casein-kinase-ii and resorufin

Protein kinase CK2 is involved in several cellular processes and has lately also been linked to the DNA damage response through phosphorylations and interactions. Herein, we have analysed two sets of mouse cell lines, one pair, which is proficient and deficient in ATM and the other set expressing or lacking a functional catalytic subunit of the DNA dependent protein kinase (DNA-PKcs). Both kinases are implicated in the downstream phosphorylation of the signaling molecules such as BID and AKT1 in response to DNA damage. BID and AKT1 are also targets of CK2, hence the four cell lines were treated with the three established CK2 inhibitors DMAT, TBB and resorufin in the absence and presence of the radiomimetic drug neocarzinostatin, which induces DNA double-strand breaks. We show that there are differences with respect to the effect of the CK2 inhibitors on the phosphorylation of AKT1 at S473 and its downstream target GSK3β as well as between the two sets of cell lines. However, no such change was seen with BID phosphorylation. The most notably difference was the higher expression of CK2α' and CK2β in DNA-PKcs defective cells compared to the DNA-PKcs proficient cells.

Topics: Animals; Ataxia Telangiectasia Mutated Proteins; Benzimidazoles; BH3 Interacting Domain Death Agonist Protein; Casein Kinase II; Cell Cycle Proteins; Cells, Cultured; DNA Breaks, Double-Stranded; DNA-Activated Protein Kinase; DNA-Binding Proteins; Fibroblasts; Gene Knockout Techniques; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Mice; Mice, Inbred BALB C; Mice, SCID; Nuclear Proteins; Oxazines; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Tumor Suppressor Proteins; Zinostatin

A low-resolution structure of the catalytic subunit CK2α of human protein kinase CK2 (formerly known as casein kinase 2) in complex with the ATP-competitive inhibitor resorufin is presented. The structure supplements previous human CK2α structures in which the interdomain hinge/helix αD region adopts a closed conformation correlating to a canonically established catalytic spine as is typical for eukaryotic protein kinases. In the corresponding crystal packing the hinge/helix αD region is nearly unaffected by crystal contacts, so that largely unbiased conformational adaptions are possible. This is documented by published human CK2α structures with the same crystal packing but with an open hinge/helix αD region, one of which has been redetermined here with a higher symmetry. An overview of all published human CK2α crystal packings serves as the basis for a discussion of the factors that determine whether the open or the closed hinge/helix αD conformation is adopted. Lyotropic salts in crystallization support the closed conformation, in which the Phe121 side chain complements the hydrophobic catalytic spine ensemble. Consequently, genuine ligand effects on the hinge/helix αD conformation can be best studied under moderate salt conditions. Ligands that stabilize either the open or the closed conformation by hydrogen bonds are known, but a general rule is not yet apparent.

Topics: Adenosine Triphosphate; Arginine; Casein Kinase II; Catalytic Domain; Crystallography, X-Ray; Databases, Protein; Humans; Ligands; Models, Chemical; Models, Molecular; Molecular Conformation; Oxazines; Protein Binding; Protein Conformation; Protein Structure, Secondary; Salts; Solvents

Screening a natural compound library led to the identification of resorufin as a highly selective and potent inhibitor of protein kinase CK2. Out of 52 kinases tested, only CK2 was inhibited, in contrast to emodin, a structurally related, known CK2 inhibitor that, in addition to CK2, inhibited ten other kinases by 90%. The IC50 values determined for the CK2 holoenzymes were 1.5 mol/l and for the free catalytic subunits ca. 4 mol/l. Altogether four cell lines were subjected to resorufin and emodin treatment. In the case of the three prostate carcinoma cell lines (PC-3, DU-145, LNCaP), 24 h treatment with 40 mol/l resorufin led to 15-20% dead cells; however, no caspase-mediated apoptosis was observed. In the case of the colorectal carcinoma HCT116 cell line, a similar picture was obtained, yet, when resorufin was administered to cells treated with doxorubicin, apoptosis was strongly induced within 24 h. Endogenous protein kinase CK2 was inhibited by resorufin by ca. 80% in the three prostate cell lines. In the case of the HCT116 cells, the inhibition was only 40% supporting the notion of cell line-specific selectivity. Moreover, we analysed the effect of resorufin and emodin on selected signalling molecules in the cell lines under investigation.

Topics: Apoptosis; Casein Kinase II; Cell Line, Tumor; Colorectal Neoplasms; Emodin; Humans; Inhibitory Concentration 50; Male; Oxazines; Prostatic Neoplasms; Protein Kinase Inhibitors; Signal Transduction

Targeting protein kinases as a therapeutic approach to treat various diseases, especially cancer is currently a fast growing business. Although many inhibitors are available, exhibiting remarkable potency, the major challenge is their selectivity. Here we show that the protein kinase CK2 inhibitors DMAT, TBB and resorufin differ in their selectivity against PI3K family members, since PI3K and DNA-PK are subject to inhibition by DMAT and TBB, however, not by resorufin. TBB and DMAT treatment together with cisplatin lead to an inhibition of cisplatin-induced stress signaling (as detected by phosphorylation of JNK and H2AX). In the case of resorufin no interference with the stress-signaling pathway is observed, supporting the notion that TBB and DMAT interfere with upstream molecules involved in genotoxic stress signaling. We have also tested the protein kinase CK2 inhibitors with respect to cell viability and inhibition of endogenous CK2 activity in the absence and presence of the anti-cancer drug cisplatin. The strongest effect on viability was observed with resorufin. In contrast to resorufin, TBB protected cells from cisplatin-induced cell killing. Furthermore, the inhibition of endogenous CK2 activity was cell type-dependent as endogenous CK2 was inhibited to different degrees in the cell lines.

Topics: Antineoplastic Agents; Benzimidazoles; Blotting, Western; Casein Kinase II; Cell Line, Tumor; Cell Survival; Cisplatin; Enzyme Inhibitors; Humans; Oxazines; Signal Transduction; Stress, Physiological; Triazoles