Identification of Novel CK2 Kinase Substrates Using a Versatile Biochemical Approach

Summary

The objective of this protocol is to label, enrich, and identify substrates of protein kinase CK2 from a complex biological sample such as a cell lysate or tissue homogenate. This method leverages unique aspects of CK2 biology for this purpose.

Abstract

The study of kinase-substrate relationships is essential to gain a complete understanding of the functions of these enzymes and their downstream targets in both physiological and pathological states. CK2 is an evolutionarily conserved serine/threonine kinase with a growing list of hundreds of substrates involved in multiple cellular processes. Due to its pleiotropic properties, identifying and characterizing a comprehensive set of CK2 substrates has been particularly challenging and remains a hurdle in the study of this important enzyme. To address this challenge, we have devised a versatile experimental strategy that enables the targeted enrichment and identification of putative CK2 substrates. This protocol takes advantage of the unique dual co-substrate specificity of CK2 allowing for specific thiophosphorylation of its substrates in a cell or tissue lysate. These substrate proteins are subsequently alkylated, immunoprecipitated, and identified by liquid chromatography/tandem mass spectrometry (LC-MS/MS). We have previously used this approach to successfully identify CK2 substrates from Drosophila ovaries and here we extend the application of this protocol to human glioblastoma cells, illustrating the adaptability of this method to investigate the biological roles of this kinase in various model organisms and experimental systems.

Introduction

Protein kinases are key components of signal transduction cascades. Phosphorylation of substrate proteins by these enzymes elicits biological responses that regulate critical events controlling cell division, metabolism, and differentiation, among others. CK2 is a ubiquitously expressed, acidophilic serine/threonine kinase that is conserved from yeast to humans and that plays important roles in many cellular processes ranging from transcriptional regulation to cell cycle progression to apoptosis^1,2,3. The enzyme is a heterotetramer composed of two catalytic α (or α') subunits and two regulatory β subunits⁴. In addition to being highly pleiotropic, CK2 exhibits two other unusual characteristics that complicate its analysis, namely constitutive activity⁵ and dual co-substrate specificity⁶. This latter property endows CK2 with the ability to use GTP as well as ATP for phosphorylation of substrate proteins.

Genetic deletion of the catalytic or regulatory subunits of CK2 in mice results in embryonic lethality indicating that it plays crucial roles during development and organogenesis^7,8. CK2 is also overexpressed in several types of cancer and thus represents a promising therapeutic target^9,10,11. Indeed, specific inhibitors that target CK2 kinase activity are currently under investigation for this purpose^12,13,14. While inhibition of CK2 is a viable option, given its pleiotropic nature, an alternative and perhaps more rational approach would be to target critical CK2 substrates that underlie the progression of certain cancers. Therefore, the comprehensive identification and characterization of CK2 substrate proteins would be of significant benefit for elucidating the specific function(s) of this kinase within a particular tissue or tumor type.

Here, we describe a versatile biochemical method for identifying CK2 substrates from a complex biological sample such as a cell or tissue lysate. This protocol takes advantage of the dual co-substrate specificity of CK2 by use of the GTP analogue GTPγS (guanosine 5'-[γ-thio]triphosphate) that other endogenous kinases cannot use. This effectively allows the kinase to "label" its substrates within this sample for subsequent isolation and identification.

Protocol

NOTE: Ensure that the required materials are available and properly prepared (see Table of Materials).

1. Preparation

1. Mechanically lyse tissue sample (1-2 mg of tissue in 100 µL of lysis buffer, Table 1) or cultured cells (10 cm plate that is 80-90% confluent in 350 µL of lysis buffer), with the goal being to collect a total of 900 µL of sample for the experiment. Note that this volume is in slight excess of what is required for the experiment described below.
2. Spin down the samples by centrifugation at 17,500 x g for 3 minutes at 4 °C. Upon completion, transfer 270 µL of the supernatant to each of three new 1.7 mL microcentrifuge tubes. There will be approximately 90 µL remaining. Remove 40 µL to be used as an “input control” and place in a new tube. Place all samples on ice.

