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Protein kinase CK2 is a ubiquitous pro-survival kinase whose substrate targets are involved in various cellular processes. Crystal structure analysis confirmed constitutive activity of the kinase, yet CK2 activity regulation in the cell is still obscure. In-vitro studies suggest autoinhibitory aggregation of the hetero-tetrameric CK2 holoenzyme as a basis for CK2 regulation. In this study, we applied bioluminescent resonance energy transfer (BRET) technology to investigate CK2 holoenzyme aggregation in living cells. We designed a BRET(2) pair consisting of the fusion proteins CK2α-Rluc8 and CK2α-GFP(2). This BRET(2) sensor reported specific interaction of CK2 holoenzyme complexes. Furthermore, the BRET(2) sensor was applied to study modulators of CK2 aggregation. We found that CK2 aggregation is not static and can be influenced by the CK2-binding protein alpha subunit of the heterotrimeric G-protein that stimulates adenylyl cyclase (Gαs) and the polycationic compound polylysine. Gαs, but not the CK2 substrate β-arrestin2, decreased the BRET(2) signal by up to 50%. Likewise polylysine, but not the CK2 inhibitor DRB, decreased the signal in a dose-dependent manner up to 50%. For the first time, we present direct experimental evidence for CK2 holoenzyme aggregates in the cell. Our data suggest that CK2 activity may be controlled by holoenzyme aggregation, to our knowledge a novel mechanism for protein kinase regulation. Moreover, the BRET(2) sensor used in our study is a novel tool for studying CK2 regulation by aggregation and pharmacological screening for novel allosteric CK2 effectors.

Human topoisomerase I has been suggested to be implicated in the maintenance of genomic stability via its ability to regulate genome topology during transcription and replication. In the present study, we demonstrate by whole-genome array comparative genomic hybridization (aCGH) and fluorescence in situ hybridisation (FISH) analysis that topoisomerase I deficiency results in chromosome 5p gain in the cervical cancer cell line, HeLa-CCL2. In contrast, chromosome 5p copy number remained unaffected by topoisomerase I down-regulation in the non-cancer cell line HEK293T, as demonstrated by FISH analysis. Chromosome 5p gain is the most frequent genetic alteration in invasive cervical cancer, which leads to overexpression of genes involved in proliferation and occurs primarily at late stages in cancer development. The amplification of this region upon topoisomerase I down-regulation specifically in HeLa-CCL2 cells may indicate an important role of topoisomerase I in preventing malignant progression of precancerous lesions in the cervix.

An electrophile caught like a mouse in a trap! An anionic stopper-wheel complex acts as a supramolecular nucleophile in an almost quantitative synthesis of a phenyl ether rotaxane. The electrophilic semiaxle has to thread through the macrocycle in order to contact the bulky phenolate group that is positioned on the other side, and probably tightly held in place by hydrogen bonds., (© 1999 WILEY-VCH Verlag GmbH, Weinheim, Fed. Rep. of Germany.)