Identification of Candidate Casein Kinase 2 Substrates in Mitosis by Quantitative Phosphoproteomics

Abstract

Protein phosphorylation is a crucial regulatory mechanism that controls many aspects of cellular signaling. Casein kinase 2 (CK2), a constitutively expressed and active kinase, plays key roles in an array of cellular events including transcription and translation, ribosome biogenesis, cell cycle progression, and apoptosis. CK2 is implicated in cancerous transformation and is a therapeutic target in anti-cancer therapy. The specific and selective CK2 ATP competitive inhibitor, CX-4945 (silmitaseratib), is currently in phase 2 clinical trials. While many substrates and interactors of CK2 have been identified, less is known about CK2 substrates in mitosis. In the present work, we utilize CX-4945 and quantitative phosphoproteomics to inhibit CK2 activity in mitotically arrested HeLa cells and determine candidate CK2 substrates. We identify 330 phosphorylation sites on 202 proteins as significantly decreased in abundance upon inhibition of CK2 activity. Motif analysis of decreased sites reveals a linear kinase motif with aspartic and glutamic amino acids downstream of the phosphorylated residues, which is consistent with known substrate preferences for CK2. To validate specific candidate CK2 substrates, we perform in vitro kinase assays using purified components. Furthermore, we identified CK2 interacting proteins by affinity purification-mass spectrometry (AP-MS). To investigate the biological processes regulated by CK2 in mitosis, we perform network analysis and identify an enrichment of proteins involved in chromosome condensation, chromatin organization, and RNA processing. We demonstrate that overexpression of CK2 in HeLa cells affects proper chromosome condensation. Previously, we found that phosphoprotein phosphatase 6 (PP6), but not phosphoprotein phosphatase 2A (PP2A), opposes CK2 phosphorylation of the condensin I complex, which is essential for chromosome condensation. Here, we extend this observation and demonstrate that PP6 opposition of CK2 is a more general cellular regulatory mechanism.

Protein kinase CK2 phosphorylation of SAPS3 subunit increases PP6 phosphatase activity with Aurora A kinase

“…These had all been previously mapped by shotgun phosphoproteomics (see Figure 1B). Though these sites have an acidic residue at the p + 2 position in the sequence, as expected for protein kinase CK2 substrates, two sites had the phosphorylated residue followed by a proline (FpT 631 PEpSQR and SpS 853 PEQR), which has never been reported previously for protein kinase CK2 (see [21] and [33]).…”

“…Kettenbach and co-workers have highlighted the preference of PP6 for reaction with sites of phosphorylation adjacent to acidic residues [21,22], such as those reactive with protein kinases CK1 and CK2. This pairing of kinase and phosphatase may include opposing one other in regulating processes in cells.…”

“…This pairing of kinase and phosphatase may include opposing one other in regulating processes in cells. Examples of substrates common for protein kinase CK2 and PP6 include cohesion, EF1D, HAP28 and S30BP [21]. Another example is E-cadherin, where PP6C binds to the cytoplasmic tail and negatively regulates phosphorylation of S846 (a CK1 site) that promotes internalization of E-cadherin from adherens junctions of epithelial cells [39].…”

“…These agents were chosen for being relatively specific for protein kinase CK2 yet structurally different [18][19][20], making it less likely that effects would be off-target and not due to inhibition of protein kinase CK2. We used the same dose of each drug and an incubation time previously shown to effectively suppress protein kinase CK2 activity in HeLa cells [21]. When added to live HeLa cells for 2 h both TBB and CX-4945 significantly increased by ∼40% the pT288 in endogenous AURKA analyzed by immunoblotting (Figure 4).…”

Quantitative phosphoproteomics to unravel the cellular response to chemical stressors with different modes of action

“…Table inlet displays an overview of the responsive phosphosites ◂ 2013). Consistently, phosphorylation of several putative substrates of CK2 kinase (Rusin et al 2017) including Dkc1 pS453 and Hdac1 pS423 was enhanced after CsA and ETO ( Supplementary Fig. S8).…”