2. Kinase assay: thiophosphorylation and alkylation

1. Label the three tubes containing 270 µL each as follows: “kinase rxn”, “GTPγS only”, and “PNBM (p-nitrobezyl mesylate) only”. Prepare the kinase reactions.
   1. To the “kinase rxn” tube, add 2.7 µL (equivalent to 1,350 U) of CK2, then add 2.7 µL of 2.5 mM GTPγS.
   2. To the “GTPγS only” tube, add 2.7 µL of 2.5 mM GTPγS, and then add 2.7 µL of lysis buffer.
   3. To the “PNBM only” tube, add 5.4 µL of lysis buffer. Flick all tubes to mix and then immediately place on ice.
2. Incubate all three tubes for 1 min in a 30 °C water bath. Following incubation, add 13.5 µL of 12 mg/mL PNBM to all three tubes. Invert to mix samples. Incubate these samples at room temperature for 1 h.
3. After starting the incubation in step 2.2, prepare the desalting columns as soon as possible as the process takes approximately 45 min.

3. Preparation of desalting columns

1. Initially prepare columns (3 for this example experiment) by inverting each column several times to re-suspend the Sephadex G-25 resin in the storage buffer. Allow the resin to settle by attaching each column to a clamp stand and letting it sit undisturbed for approximately 5 min.
2. Following settling of the Sephadex G-25 resin, remove the caps from both the top and bottom of the column to allow the storage buffer to drain by gravity and have a tube placed below the bottom opening to collect the flow-through for discard.
3. Once storage buffer is depleted, add approximately 2.7 mL of lysis buffer in order to equilibrate the columns. Collect the flow-through and discard. Repeat 3 times. Following the final equilibration, the columns are ready for step 4.1.

4. Removal of PNBM

1. After the 1 h incubation (step 2.2) and column preparation (step 3) are complete, apply the samples to the columns. Label each column as follows: “kinase rxn”, “GTPγS only”, and “PNBM only”. Load all of each sample onto its respective column. Collect and discard the flow-through.
2. Wash samples by adding 420 µL of lysis buffer to each column. Allow lysis buffer to filter through the column and collect the flow-through for discard. Following this wash step, place tubes in position for collection of samples.
3. Elute samples by adding 500 µL of lysis buffer to each column. Collect the flow-through that now contains thiophosphorylated and alkylated CK2 substrates.

5. Immunoprecipitation: Part I

1. Prepare samples for immunoprecipitation by first removing 80 µL for an “elution input control” sample from each of the respective elutions (“kinase rxn”, “GTPγS only”, and “PNBM only”) collected in step 4.3. Following removal of 80 µL from each sample, there will be approximately 420 µL remaining per sample.
   1. Split each sample into 2 tubes containing 200 µL each. Label each respective tube: “kinase rxn anti-thiophosphate ester”, “kinase rxn IgG”, “GTPγS anti-thiophosphate ester”, “GTPγS IgG”, “PNBM anti-thiophosphate ester”, and “PNBM IgG”.
2. Add 2.8 µg of anti-thiophosphate ester antibody to each of the anti-thiophosphate ester-labeled tubes and 2.8 µg of isotype control antibody to each of the IgG-labeled tubes. Place tubes on a rotator at 4 °C for 2 h.
3. Begin preparation of protein A/G bead during the last 15 min of the 2 h incubation in step 5.2.

6. Protein A/G agarose bead preparation

1. Briefly vortex the storage tube to ensure beads are completely re-suspended. Cut off the end of a P200 pipette tip using a clean razor blade in order to increase gauge size. Pipet 100 µL of the bead slurry per immunoprecipitation into a new 1.7 mL microcentrifuge tube. For this example, a total of 6 tubes are needed.
2. Centrifuge the tubes at 17,500 x g for 1 min at 4 °C. Remove the supernatant and discard. Re-suspend the beads in 200 µL of lysis buffer and briefly vortex. Repeat the spin and wash steps 3 times.
3. Following the final wash, place the beads on ice until the incubation in step 5.2 is complete.

7. Immunoprecipitation: Part II

1. After the 2 h incubation from step 5.2, spin down the samples at 17,500 x g for 3 min at 4 °C. Following centrifugation add 200 µL from each sample to the tubes with the washed beads. Place the tubes on a rotator at 4 °C for 1 h.
2. Following the 1 h incubation, centrifuge the tubes at 17,500 x g for 1 min at 4 °C. Next remove 40 µL of supernatant from each sample and save as a “depletion control” (total 6 tubes). Remove the remainder of the supernatant and discard. Take care not to disturb the beads.
3. Wash the samples by adding 200 µL of lysis buffer and vortexing briefly. Then centrifuge at 17,500 x g for 1 min at 4 °C. Remove the supernatant and discard. Repeat the wash and spin steps 3 times. Take care not to disturb the beads during these steps.
4. After completing the wash steps, add 50 µL of 2x sample buffer to each sample containing beads. For all other samples, add 8 µL of 6x sample buffer: “input control”, “elution input controls”, and “depletion controls”.
5. Once buffer is added to the tubes, pipet up and down to mix, and heat all samples at 95 °C for 5 min before proceeding with SDS-PAGE.

