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CK2␣ is one of two isoforms of protein kinase CK2, a highly conserved, ubiquitous, and vital phosphotransferase whose expression is kept at constant cellular levels and whose dysregulated expression has been linked to malignant diseases. The upstream sequence of the gene coding for human CK2␣ (CSNK1A1, chromosomal location 20p13) has been examined for promoter location and transcription factor interactions using reporter gene assays (luciferase; HeLa cells), site-directed mutagenesis, electrophoretic mobility shift assays, super-shifts, UV cross-linking, Western blotting, and DNA affinity chromatography. Highest promoter activity has been found in a region comprising positions ؊9 to 46. Factors Sp1, Ets-1, and NF-B have been identified as interaction partners and, by mutation of individual sites and simultaneous mutations of two or more sites, shown to cross-talk to each other. At least two of the factors (Sp1; NF-B) were susceptible to phosphorylation by CK2 holoenzyme, a tetramer composed of two CK2␣ and two regulatory CK2␤ proteins, but not by individual CK2␣. Because the phosphorylation decreases promoter binding and repeated immunoprecipitation reveals presence of "free" CK2␤ in cell extracts, it is tempting to speculate that the gene product CK2␣ might readily form CK2 holoenzyme and feed back onto gene transcription. The data represent the first promoter control analysis of a mammalian CK2␣ gene and provide a hypothesis of how the constant expression level of CK2␣ may be achieved. Protein kinase CK21 (also named casein kinase II) is a pleiotropic, ubiquitous, and conserved Ser/Thr kinase that is essential for viability of eukaryotes. CK2 occurs in two highly related isoforms, CK2␣ and CK2␣Ј. Both of these occur as tetrameric holoenzymes complexed stoichiometrically to regulatory CK2␤ proteins. This tetrameric structure is also highly conserved and required for appropriate control of substrate specificity. Although a considerable number of substrates has been documented, comprising proteins involved in processes such as transcription, replication, translation, and signaling, the exact physiological role of CK2 remains poorly understood. However, CK2 has been linked to proliferation, transformation, and cell cycle regulation (reviewed in Refs.

Human casein kinase I1 (CKII) is a ubiquitous and multipotential Ser/Thr kinase involved in the regulation of cell growth and differentiation. Biochemically, two characteristics are particularly notable; first, the tetrameric composition of two catalytic subunits ( a and/or a') and two regulatory subunits @); second, the autophosphorylation of the holoenzyme at the N-terminus of CKIIP, suspected to be involved in tuning of the kinase activity. Whether CKIIa and CKIIa' reconstitute comparably with CKIIP to form holoenzyme is unclear. For a systematic investigation, the complete set of recombinant CKII subunits and of autophosphorylation mutants of C H I P were expressed in Escherichiu coli and comparative reconstitutions carried out. At 1 : 1 molar ratio, CKIIP stimulated both catalytic subunits roughly fivefold with phosvitin as a substrate. The level of activity reached with both of the reconstituted CKII isoforms was of the same order of magnitude as that of holoenzyme isolated from human placenta. It was also similar to a recombinant a,p2 holoenzyme whose expression had been attained in E. coli with a bicistronic construct containing the coding regions of CKIIP and CKIIa in a tandem arrangement. Both Ser2 and Ser3 were identified as the autophosphorylation sites ; replacement of one of these with Ala by oligonucleotide-mediated site-directed mutagenesis influenced only the extent of CKIIP autophosphorylation, replacement of both resulted in a loss of autophosphorylation. Despite these differences, the stimulatory effect of all the CKIIP mutants was comparable both to each other and to that of wild-type CKIIP. This was also obtained when substrates other than phosvitin were employed such as tubulin, or upstream-binding factor (UBF). However, the degree of stimulation was substrate specific and ranged from 2-5-fold with no major differences between CKIIa and CKIIa' stimulation. Calmodulin phosphorylation by both CKIIa and CKIIa' was decreased similarly by CKIIP and the CKIIP mutants. Proteins such as CAMP-1-esponsive-element-binding protein (CREB), HPVl6 E7 or Jun were not phosphorylated by either catalytic subunit but became substrates of both in the presence of CKIIP or CKIIP mutants. The data suggest that CKIIa and CKIIa' form similar CKII holoenzymes and that the tuning of holoenzyme activity is independent of the autophosphorylation status of CKIIP.Casein kinase type I1 (CKII) is one of three protein kinases named for their ability to phosphorylate acidic proteins such as casein. These are the authentic, mammary-gland-specific Golgi-located casein kinase and two operationally defined ubiquitous kinases, the casein kinase type I (CKI) and CKII. While Golgi-located CK definitely phosphorylates casein in vivo, CKI and CKII do not; these phosphorylate a vast array of cellular proteins including key factors of cell Correspondence to W. Pyerin, Biochemical Cell Physiology,

