways [9, 10].
Phosphorylation is usually considered as a binary chemical
switch between the phosphorylated and unphosphorylated states,
enabling or disabling PPIs thus resulting in drastic changes in
binding affinities [11]. However, controlling signaling pathways
Jean-Paul Borg (ed.), PDZ Mediated Interactions: Methods and Protocols, Methods in Molecular Biology, vol. 2256,
https://doi.org/10.1007/978-1-0716-1166-1_11, © Springer Science+Business Media, LLC, part of Springer Nature 2021
179
Dynamic Control of Signaling by Phosphorylation of PDZ
Binding Motifs
Ma ´ rton A. Simon and La ´ szlo ´ Nyitray
Abstract
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.
Key words PDZ domain, Fluorescence polarization, Isothermal titration calorimetry, Bimolecular
fragment complementation assay, NanoBiT, Protein–protein interaction
1 Introduction
Dynamic regulation of protein–protein interactions (PPIs) is essential for maintaining cellular physiology [1, 2]. One of the key
mechanisms for such regulation, reversible phosphorylation, frequently occurs in disordered protein regions and can alter interactions with structured domains, controlling the functional network
of the proteome [3–5]. An example of this scenario, the globular
PDZ (PSD95/DLG1/ZO-1) domains interact with disordered
binding regions, called PDZ-binding motifs (PBMs) that are usually located at the disordered C-terminus of their targets [6–8]. The
interactions between PDZ domains and their partners containing
the PBM are affected by phosphorylation events, exerting a
dynamic control of signaling pathways [9, 10].
Phosphorylation is usually considered as a binary chemical
switch between the phosphorylated and unphosphorylated states,
enabling or disabling PPIs thus resulting in drastic changes in
binding affinities [11]. However, controlling signaling pathways
Jean-Paul Borg (ed.), PDZ Mediated Interactions: Methods and Protocols, Methods in Molecular Biology, vol. 2256,
https://doi.org/10.1007/978-1-0716-1166-1_11, © Springer Science+Business Media, LLC, part of Springer Nature 2021
179