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Andrei A. Kramerov, Alexander V. Ljubimov; Protein Kinase CK2 Inhibition Affects Actin-Myosin Cytoskeleton In Cultured Human Astrocytes And Vascular Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2014.
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Protein kinase CK2 participates in the regulation of cellular morphology and migration, and could be an important angiogenesis mediator. CK2 localized in the retinal tissue mostly in astrocytes and vascular endothelial cells (EC), and CK2 inhibitors reduced retinal neovascularization and stem cell recruitment in the mouse model of retinopathy. Recently, we have co-localized CK2 and F-actin in contractile stress fibers in microvascular EC. The purpose was to examine a possible role of CK2 in the regulation of actin-myosin-based contractility in astrocytes and EC.
Cultured human astrocytes and vascular EC were treated by specific inhibitors of CK2 (TBB, TBCA) alone, or in combination with inhibitors of Rho- kinase (hydroxyfasudil) or myosin light chain (MLC)-kinase (ML7). After 1-18 hr of treatment, cultured cells were fixed in 4% formaldehyde, permeabilized in 0.1% Triton X100 and incubated with mouse anti-CK2 antibody followed by secondary antibodies conjugated with fluorescein, or phalloidin conjugated with rhodamine. For Western blotting of the cultured cell extracts, rabbit antibodies to phospho-Ser19-MLC were used.
We found that CK2 inhibitors caused disassembly of actin-myosin stress fibers and cell shape changes, including cytoplasmic retraction and generation of processes. Stress fiber formation is mainly regulated by phosphorylation of MLC that leads to an increase in myosin II activity, which cross-links actin filaments and generates contractile force. Interestingly, we discovered that low doses of inhibitors of Rho-kinase and MLC-kinase that both phosphorylate MLC, potentiated the effect on cell shape of suboptimal CK2 inhibition. Such striking morphological effect of the combined action of the protein kinase inhibitors was accompanied by decreased level of phospho-MLC, thus implicating CK2 in the regulation of actin-myosin cytoskeleton.
These results suggest an important role of CK2 in control of contractility and cell motility, which may account for suppressing effect of CK2 inhibition on retinal neovascularization in the mouse retinopathy model.
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