often requires fine-tuning, resulting in minor alterations in thermodynamics and kinetics to regulate cellular processes more effectively
[3, 12]. Our recent study provided evidence that in the PDZ
interactome of RSK1, phosphorylation does not usually act as
hard switch, but its effect rather can be described as a fine-tuning
regulatory mechanism (dimming) [13, 14].
Although phosphorylation events usually involve drastic
changes in the interactome, PDZ interactions are mostly considered weak, in the μM range, therefore the effects triggered by
phosphorylation are subtle. To measure such small effects on
weak interactions with high precision, robust and sensitive methods
are required. Thus, these interactions should be characterized
either by robust high-throughput (HTP) methods such as Holdup
assay [15], surface plasmon resonance [16], micro-scale thermophoresis [10] and fluorescence polarization (FP) [14]; or with
accurate low-throughput structural/biophysical methods such as
isothermal titration calorimetry (ITC) [9], nuclear magnetic resonance spectroscopy [17], X-ray diffraction [9]; or to be closer to
the in vivo system, with cell-based approaches such as coimmunoprecipitation [18], proximity ligation assay [19], or proteinfragment complementation assay (PCA) [14].
Here we present a detailed workflow to investigate the impact
of phosphorylation on RSK1-PDZ domain interactions as a proof
of the concept for studying phosphorylation regulation of signaling
pathways. This chapter covers in vitro biochemical and biophysical
methods using isolated PDZ domains and RSK1 peptides as well as
a cell-based approach using full length PDZ domains and RSK1 to
extensively characterize the effect of phosphorylation of PBMs on
the PDZ interactome. We chose to describe in detail FP assays since
it is a high-throughput, simple, sensitive, and cost-effective method
to obtain dissociation constants of PPIs. ITC, a robust biophysical
method, which provides more thermodynamic data, and a novel
cell-based protein-fragment complementation assay (NanoBit)
were chosen mainly because they are effective validation methods
for HTP data acquired by FP measurements. The scheme of RSK1
and their peptides used for the experiments is shown in Fig. 1.
2 Materials
2.1 In Vitro
Phosphorylation
of RSK1 683–735
1. Phosphorylation buffer (buffer A): 150 mM NaCl, 5 mM
MgCl2, 100 μM TCEP, 20 mM HEPES–NaOH pH 7.5. Dissolve 0.88 g NaCl, 0.48 g HEPES, and 48 mg MgCl 2 in 95 ml
Milli-Q water. Add 20 μl 0.5 M TCEP solution, then fill the
volume to 100 ml and adjust the pH to 7.5 with sodium
hydroxide solution. Store at À20
C in 10 ml aliquots.
180
Ma ´ rton A. Simon and La ´ szlo ´ Nyitray
[3, 12]. Our recent study provided evidence that in the PDZ
interactome of RSK1, phosphorylation does not usually act as
hard switch, but its effect rather can be described as a fine-tuning
regulatory mechanism (dimming) [13, 14].
Although phosphorylation events usually involve drastic
changes in the interactome, PDZ interactions are mostly considered weak, in the μM range, therefore the effects triggered by
phosphorylation are subtle. To measure such small effects on
weak interactions with high precision, robust and sensitive methods
are required. Thus, these interactions should be characterized
either by robust high-throughput (HTP) methods such as Holdup
assay [15], surface plasmon resonance [16], micro-scale thermophoresis [10] and fluorescence polarization (FP) [14]; or with
accurate low-throughput structural/biophysical methods such as
isothermal titration calorimetry (ITC) [9], nuclear magnetic resonance spectroscopy [17], X-ray diffraction [9]; or to be closer to
the in vivo system, with cell-based approaches such as coimmunoprecipitation [18], proximity ligation assay [19], or proteinfragment complementation assay (PCA) [14].
Here we present a detailed workflow to investigate the impact
of phosphorylation on RSK1-PDZ domain interactions as a proof
of the concept for studying phosphorylation regulation of signaling
pathways. This chapter covers in vitro biochemical and biophysical
methods using isolated PDZ domains and RSK1 peptides as well as
a cell-based approach using full length PDZ domains and RSK1 to
extensively characterize the effect of phosphorylation of PBMs on
the PDZ interactome. We chose to describe in detail FP assays since
it is a high-throughput, simple, sensitive, and cost-effective method
to obtain dissociation constants of PPIs. ITC, a robust biophysical
method, which provides more thermodynamic data, and a novel
cell-based protein-fragment complementation assay (NanoBit)
were chosen mainly because they are effective validation methods
for HTP data acquired by FP measurements. The scheme of RSK1
and their peptides used for the experiments is shown in Fig. 1.
2 Materials
2.1 In Vitro
Phosphorylation
of RSK1 683–735
1. Phosphorylation buffer (buffer A): 150 mM NaCl, 5 mM
MgCl2, 100 μM TCEP, 20 mM HEPES–NaOH pH 7.5. Dissolve 0.88 g NaCl, 0.48 g HEPES, and 48 mg MgCl 2 in 95 ml
Milli-Q water. Add 20 μl 0.5 M TCEP solution, then fill the
volume to 100 ml and adjust the pH to 7.5 with sodium
hydroxide solution. Store at À20
C in 10 ml aliquots.
180
Ma ´ rton A. Simon and La ´ szlo ´ Nyitray