Regulation of DNA-dependent protein kinase by protein kinase CK2 in human glioblastoma cells.

The DNA-dependent protein kinase (DNA-PK) is a nuclear serine/threonine protein kinase composed of a large catalytic subunit (DNA-PKcs) and a heterodimeric DNA-targeting subunit Ku. DNA-PK is a major component of the nonhomologous end-joining pathway of DNA double-strand breaks repair. Although DNA-PK has been biochemically characterized in vitro, relatively little is known about its functions in the context of DNA repair and how its kinase activity is precisely regulated in vivo. Here, we report that cellular depletion of the individual catalytic subunits of protein kinase CK2 by RNA interference leads to significant cell death in M059K human glioblastoma cells expressing DNA-PKcs, but not in their isogenic counterpart, that is M059J cells, devoid of DNA-PKcs. The lack of CK2 results in enhanced DNA-PKcs activity and strongly inhibits DNA damage-induced autophosphorylation of DNA-PKcs at S2056 as well as repair of DNA double-strand breaks. By the application of the in situ proximity ligation assay, we show that CK2 interacts with DNA-PKcs in normal growing cells and that the association increases upon DNA damage. These results indicate that CK2 has an important role in the modulation of DNA-PKcs activity and its phosphorylation status providing important insights into the mechanisms by which DNA-PKcs is regulated in vivo.

The regulatory beta-subunit of protein kinase CK2 regulates cell-cycle progression at the onset of mitosis.

Cell-cycle transition from the G(2) phase into mitosis is regulated by the cyclin-dependent protein kinase 1 (CDK1) in complex with cyclin B. CDK1 activity is controlled by both inhibitory phosphorylation, catalysed by the Myt1 and Wee1 kinases, and activating dephosphorylation, mediated by the CDC25 dual-specificity phosphatase family members. In somatic cells, Wee1 is downregulated by phosphorylation and ubiquitin-mediated degradation to ensure rapid activation of CDK1 at the beginning of M phase. Here, we show that downregulation of the regulatory beta-subunit of protein kinase CK2 by RNA interference results in delayed cell-cycle progression at the onset of mitosis. Knockdown of CK2beta causes stabilization of Wee1 and increased phosphorylation of CDK1 at the inhibitory Tyr15. PLK1-Wee1 association is an essential event in the degradation of Wee1 in unperturbed cell cycle. We have found that CK2beta participates in PLK1-Wee1 complex formation whereas its cellular depletion leads to disruption of PLK1-Wee1 interaction and reduced Wee1 phosphorylation at Ser53 and 121. The data reported here reinforce the notion that CK2beta has functions that are independent of its role as the CK2 regulatory subunit, identifying it as a new component of signaling pathways that regulate cell-cycle progression at the entry of mitosis.

UV light blocks EGFR signalling in human cancer cell lines.

UV light excites aromatic residues, causing these to disrupt nearby disulphide bridges. The EGF receptor is rich in aromatic residues near the disulphide bridges. Herein we show that laser-pulsed UV illumination of two different skin-derived cancer cell lines i.e. Cal-39 and A431, which both overexpress the EGF receptor, leads to arrest of the EGFR signaling pathway. The phosphorylation status of the receptor and the level of phosphorylated downstream signaling molecules i.e. AKT and the mitogen activated protein kinases (MAPKs) ERK1 and 2 is detected by Western blotting using phosphospecific antibodies. There was a threshold level, below which the receptor could not be blocked. In addition, illumination caused the cells to upregulate the cyclin-dependent kinase inhibitor p21WAF1, irrespective of the p53 status. Since the EGF receptor is often overexpressed in cancers and other proliferative skin disorders, it might be possible to significantly reduce the proliferative potential of these cells making them good targets for laser-pulsed UV light treatment.