Activation of human topoisomerase I by protein kinase CK2

The enzymatic studies were performed to reveal a mode of activation of human topoisomerase I by a direct interaction with protein kinase CK2. In the absence of ATP CK2 kinase activated DNA relaxation about twofold. CK2 subunit was identified as solely responsible for the stimulation of relaxing activity by CK2 kinase. CK2 activated the relaxation only at the excess of the substrate over topoisomerase I. At the equimolar ratio of the substrate DNA and topoisomerase I the activation was not observed. There was also no effect of CK2 on camptothecin-induced cleavage of DNA by htopo I. These results identify an accelerated movement of topoisomerase I between substrate molecules as a cause of the activation of DNA relaxation by CK2 kinase.     

Interactions of protein kinase CK2 subunits

Several approaches have been used to study the interactions of the subunits of protein kinase CK2. The inactive mutant of CK2 that has Asp 156 mutated to Ala (CK2A156) is able to bind the CK2 subunit and to compete effectively in this binding with wild-type subunits and . The interaction between CK2A156 and CK2 was also demonstrated by transfection of epitope-tagged cDNA constructs into COS-7 cells. Immunoprecipitation of epitope-tagged CK2A156 coprecipitated the subunit and vice-versa. The assay of the CK2 activity of the extracts obtained from cells transiently transfected with these different subunits yielded some surprising results: The CK2 specific phosphorylating activity of these cells transfected with the inactive CK2A156 was considerably higher than the control cells transfected with vectors alone. Assays of the immunoprecipitated CK2A156 expressed in these cells, however, demonstrated that the mutant was indeed inactive. It can be concluded that transfection of the inactive CK2A156 affects the endogenous activity of CK2. Transfection experiments with CK2 and subunits and CK2A156 were also used to confirm the interaction of CK2 with the general CDK inhibitor p21WAF1/CIP1 co-transfected into these cells. Finally a search in the SwissProt databank for proteins with properties similar to those derived from the amino acid composition of CK2 indicated that CK2 is related to protein phosphatase 2A and to other phosphatases as well as to a subunit of some ion-transport ATPases.     

Expression and localization of epitope-tagged protein kinase CK2

Protein kinase CK2, formerly known as casein kinase II, is a ubiquitous protein serine/threonine kinase. The enzyme exists in tetrameric complexes composed of two catalytic (CK2α and/or CK2α′) subunits and two subunits (CK2β) that appear to have a role in modulating the activity of the catalytic subunits. With the exception of their unrelated carboxy-terminal domains, the two isozymic forms of mammalian CK2 display extensive sequence identity. Furthermore, CK2α and CK2α′ exhibit remarkable conservation between species, suggesting that they may have unique functions. In the present study, the cDNAs encoding CK2α and CK2α′ were modified by addition of the hemagglutinin tag of the influenza virus at the amino terminus of the respective proteins. The epitope-tagged proteins were transfected into Cos-7 cells and the localization of the expressed proteins determined by indirect immunofluorescence using monoclonal antibodies specific for the epitope tag. The use of transfection favors the formation of homotetrameric complexes (i.e., α₂β₂, α′₂β₂) instead of heterotetrameric complexes (i.e., αα′β₂) that are present in many cells. Epitope-tagged CK2α and CK2α′ displayed kinase activity and the ability to form complexes with CK2β. The results of these studies also indicate definitively that CK2α and CK2α′ are both localized predominantly within the nucleus. Mutation of conserved lysine residues within the ATP binding domains of CK2α and CK2α′ resulted in loss of kinase activity. However, examination of these mutants indicates that kinase activity is not essential for formation of complexes between subunits of CK2 and is not required for nuclear localization of CK2. J. Cell. Biochem. 64: 525–537. © 1997 Wiley-Liss, Inc.     

Allosteric transcription stimulation by RNA polymerase II super elongation complex

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Phosphorylation of human high mobility group N1 protein by protein kinase CK2

High mobility group (HMG) N1 protein, formerly known as HMG 14, is a member of the chromosomal HMG protein family. Protein kinase CK2 was previously reported to be able to phosphorylate bovine HMGN1 in vitro; Ser89 and Ser99, corresponding to Ser88 and Ser98 in human HMGN1, were shown to be major and minor recognition sites, respectively. In this report, we employed mass spectrometry and examined both the extent and the sites of phosphorylation in HMGN1 protein catalyzed by recombinant human protein kinase CK2. We found that five serine residues, i.e., Ser6, Ser7, Ser85, Ser88, and Ser98, in HMGN1 can be phosphorylated by the kinase in vitro. All five sites were previously shown to be phosphorylated in MCF-7 human breast cancer cells in vivo. Among these five sites, Ser6, Ser7, and Ser85 were new sites of phosphorylation induced by protein kinase CK2 in vitro.     

Localization of individual subunits of protein kinase CK2 to the endoplasmic reticulum and to the Golgi apparatus

The protein kinase CK2 is composed of two catalytic - or - and two regulatory -subunits. In mammalian cells there is ample evidence for the presence of individual CK2 subunits beside the holoenzyme. By immunofluorescence studies using peptide antibodies which allow us to detect the CK2-, - and -subunits we found all three subunits to be co-localized with a 58 KDa Golgi protein which is specific for the Golgi complex. Subfractionation studies using dog pancreas cells revealed the presence of all three subunits of CK2 at the smooth endoplasmic reticulum (sER)/Golgi fraction whereas the rough endoplasmic reticulum (rER) harboured only the catalytic - and -subunits. We found that the microsomal preparation from dog pancreas cells contained CK2 which phosphorylated a CK2 specific synthetic peptide and which was heparin sensitive. Furthermore, we could immunoprecipitate the CK2-subunit that exhibited a kinase activity which phosphorylated a CK2 specific substrate and which was heparin sensitive. Protease digestion experiments revealed that the CK2 subunits were located on the cytosolic side of the rER and the sER/Golgi complex. Thus, we could demonstrate an asymmetric distribution of the CK2 subunits at the rER and sER/Golgi complex. Since the CK2- and -subunits exhibit a substrate specificity which is different from the CK2 holoenzyme one might speculate that the asymmetric distribution of the CK2 holoenzyme and the CK2 catalytic subunits may have regulatory functions.