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&....

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 .......

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