Chemical Genetic Validation of CSNK2 Substrates Using an Inhibitor-Resistant Mutant in Combination with Triple SILAC Quantitative Phosphoproteomics

Abstract

Casein Kinase 2 (CSNK2) is an extremely pleiotropic, ubiquitously expressed protein kinase involved in the regulation of numerous key biological processes. Mapping the CSNK2-dependent phosphoproteome is necessary for better characterization of its fundamental role in cellular signalling. While ATP-competitive inhibitors have enabled the identification of many putative kinase substrates, compounds targeting the highly conserved ATP-binding pocket often exhibit off-target effects limiting their utility for definitive kinase-substrate assignment. To overcome this limitation, we devised a strategy combining chemical genetics and quantitative phosphoproteomics to identify and validate CSNK2 substrates. We engineered U2OS cells expressing exogenous wild type CSNK2A1 (WT) or a triple mutant (TM, V66A/H160D/I174A) with substitutions at residues important for inhibitor binding. These cells were treated with CX-4945, a clinical-stage inhibitor of CSNK2, and analyzed using large-scale triple SILAC (Stable Isotope Labelling of Amino Acids in Cell Culture) quantitative phosphoproteomics. In contrast to wild-type CSNK2A1, CSNK2A1-TM retained activity in the presence of CX-4945 enabling identification and validation of several CSNK2 substrates on the basis of their increased phosphorylation in cells expressing CSNK2A1-TM. Based on high conservation within the kinase family, we expect that this strategy can be broadly adapted for identification of other kinase-substrate relationships.

Keywords: CSNK2; CX-4945; SILAC; chemical genetics; kinase-substrate relationship validation; mass spectrometry; phosphoproteomics; protein kinase CK2.

FIGURE 1

Comparing the efficacy of CSNK2 inhibitors in U2OS cells. (A) Schematic illustration of the CSNK2 inhibitors that were evaluated in this study with reported in vitro IC₅₀ values (B) CSNK2 inhibition was assessed by immunoblotting with the indicated phospho-specific antibodies after 24 h of inhibitor CX-4945 treatment at the indicated concentrations in U2OS cells. Lysis buffer of lysates marked with * did not contain phosphatase inhibitors. DMSO treated lysates +/-λ-phosphatase were used as immunoblotting controls for the phospho-specific antibodies. Results are representative of two independent experiments.

FIGURE 2

Use of a triple mutant CSNK2A1 for validation of CSNK2 substrates. (A) Crystal structure of CX-4945 interacting with the ATP-binding site of CSNK2A1 (PDB ID: 3PE1). Highlighted are the residues mutated in CSNK2A1-TM (V66A/H160D/I174A) to create an inhibitor tolerant/resistant kinase. (B) Immunoblots demonstrate the inhibition and rescue of CSNK2-dependent phosphorylation using Flp-In T-REx U2OS cell lines stably expressing CSNK2A-HA WT or CSNK2A-HA TM with tight Tetracycline (Tet-ON) regulation. Cells were treated with 30 µM of CX-4945 for the indicated times. (C) Bar charts demonstrate residual CSNK2 activity based on EIF2S2 pS2/total EIF2S2 or EEF1D pS162/GAPDH ratios, with CSNK2 activity in the DMSO control defined as 100%. Each column represents the mean value of two independent experiments with range bars displayed. Band intensities on blots were quantified with LiCor Odyssey v3.0 software. Results are representative of two independent experiments (D) Overview of large-scale identification and validation of CSNK2 substrates using a chemical genetics approach combined with triple SILAC quantitative phosphoproteomics.

FIGURE 3

Filtering criteria utilized in CSNK2 substrate identification and validation. (A) Filtering criteria utilized to identify CSNK2-dependent phosphopeptides. The number of distinct phosphopeptides that remained after each filtering step is displayed in the Venn diagram variation. WebLogo consensus sequence analysis was conducted on phosphosites identified (B) prior to filtering, (C) after filtering for phosphopeptides significantly inhibited, (D) after filtering for phosphopeptides demonstrating partial rescue, and (E) after filtering for phosphopeptides demonstrating rescue. The n values represent the number of phosphosites used for consensus sequence analysis.

FIGURE 4

Inhibition and rescue profile of CSNK2 substrates. Differential expression of (A) phosphopeptides and (C) proteins after treating U2OS cells expressing CSNK2A1-WT with 30uM of CX-4945 for 4 h. Phosphopeptides/proteins highlighted in gold are significantly down- or upregulated 1.5-fold. Phosphopeptides/proteins highlighted in purple demonstrate rescue in U2OS cells expressing CSNK2A1-TM with 30uM of CX-4945 for 4 h. Rescued phosphopeptides are labelled with the gene name of the corresponding protein. Vertical dashed lines (−/+ 0.585) represent 1.5-fold down- and upregulation, respectively. The horizontal dashed line represents an adjusted p-value cutoff of 0.05 (B) Inhibition and rescue profiles of CSNK2 substrate phosphopeptides. The horizontal dashed line is representative of significant inhibition (-0.585).

FIGURE 5

Immunoblot evaluation of phosphospecific antibodies recognizing SSB pS366, SSB S366, and LIG1 pS36. Flp-In T-REx U2OS cell lines stably expressing CSNK2A-HA WT or CSNK2A-HA TM with tight Tetracycline (Tet-ON) regulation were treated with 30 µM of CX-4945 for the indicated times and blotted with the indicated antibodies recognizing total protein or specific sites. Results are representative of two independent experiments.

FIGURE 6

CSNK2A1 Interaction Network. (A) Rescued CSNK2 protein substrates and related proteins are depicted in a GeneMANIA network. Previous database information regarding physical interactions (red), co-expression (purple), predicted interactions (orange), genetic interactions (green), co-localization (blue), and shared protein domains (yellow) were employed in network creation in an “equal by data type” weighting approach. Our own list of rescued substrates was utilized in network creation and each assigned a value of 1. If a phosphopeptide was not unique and could have originated from several proteins, all potential proteins were assigned a value of 1. Hashed gene names indicate proteins included in our query list, while those with a solid background are related proteins inserted during GeneMANIA network creation. Proteins implicated in Gene Ontology (GO) biological processes are labelled. (B) GO terms enriched amongst proteins displayed in the GeneMANIA network. Coverage represents the proportion of displayed proteins which are annotated with a certain GO term when compared to all genes in the genome annotated with that same term.