Brigitte Boldyreff

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.

Cloning and sequencing of the casein kinase 2 α subunit from Zea mays

The nucleotide sequence of the cDNA coding for the alpha subunit of casein kinase 2 of Zea mays has been determined. The cDNA clone contains an open reading frame of 996 nucleotides encoding a polypeptide comprising 332 amino acids. The primary amino acid sequence exhibits 75% identity to the alpha subunit and 71% identity to the alpha subunit of human casein kinase 2.

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.

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.

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.

The autophosphorylation and p34cdc2 phosphorylation sites of casein kinase-2 β-subunit are not essential for reconstituting the fully-active heterotetrameric holoenzyme

Two mutants of human casein kinase-2 beta-subunit with short deletions at either their amino (delta 1-4) or carboxy (delta 209-215) terminal side have been created that have lost the capability to undergo autophosphorylation and p34cdc2 mediated phosphorylation, respectively. Both mutants give rise to reconstituted CK2 holoenzymes displaying basal catalytic activity, thermostability and responsiveness to polylysine, identical to those of wild-type holoenzyme, whose reconstitution, moreover, is not affected by previous phosphorylation of the beta-subunit at either its N-terminal or C-terminal sites. Unlike the wild-type beta and beta(delta 209-215), however, beta(delta 1-4) fails to confer to the reconstituted holoenzyme the typical responsiveness to NaCl stimulation. These results suggest that while neither the autophosphorylation nor the p34cdc2 phosphorylation sites are required for conferring a stable structure and full catalytic activity to CK2, the autophosphorylation site is implicated in the NaCl-dependent fine tuning of CK2 activity.

Phosphorylation of the Epstein-Barr virus nuclear antigen 2

A major in vivo phosphorylation site of the Epstein-Barr virus nuclear antigen 2 (EBNA-2) was found to be localized at the C-terminus of the protein. In vitro phosphorylation studies using casein kinase 1 (CK-1) and casein kinase 2 (CK-2) revealed that EBNA-2 is a substrate for CK-2, but not for CK-1. The CK-2 specific phosphorylation site was localized in the 140 C-terminal amino acids using a recombinant trpE-C-terminal fusion protein. In a similar experiment, the 58 N-terminal amino acids expressed as a recombinant trpE-fusion protein were not phosphorylated. Phosphorylation of a synthetic peptide corresponding to amino acids 464-476 of EBNA-2 as a substrate led to the incorporation of 0.69 mol phosphate/mol peptide indicating that only one of three potential phosphorylation sites within the peptide was modified. The most likely amino acid residues for phosphorylation by CK-2 are Ser469 and Ser470.

Dissection of the dual function of the β‐subunit of protein kinase CK2 (‘casein kinase‐2’): a synthetic peptide reproducing the carboxyl‐terminal domain mimicks the positive but not the negative effects of the whole protein

The dual function of the regulatory β‐subunit of protein kinase CK2 is highlighted by its ability to abolish calmodulin phosphorylation in contrast to its stimulatory effect on the phosphorylation of peptide substrates. Here we show that a synthetic peptide reproducing the C‐terminal region of the β‐subunit (β[170–215]) stimulates to a similar extent the phosphorylation of either the peptide substrate or calmodulin and also protects the catalytic α‐subunit against thermal inactivation as efficiently as full‐length β‐subunit. These data show that the positive and negative functions of the β‐subunit reside in physically separated domains and that the elements responsible for positive regulation are located in the C‐terminal region.

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.

Assignment of casein kinase 2 alpha sequences to two different human chromosomes.

SummaryHuman casein kinase 2 alpha gene (CK-2-alpha) sequences have been localized within the human genome by in situ hybridization and somatic cell hybrid analysis using a CK-2 alpha cDNA as a probe. By in situ hybridization, the CK-2 alpha cDNA could be assigned to two different loci, one on 11p15.1-ter and one on 20p13. The existence of two separate chromosomal loci suggests that CK-2 alpha is a member of a gene family. Only the locus on chromosome 11 was confirmed by somatic cell hybrid analysis. The analysis was based on the presence of a CK-2-alpha-specific 20-kb fragment. However, the CK-2 alpha cDNA hybridizes to several additional fragments in total human DNA.