CK2β Is a Gatekeeper of Focal Adhesions Regulating Cell Spreading

Abstract

CK2 is a hetero-tetrameric serine/threonine protein kinase made up of two CK2α/α' catalytic subunits and two CK2β regulatory subunits. The free CK2α subunit and the tetrameric holoenzyme have distinct substrate specificity profiles, suggesting that the spatiotemporal organization of the individual CK2 subunits observed in living cells is crucial in the control of the many cellular processes that are governed by this pleiotropic kinase. Indeed, previous studies reported that the unbalanced expression of CK2 subunits is sufficient to drive epithelial to mesenchymal transition (EMT), a process involved in cancer invasion and metastasis. Moreover, sub-stoichiometric expression of CK2β compared to CK2α in a subset of breast cancer tumors was correlated with the induction of EMT markers and increased epithelial cell plasticity in breast carcinoma progression. Phenotypic changes of epithelial cells are often associated with the activation of phosphotyrosine signaling. Herein, using phosphotyrosine enrichment coupled with affinity capture and proteomic analysis, we show that decreased expression of CK2β in MCF10A mammary epithelial cells triggers the phosphorylation of a number of proteins on tyrosine residues and promotes the striking activation of the FAK1-Src-PAX1 signaling pathway. Moreover, morphometric analyses also reveal that CK2β loss increases the number and the spatial distribution of focal adhesion signaling complexes that coordinate the adhesive and migratory processes. Together, our findings allow positioning CK2β as a gatekeeper for cell spreading by restraining focal adhesion formation and invasion of mammary epithelial cells.

Keywords: CK2β depletion; EMT; FAK1-Src-PAX1 signaling pathway; epithelial cell spreading; focal adhesions; tyrosine-phosphorylated proteins.

FIGURE 1

Changes in PTK activity in ΔCK2β MCF10A cells compared with WT cells. (A) Functional kinome profiling using the PamChips, which contain multiple copies of individual peptides. Sequence-specific tyrosine phosphorylation is detected by fluorochrome-coupled phospho-specific antibodies and CCD camera. The volcano plot visualizes the result of the tests by plotting, for each test, the effect size (x-axis, LFC or delta) versus significance (y-axis, –log10 (p-value)) of the test. Red spots are peptides that show significant differences compared to control (p < 0.05). (B) PTK score plot highlighting kinases that were modulated in ΔCK2β MCF10A cells compared with WT cells. The plot shows results from the upstream kinase analysis (UKA) tool, a functional scoring tool which rank-orders the top kinases that are differentially active between the two groups. The ranking factor is the final (median) kinase score. This score is based on a combined sensitivity score (difference between “treatment” and “control” groups) and specificity score (for a set of peptides to kinase relationship derived from current databases). The kinase statistics indicates the group differences, with effect size (values) and direction (+ or >0 = activation; – or 0 = inhibition). (C) Signaling pathways and biological process analysis in ΔCK2β MCF10A cells compared with WT cells (GProfiler, Gene Ontology Biological Processes).

FIGURE 2

LC-MS/MS and GSEA analysis of differentially elevated levels of Tyr-phosphorylated proteins in WT and ΔCK2β MCF10A cells. (A) Proteins immunopurified from WT and ΔCK2β MCF10A cell lysates were analyzed by Western blotting for pTyr proteins using the 4G10 antibody. (B) Venn diagram showing differentially abundant proteins between WT and CK2β-depleted cells in samples obtained by immunoaffinity purification of Tyr-phosphorylated proteins (Supplementary Table S1). (C) Venn diagram showing the proteins in which differentially abundant pTyr-phosphopeptides between WT and CK2β-depleted cells were identified (Supplementary Table S2). These pTyr-phosphopeptides were identified and quantified from the LC-MS/MS analyses of peptide samples coming from a double enrichment starting from the two cell types: immunoaffinity purification of Tyr-phosphorylated proteins followed by the enrichment of their tryptic peptides on a TiO₂ resin. (D) Significant gene ontology cellular components enriched from proteomics quantification of phosphoproteins (Supplementary Table S3) according to corrected p-values by the Benjamini–Hochberg method <0.05. Cellular component names are displayed as yellow nodes and bound to core phosphoproteins that led to a significant enrichment by GSEA. The core phosphoproteins are the nodes colored in red according to their log2 fold change value.

FIGURE 3

Upregulation of pTyr proteins in lysates of ΔCK2β MCF10A cells. (A) Lysates from WT and ΔCK2β MCF10A cells were analyzed for pTyr-containing proteins using the 4G10 antibody. GAPDH was used as a loading control. (B) Cell extracts (500 μg of protein) of WT and ΔCK2β MCF10A cells were loaded on 4G10 resin. After extensive washing, the bound proteins were eluted with 100 mM phenyl phosphate. The collected fractions were analyzed by Western blotting with the 4G10 antibody. (C) Proteins immunopurified from WT and ΔCK2β MCF10A cell lysates were analyzed by Western blotting for p-FAK (Y576/577), p-Src (Y418), p-PAX1(Y118), p-p130CAS (Y410), FAK, Src, PAX1, and p130CAS and (D) for p-EGFR (Y1173), EGFR, p-PLCγ (Y771), and PLCγ.

FIGURE 4

Cell spreading and size of focal adhesions are dependent on CK2β. (A) Staining of pTyr and actin was carried out on cells spread on vitronectin-coated cover glass for 24 h. Scale bar represents 10 µm. (B) Quantification of the cell area. (C) Quantification of the number of FAs normalized to the cell area. (D) Quantification of the FA average area. (E) Quantification of the FA area, normalized to the cell area. Error bars represent standard deviation. ***p-value ≤0.0005.

FIGURE 5

CK2 catalytic activity does not contribute to enhanced Tyr phosphorylation in ΔCK2β MCF10A cells. (A) CK2 kinase activity in cell lysates from WT or ΔCK2β MCF10A cells treated for 5 h in the absence or presence of 10 μM CX-4945. (B) Cell lysates as in (A) were Western blotted with 4G10 antibody for the presence of pTyr proteins. GAPDH was used as a loading control. (C) ΔCK2β MCF10A cell lysates were also analyzed by Western blotting for p-FAK (Y576/577), p-Src (Y418), p-PAX1 (Y118), p-p130CAS (Y410), FAK, Src, PAX1, and p130CAS.