Protein kinase CK2, a heterotetramer composed of two catalytic subunits (alpha and/or alpha') and two regulatory subunits (beta), has been examined for intermolecular contact sites by methods that allow investigation of the native, unaltered proteins. Antibodies were raised against a series of 11 subunit peptides, affinity purified, and ensured for site specific binding by peptide competition. Chemical cross-linking of CK2 subunits with a hydrophilic carbodiimide and analysis of fused subunits and of CNBr-digested fusion products by immunoblotting with the sequence specific antibodies identified a tight interaction between positions beta55-70 and alpha65-80 (alpha'66-81) of subunits beta and alpha (alpha'), respectively. This was corroborated by cross-linking of subunits with peptides alpha65-80 and beta55-70 by a peptide-based enzyme-linked immunosorbent assay in which peptides bound to wells via C-10 spacer arms are probed for complexing individual subunits and immunoprecipitation with antibodies anti-alpha65-80 and anti-beta55-70, resulting in precipitation but not coprecipitation of subunits. This alpha-beta (alpha'-beta) interaction site obviously is also of functional importance since subunits with attached antibodies cannot reconstitute to the fully active holoenzyme. Indeed, sites beta55-70 and alpha65-80 (alpha'66-81) correspond to an acidic (beta) and a basic (alpha or alpha') domain involved in activity and stability control and in substrate and cosubstrate binding (kinase domain II/III), respectively. By contrast, a number of suspected contact sites were found to be rather loose and not essential for enzyme control as concluded from precipitation behavior of respective antibodies and the toleration of attached antibodies when active holoenzymes were being constituted. At subunit beta, these include the terminal positions beta2-14 and beta204-213, the positions beta97-105 and beta140-156, and, surprisingly, also beta171-186 which have been shown by deletion mutation and peptide replacement studies to represent a positively affecting interaction site. At subunits alpha and alpha', these are the C-terminal positions alpha329 -343 and alpha'336-350. Binding of antibodies to the positions alpha15-27 (alpha'16-28) and position alpha151-166(alpha'152-167), on the other hand, inhibits activity.

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Sites essential for tight physical intersubunit interaction in protein kinase CK2, a tetramer composed of two catalytic (A and/or A′) and two regulatory subunits (β), have been assigned to the C-terminal part of subunit β. Mutational analysis suggests region 171Ϫ181 of β to be one of these but this is not consistent with the observation of coprecipitation of catalytic subunits by antibodies directed specifically to this β segment which indicates that this region is accessible to antibodies even if the β subunit is associated with the A subunit. In an attempt to clarify the apparent contradiction, we have subdivided β-(155Ϫ181)-peptide, which includes the fragment of β and that both binds to catalytic subunits and stimulates kinase activity, into six more or less overlapping peptides with a length of 9Ϫ16 amino acid residues and performed peptide competition and A subunit binding assays. The kinase-stimulating effect of β-(155Ϫ 181)-peptide was counteracted significantly by β-(162Ϫ175)-peptide while the others had less or no effect. In an ELISA assay with the peptides covalently bound to wells via a C 10 spacer arm, binding of A occurred besides β-(155Ϫ181)-peptide only with β-(162Ϫ175)-peptide and β-(165Ϫ175)-peptide.The data provide the first evidence that the contact and the stimulation competences of subunit β represent separable functions and suggest further that a tight physical contact to catalytic subunits is located at position 162Ϫ175 consisting of a hydrophobic stretch (position 162Ϫ171) and a hydrophilic binding motif, PEY (position 172Ϫ174), with the adjoining downstream part allowing for surface exposure and antibody binding. The presence of several Pro residues within that region might relate to the structural basis for both the AϪβ interaction and the surface orientation.Keywords : protein kinase CK2; intersubunit contact; holoenzyme structure; human.The pleiotropic Ser/Thr protein kinase CK2 (also named various laboratories and various methods. Chemical cross-linking and peptide-based immunomapping of native, unaltered subcasein kinase II) is a vital, ubiquitously occurring enzyme (reviewed by Pinna, 1990; Tuazon and Traugh, 1991; Litchfield unit proteins provided direct evidence for functional contact between position 55Ϫ70 of subunit β and position 65Ϫ80 of sub-

Protein kinase CK2 is a conserved and vital Ser/Thr phosphotransferase with various links to malignant diseases, occurring as a tetramer composed of two catalytically active (CK2α and/or CK2α′) and two regulatory subunits (CK2β). There is balanced availability of CK2α and CK2β transcripts in proliferating and differentiating cultured cells. Examination of the human CK2β gene for transcriptionally active regions by systematic deletions and reporter gene assays indicates strong promoter activity at positions −42 to 14 and 12 to 72 containing transcription start sites 1 and 2 of the gene (positions +1 and 33), respectively, an upstream and a downstream enhancer activity at positions −241 to −168 and 123 to 677, respectively, and silencer activity at positions −241 to −261. Of the various transcription factor binding motifs present in those regions, Ets1 and CAAT‐related motifs turned out to be of particular importance, Ets1 for promoter activation and CAAT‐related motifs for enhancer activation. In addition, there are contributions by Sp1. Most strikingly, the Ets1 region representing two adjoining consensus motifs also occurs with complete identity in the recently characterized promoter of the CK2α gene [Krehan, A., Ansuini, H., Böcher, O., Grein, S., Wirkner, U. & Pyerin, W. (2001) J. Biol. Chem.275, 18327–18336], and affects comparably, when assayed in parallel, the promoters of both CK2 genes, both by motif mutations and by Ets1 overexpression. The data strongly support the hypothesis that Ets1 acts as a common regulatory element of the CK2α and CK2β genes involved in directing coordinate transcription and contributing to the balanced availability of transcripts.