SILAC-based Quantitative Phosphoproteome Analysis by PEAKS

Case Study: Background & Aim

Casein Kinase 2 (CSNK2) has been shown to be upregulated in many cancers, wherein its elevated expression has been linked to poor prognosis. This has deemed CSNK2 a potential target for anti-cancer therapy.

Gyenis and his team [1] devised an approach for the large-scale identification and validation of CSNK2 substrates, using an inhibitor-resistant mutant kinase to clarify differential phosphorylation resulting from on- versus off-target effects of inhibitors. The mutant kinase remains active in the presence of the inhibitor and therefore, bona fide sites remain phosphorylated when compared with the inhibitor-sensitive wild-type kinase. Phosphosites which are downregulated due to off-target effects remain downregulated in cells expressing the inhibitor-resistant mutant.

Experimental Design

Inhibition and rescue of CSNK2-dependent phosphorylation was demonstrated using Flp-In T-REx U2OS cell lines stably expressing CSNK2A-HA WT or CSNK2A-HA TM with tight Tetracycline (Tet-ON) regulation [2]. CSNK2A-HA TM was assigned a Heavy label was a chemically altered cell line expressing a triple mutant CK2A1, which has certain residues substituted to resist inhibitor drug binding and keep the catalytic activity.

Inhibitor efficacy and specificity of two inhibitor drugs, CX-4945 or SGC-CK2-1, at three time points (0 hours, 4 hours, and 24 hours) was assessed using a triple SILAC phosphoproteomic strategy:

1. Control – CSNK2A-HA WT was assigned a Light label for DMSO control,
2. Inhibition – CSNK2A-HA WT was assigned a Medium label (K4, R6) and treated with the inhibitor drug,
3. Rescue – CSNK2A-HA TM was assigned a Heavy label (K8, R10) and treated with the inhibitor drug.

Result & Conclusion

PEAKS was used for sensitive identification of phosphorylated peptides and proteins followed by accurate quantification using a built-in triple SILAC method. Here, we demonstrated a comparison of efficacy of the two CSNK2 inhibitor drugs, CX-4945 and SGC-CK2-1, where the later appeared to be a significantly more selective inhibitor of CSNK2 as reported in the publication. The majority of SGC-CK2-1-induced decrease in phosphorylation was rescued with CSNK2A1-TM and thus determined to be entirely CSNK2A1-dependent. PEAKS was shown to clearly capture the profiles of Light, Medium, and Heavy conditions as hypothesized. This in turn allowed for a comprehensive investigation and validation of the CSNK2 dependent Inhibition (Medium) and Rescue (Heavy) at 4- and 24-hours treatment time points.