Research Explained: Rapid method for evaluation of CK2 enzymatic activity and CK2alpha/CK2beta-interaction in Escherichia coli cell lysates

Gast, A. et al.

2025

Research Explained by: Lindsay Bass, CSNK2A1 Foundation Intern

Edited by: Gabrielle Rushing, PhD, CSNK2A1 Foundation Chief Scientific Officer

Reviewed by Parent Advisory Board members: Michelle Proctor-Simms and Jessica Wilfong

CK2 is a protein kinase that adds phosphate groups to various proteins to impact their function. CK2 consists of two activating CK2α or CK2α’ subunits and two regulatory CK2β subunits. Mutations in the gene that encodes the CK2α subunit can lead to Okur-Chung Neurodevelopmental Syndrome (OCNDS). Of the 68 different mutations associated with OCNDS described currently, many have shown an impact on CK2 kinase activity (or the ability to add phosphates to proteins, also called phosphorylation); yet it is not known how these mutations impact the interaction of CK2α with CK2β, which could provide insight into future treatment strategies.

Gast et al. developed an efficient method to evaluate the impact of CK2α mutations on binding interactions with CK2β and kinase activity using bacterial E. coli cells to express CK2α protein. The function of CK2α mutant proteins (or variants) can be studied by breaking open the bacterial cells and testing the unpurified CK2α in the cell contents (or lysate). However, the amount of CK2α present must be accounted for. To do so, Gast et al. fused a red fluorescent protein called mScarlet to a fully functional shortened version of CK2α (called CK2α^1-335) to measure the amount of CK2α^1-335 present during analyses. Gast et al. confirmed that the CK2α^1-335-mScarlet protein maintained the same functional capacity as the non-labeled (normal) CK2α^1-335. Two CK2α variants described in individuals with OCNDS, CK2α^R47G and CK2α^R191Q, were expressed with the fluorescent mScarlet, and CK2α^1-335 activity was determined. Briefly, wildtype and variant CK2α^1-335 were respectively combined with a substrate peptide (a small protein fragment that the phosphate can be added to) and ATP (the source of phosphate). The amount of phosphorylated and non-phosphorylated protein was determined by a process called capillary electrophoresis. The results showed that CK2α^{1-335, R47G} and CK2α^{1-335, R191Q} exhibited decreased kinase activity when evaluated on their own and in complex with the regulatory subunit CK2β^1-193, compared to the wildtype CK2α.

Measurement of the increased activity of CK2α^1-335 with increasing amounts of CK2β^1-193 was used to calculate the binding strength of CK2α to CK2β, as determined by the dissociation constant (KD). A lower KD value indicates stronger binding of CK2α to CK2β. Modeling was used to evaluate the uncertainty of the determined KD values. Through this method, the CK2α^R191Q variant showed decreased kinase activity and decreased binding strength to CK2β compared to wildtype.

This method to study CK2α activity in E. coli cell lysates is time- and cost-effective and limits potential background signal, as E. coli does not express its own CK2 protein. One substrate peptide was tested here, but this method could be used to evaluate phosphorylation of other protein substrates especially since CK2 is known to have many substrates. Additionally, other protein-protein or small molecule interactions with CK2α could be tested using this method in the future. Using this method to understand the functional impact of CK2 mutations could provide insight into treatment strategies, whether that be identifying potential drug candidates for mutations with shared functional impacts, or even grouping OCNDS individuals that might be more or less likely to respond to a specific treatment based on how their mutation impacts CK2 function.

Glossary

Adenosine triphosphate (ATP):  a major energy source for all living cells that transfers energy by donating a phosphate group

Capillary electrophoresis:  a lab method that separates molecules (such as DNA or proteins) based on how fast they move through a tiny tube, which is determined by their size and electric charge.

Link: Figure 4, https://www.technologynetworks.com/analysis/articles/an-introduction-to-capillary-electrophoresis-theory-practice-and-applications-378737

CK2α^R47G:   a CK2α variant. The amino acid (building block of a protein) arginine (R) is at position 47 of the CK2α protein structure, but in some individuals with OCNDS, this amino acid is changed to glycine (G). This change (or mutation) can impact the function of the CK2 protein.

CK2α^R191Q:  a CK2α variant. The amino acid arginine (R) is at position 191 of the CK2α protein structure, but in some individuals with OCNDS, this amino acid is changed to glutamine (Q). This change (or mutation) can impact the function of the CK2 protein.

Dissociation constant (KD):  a number that explains how strongly one molecule binds or sticks to another. A lower KD value indicates stronger binding.

E. coli:  a type of bacteria, or single cell living organism. Some types of E. coli are harmful to humans, but other types are not harmful and are often used in scientific research because the cells are easy to grow, can survive in various conditions, and are easy to manipulate. These cells are often used to produce genes or proteins of interest for research use.

Fluorescent protein:  a special kind of protein that glows a specific color under a certain type of light which can be used to detect or track a protein of interest in research experiments.

Substrate peptide:  a protein fragment that is modified by an enzyme (protein that makes a process go faster). In this case, the CK2α enzyme adds a phosphate group from ATP (see definition) to the substrate peptide.

Wildtype:  the normal version of a gene, protein or organism (often compared to a mutated or modified version).