8. Analysis/Validation of results

1. Validate successful CK2-dependent thiophosphorylation and alkylation.
   1. To determine the efficacy of CK2-mediated thiophosphorylation in step 2.2, perform SDS-PAGE and Western blotting by running 15-20 µL of the “elution input controls” collected in step 5.1 on a 12.5% polyacrylamide gel.
   2. Probe membranes with the following antibodies: anti-thiophosphate ester, anti-CK2α, and anti-GAPDH (or other appropriate loading control). If this step was successful, an enhanced anti-thiophosphate ester signal should be apparent in the “kinase rxn” lane compared to the other two lanes (Figure 2).
2. Visualize enriched putative substrates of CK2 and determine protein identity.
   1. To assess if the immunoprecipitation steps were successful, run 25-30 µL of the samples eluted from the beads in step 7.4 on a separate 12.5% polyacrylamide gel. Ensure that all equipment is clean and wear gloves at all times during this step to minimize contamination.
   2. Stain the gel with Coomassie blue to visualize enriched proteins from various stages of the experimental protocol (Figure 3). Using new razorblades, carefully excise unique bands present in the “kinase rxn anti-thiophosphate ester IP” lane, noting their approximate molecular weights.
   3. Submit these bands for protein identification by liquid chromatography/tandem mass spectrometry (LC-MS/MS) (Figure 4). If antibodies directed against the identified proteins are available, confirm the results of mass spectrometry by SDS-PAGE and immunoblotting of input, depleted, and IP fractions collected during the course of the protocol (Figure 4).

Results

A schematic diagram of the experimental procedure is provided in Figure 1. The underlying basis of the technique is the unusual ability of CK2 to use GTP for phosphoryl group transfer. Addition of exogenous CK2 holoenzyme along with the GTP analogue, GTPγS, to a cell lysate results in thiophosphorylation of endogenous CK2 substrates. Subsequent treatment of the lysate with the alkylating reagent p-nitrobenzyl mesylate (PNBM) generates a thiophos...

Discussion

Here, we describe a relatively simple biochemical method for identifying substrates of protein kinase CK2 from a complex biological sample. The critical steps of this protocol are based on the unusual enzymatic properties of CK2 and include CK2-dependent thiophosphorylation of specific substrate proteins using GTPγS and their subsequent immunoprecipitation and identification. With these results, we have demonstrated the utility and versatility of this approach as we have now applied this strategy in both hu...

Materials

Name	Company	Catalog Number	Comments
12 mg/mL PNBM	Abcam	ab138910	40.5 µL
2.5 mM GTPγS	Sigma-Aldrich	G8634-1MG	5.4 µL
Anti-CK2α (E-7) mouse monoclonal antibody	Santa Cruz Biotechnology	sc-373894	1:1000 for Western blotting
Anti-GAPDH (6C5) mouse monoclonal antibody	Santa Cruz Biotechnology	sc-32233	1:1000 for Western blotting
Anti-nucleolin rabbit polyclonal antibody	Abcam	ab22758	1:1000 for Western blotting
Anti-thiophosphate ester [51-8] rabbit monoclonal antibody	Abcam	ab92570	Varies (final concentration 2.8 µg for each sample)
Centrifuge pre-set to 4ºC	ThermoScientific	Sorvall Legend Micro 21R Cat# 75-772-436
cOmplete Mini EDTA-Free Protease Inhibitor	Roche	11836170001
Lysis Buffer	See recipe below	See recipe below	30 mL
Normal rabbit IgG antibody (isotype control)	Cell Signaling Technology	2729S	Varies (final concentration 2.8 µg for each sample)
PD MiniTrap Column	GE Healthcare	28-9180-10	3 columns
Protein A/G Plus Agarose Beads	Santa Cruz Biotechnology	sc-2003	600 µL
Recombinant human CK2 holoenzyme	New England Biolabs	P6010S	2.7 µL
Rotator	Labnet: Mini Labroller	Mini Labroller SKU# H5500
T98G human glioblastoma cells	ATCC	CRL-1690
Water bath pre-set to 30ºC	Shel Lab	H20 Bath Series Model# SWB15