Olaf-Georg Issinger

CK2: a protein kinase in need of control

Protein kinase CK2 is a heterotetrameric alpha2beta2 Ser/Thr protein kinase with some features unusual among the eukaryotic protein kinases: (1) CK2 recognizes phosphoacceptor sites specified by several acidic determinants; (2) CK2 can use both ATP and GTP as phosphoryl donors; and (3) the regulatory properties of CK2 are poorly understood; it is insensitive to any known second messenger and displays high basal activity. To gain insight into CK2 regulation and to understand its unusual properties, site-directed mutagenesis experiments on both subunits and X-ray crystallographic studies of the catalytic alpha-subunit were performed. The noncatalytic beta-subunit has at least three functions: (1) it protects the alpha-subunit against denaturing agents or conditions; (2) it alters the substrate specificity of the alpha-subunit; and (3) it modulates the activity of the enzyme, i.e., depending on the substrate, it increases or decreases the activity of the alpha-subunit. Mutagenesis experiments revealed that an acidic stretch between amino acids 55 and 64 has a down-regulatory and autoinhibitory function. Mutational analysis of the alpha-subunit has revealed a network of unique basic residues that are responsible for the recognition of phosphoacceptor substrates and for down-regulation by the beta-subunit and by polyanionic inhibitors. The resolution of the crystal structure of Zea mays CK2 alpha-subunit has disclosed the structural features that are responsible for high basal activity and for unusual response to nucleotide analogs. The increasing knowledge of CK2 structure-function relationships will allow the design of highly selective inhibitors of this pleiotropic kinase with oncogenic potential.

Enhanced casein kinase II activity in human tumour cell cultures.

Casein kinase II (CKII) activity is enhanced as much as 2–3 fold in established and 4–5‐fold in transformed human cell lines when compared to that of fibroblasts and primary human tumour cell cultures where CKII activity never exceeded a basic level. The high activity of CKII in transformed cells and in established cell lines was reduced to about the same basic level after treatment with heparin, a highly specific inhibitor of CKII activity. The activity of the cAMP‐dependent protein kinase was virtually the same in fibroblasts and various human tumour cell lines investigated.

Casein kinase-2 structure-function relationship: Creation of a set of mutants of the β subunit that variably surrogate the wildtype β subunit function

Abstract Nine mutants of human casein kinase-2 β subunit have been created and assayed for their ability to assemble with the catalytic α subunit to give, at a 1:1 molar ratio, a fully competent CK-2 holoenzyme as judged by the following criteria: 1) the generation of an active heterotetrameric form of CK-2 exhibiting the expected sedimentation coefficient and 2) the enhancement of catalytic activity of CK-2 α. Extended deletions of 71 and 44 residues from the C-terminal end, but not a 7 residue deletion (including the cdc2 phosphorylation site) prevent both reconstitution of the holoenzyme and, consequently, stimulation of activity. This indicates that residue (s) located in the 171 – 209 sequence is essential for reconstitution. Also a four residues N-terminal deletion (removing the autophosphorylation site) and single to quintuple substitutions of alanine for the acidic residues clustered in the 55 – 70 sequence give rise to mutants that still assemble with the α subunit to give a tetrameric holoenzyme. However, in the case of the mutants A 57,59 , A 63,64 A 59–61,63,64 in vitro assembly with the CK-2 α subunit was not complete. There were also intermediate complexes, free α-subunit and β-mutants found to sediment at various positions in the sucrose density gradient. In comparison to CK-2 β + , mutants A 57,59 , A 59–61 and A 59–61,63,64 show an increased stimulation of the catalytic activity supporting the view that these residues play a crucial role in determining the basal activity of reconstituted CK-2 holoenzyme.

Increased detectability of somatic changes in the DNA from human tumours after probing with "synthetic" and "genome-derived" hypervariable multilocus probes.

SummaryDNA fingerprinting with two minisatellite (33.15, M13) and two simple repeat probes [(GACA)4, (CAC)4/ (GTG)5] was performed to screen for somatic changes in the DNA from various solid human tumours in comparison with constitutional DNA from the same patient. Loss of bands or changes in band intensitities were observed. Together the probes 33.15 and (CAC)5/(GTG)5 detected deviating fingerprint patterns in 63% of the colorectal carcinomas investigated. In mammary and stomach carcinomas, only 1/11 and 2/11 tumours, respectively, showed differences with either of the three probes, 33.15, (GACA)4 and (CAC)5/(GTG)5.

Expression of Casein Kinase 2 during Mouse Embryogenesis

This paper deals with the expression and distribution of casein kinase 2 (CK-2) subunits in mouse embryos at different developmental stages. Expression was investigated at the mRNA level of CK-2 alpha- and beta-subunits by in situ hybridization and distribution at the protein level by immunohistochemistry using CK-2-alpha- and CK-2-beta-specific antibodies, respectively. In general both methods gave similar results. In earlier stages of mouse embryonic development (day 10.5 after coitus) CK-2 was more expressed in neuroepithelia than in all other tissues. From day 11.5 after coitus on, high expression of CK-2 was detected in all epithelia. From day 16.5 on, all tissues and anlagen of the fetus involved in organogenesis revealed a higher CK-2 expression as compared with secondary mesenchyma. The only difference between in situ hybridization and immunocytochemistry was observed in the skin. Transcripts of CK-2 were found mostly in the basal layer of the epidermis and in the nuclei of keratinocytes, whereas CK-2 protein was almost exclusively found in the cytoplasm of epidermal cells.

Nucleolin (C23), a physiological substrate for casein kinase II

Nucleolin (C23), a 110 kDa phosphoprotein, which is mainly found in the nucleolus has been shown to be a physiological substrate for casein kinase II (CKII). Nucleolin was identified and characterized by immunodetection using an anti-nucleolin antibody. Phosphopeptide patterns from nucleolin phosphorylated by purified casein kinase II and of phosphorylated nucleolin which had been isolated from tumor cells grown in the presence of [32P]-o-phosphate, were identical. The partial tryptic digest revealed nine phosphopeptides. Nucleolin isolated from Krebs II mouse ascites cells was phosphorylated by purified casein kinase II with about two moles phosphate per one mole of nucleolin.

Expression and characterization of a recombinant maize CK-2 α subunit

Abstract CKIIB, one of the CK-2 like enzymes which have been isolated from maize, has been shown to be a monomeric enzyme that cross-reacts with anti CK-2 α specific antibodies suggesting a possible relationship between the two proteins (Dobrowolska et al. (1992) Eur. J. Biochem. 204, 299–303). In order to support the immunological data also by biochemical and biophysical experiments the availability of a recombinant CK-2 α from maize was a prerequisite. A maize cDNA clone of maize CK-2 α was expressed in the bacterial strain BL21 (DE3). The recombinant protein was purified to homogeneity; its molecular mass on one-dimensional SDS PAGE was estimated to be 36.5 kDa. The calculated molecular mass according to the amino acid composition is 39228 Da (332 amino acids). The recombinant maize CK-2 α (rmCK-2 α) exhibited mostly the same properties as the recombinant human CK-2 α (rhCK-2 α). In several respects it behaved differently from CKIIB, thus supporting the notion that either CKIIB is encoded by another gene or it undergoes extensive posttranscriptional and/or posttranslational alterations. Three observations in particular disprove any close relatedness between CKIIB and rmCK-2 α, namely: (a) the phosphorylation of calmodulin by CKIIB is dramatically stimulated by polylysine, whereas polylysine inhibits rather than stimulating the phosphorylation of calmodulin by rmCK-2 α (and by rhCK-2 α). (b) Addition of rhCK-2 β has no significant influence on the stimulation of the calmodulin phosphorylation by CKIIB whereas in the case of rmCK-2 α and rhCK-2 α addition of rhCK-2 β is required for optimal stimulation by polylysine. (c) CKIIB does not self-assemble with rhCK-2 β to form a high molecular mass complex as it is demonstrated for rmCK-2 α.

Characterization of casein kinase II in human colonic carcinomas after heterotransplantation into nude mice

Casein kinase II (CKII) activity in colorectal tumours was compared before and after heterotransplantation onto nude mice. The test revealed that the enzyme activity was about two-fold enhanced in the tumours isolated from the nude mice when compared to the respective primary tumours from which they were derived. Immunoblots using a polyclonal CKII-specific antibody showed that the increase of activity was due to a higher expression of the enzyme. Immunohistochemical studies on cross sections of nude mouse tumours showed that most of the CKII molecules were located at the peripheral part of the tumour; the central part did not show intense CKII-specific staining.

Growth-dependent modulation of casein kinase II and its substrate nucleolin in primary human cell cultures and HeLa cells

We have previously provided evidence that casein kinase II (CKII) and its substrate nucleolin increase concomitantly during certain development stages during embryogenesis (Schneider et al., Eur. J. Biochem. 161, 733-738). We now show that during normal growth of primary cell cultures and HeLa cells CKII activity is increased concomitant with cellular growth and that the activity declines when confluency is reached. Parallel to the CKII activity increase, nucleolin, which has been shown to be a potential substrate of CKII changes its phosphorylation status, reaching a maximum at the time when CKII activity is highest and decreasing again when CKII activity declines.

The β subunit of casein kinase II: cloning of cDNAs from murine and porcine origin and expression of the porcine sequence as a fusion protein☆

cDNAs encoding the beta subunit of pig and mouse CKII were isolated. The porcine cDNA was expressed as a fusion protein in Escherichia coli and used for the production of anti-CKII-beta subunit specific antibodies.