Protein kinase CK2: from structures to insights

European Journal …, 2000

Eur J Biochem, 2001

The crystal structure of a complex between the catalytic a subunit of Zea mays CK2 and a 23-mer peptide corresponding the C-terminal sequence 181±203 of the human CK2 regulatory b subunit has been determined at 3.16-A Ê resolution. The complex, composed of two a chains and two peptides, presents a molecular twofold axis, with each peptide interacting with both a chains. In the derived model of the holoenzyme, the regulatory subunits are positioned on the opposite side with respect to the opening of the catalytic sites, that remain accessible to substrates and cosubstrates. The b subunit can influence the catalytic activity both directly and by promoting the formation of the a 2 dimer, in which each a chain interacts with the active site of the other. Furthermore, the two active sites are so close in space that they can simultaneously bind and phosphorylate two phosphoacceptor residues of the same substrate.

The EMBO Journal, 1998

CK2α is the catalytic subunit of protein kinase CK2, an acidophilic and constitutively active eukaryotic Ser/Thr kinase involved in cell proliferation. A crystal structure, at 2.1 Å resolution, of recombinant maize CK2α (rmCK2α) in the presence of ATP and Mg 2⍣ , shows the enzyme in an active conformation stabilized by interactions of the N-terminal region with the activation segment and with a cluster of basic residues known as the substrate recognition site. The close interaction between the N-terminal region and the activation segment is unique among known protein kinase structures and probably contributes to the constitutively active nature of CK2. The active centre is occupied by a partially disordered ATP molecule with the adenine base attached to a novel binding site of low specificity. This finding explains the observation that CK2, unlike other protein kinases, can use both ATP and GTP as phosphorylating agents. Keywords: activation segment/co-substrate specificity/ N-terminal region/protein kinase CK2/X-ray crystallography

2000

CK2, one of the first protein kinase ever discovered, is an eukaryotic acidophilic Ser/Thr protein kinase. CK2 is considered a quite anomalous protein kinase for the following peculiar properties: i) it is highly pleiotropic, ii) it can use both ATP and GTP as co-substrate, iii) the target serine or threonine must be surrounded by acidic residues (with the minimal consensus sequence Ser/Thr-X-X-Asp/ Glu), and iv) the CK2α catalytic subunit is intrinsically active [1]. More than 300 substrates are known for CK2. The regulatory mechanism of this kinase is still a matter of debate, and it was the subject of extensive investigation. The catalytic subunit of this enzyme is intrinsically active. CK2 is involved in many cellular processes such as cell cycle regulation, circadian rhythms, gene expression, cell growth and differentiation, embryogenesis and apoptosis. CK2 can be considered a valuable drug target for cancer therapy essentially on the basis of the following arguments: a) at protein level, CK2 is elevated in various cancers; b) it is a potent suppressor of apoptosis and strongly promotes the survival of the cell; c) it strengthens the multi-drug resistant phenotype; d) for the previous reasons, it establishes favourable conditions for tumorigenesis [2]. An important CK2 feature that influences the inhibitor design process is constitutive activity, with the consequence that only the active conformation can be targeted. The catalytic site of CK2 displays some unique properties that can be exploited in the design of inhibitors with a high degree of specificity, as indicated by the ability to utilize both ATP and GTP and by the low sensitivity to staurosporine inhibition (IC 50 of 19.5 μM versus values in the low nanomolar range for other kinases). Actually, as described below, fairly specific, potent, and cell-permeable inhibitors of CK2 have been successfully developed in the last years [3][4][5][6][7]. From the analysis of the known maize and human CK2α co-crystal structures, it was noted that if a negatively charged moiety is present in a ligand (inhibitor or co-substrate) it tends to cluster in a well specific zone of the ATPbinding cleft, near the salt bridge Lys68-Glu81. A quantitative analysis of the electrostatic potential in the CK2α active site revealed the presence of a positively charged region located in the deeply buried area of the cavity, between the hydrophobic region I and the salt bridge formed by the fully conserved Lys68 and Asp81, with a mean positive electrostatic potential of 1.5-2.0 kcal/mol. As seen by the systematic analysis of the binding of different classes of CK2 inhibitors, the electrostatic interaction with this area is responsible for the different orientation of the ligands in the active site of CK2. A striking example of this effect is that seen for the different binding modes of the two closely related tetrabromobenzo derivatives TBB and TBI. TBB, with a pK a ~5, binds with the triazole ring inside the positive area, while TBI, with a pK a ~9, is shifted towards the hinge region and forms two halogen bonds with Glu114 and Val116, like all the other tetrabromobenzo-imidazole derivatives analysed so far.

1999

Protein kinase CK2 is characterized by a number of features, including substrate specificity, inhibition by polyanionic compounds and intrasteric down-regulation by its β-subunit, which denote a special aptitude to interact with negatively charged ligands. This situation may reflect the presence in CK2 catalytic subunits of several basic residues that are not conserved in the majority of other protein kinases. Some of these residues, notably K49 in the 'Gly rich loop', K74, K75, K76, K77, K79, R80, K83 in the 'Lys rich segment' and R191, R195, K198 in the 'p+1 loop', have been shown by mutational studies to be implicated to various extents and with distinct roles in substrate recognition, inhibition by heparin and by pseudosubstrate and instrasteric regulation. Molecular modelization based on crystallographic data provide a rationale for the biochemical observations, showing that several of these basic residues are clustered around the active site where they make contact with individual acidic residues of the peptide substrate. They can also mediate the effect of polyanionic inhibitors (e.g. heparin) and of regulatory elements present in the β-subunit, in the N terminal segment of the catalytic subunit and possibly in other proteins interacting with CK2. Our data also disclose a unique mode of binding of the phosphoacceptor substrate which bridges across the catalytic cleft making contacts with both the lower and upper lobes of CK2. (Mol Cell Biochem 191: 13-19, 1999)

Journal of Molecular Biology, 2003

Protein kinase CK2 (formerly called: casein kinase 2) is a heterotetrameric enzyme composed of two separate catalytic chains (CK2a) and a stable dimer of two non-catalytic subunits (CK2b). CK2a is a highly conserved member of the superfamily of eukaryotic protein kinases. The crystal structure of a C-terminal deletion mutant of human CK2a was solved and refined to 2.5 Å resolution. In the crystal the CK2a mutant exists as a monomer in agreement with the organization of the subunits in the CK2 holoenzyme. The refined structure shows the helix aC and the activation segment, two main regions of conformational plasticity and regulatory importance in eukaryotic protein kinases, in active conformations stabilized by extensive contacts to the N-terminal segment. This arrangement is in accordance with the constitutive activity of the enzyme. By structural superimposition of human CK2a in isolated form and embedded in the human CK2 holoenzyme the loop connecting the strands b4 and b5 and the ATP-binding loop were identified as elements of structural variability. This structural comparison suggests that the ATP-binding loop may be the key region by which the non-catalytic CK2b dimer modulates the activity of CK2a. The b4/b5 loop was found in a closed conformation in contrast to the open conformation observed for the CK2a subunits of the CK2 holoenzyme. CK2a monomers with this closed b4/b5 loop conformation are unable to bind CK2b dimers in the common way for sterical reasons, suggesting a mechanism to protect CK2a from integration into CK2 holoenzyme complexes. This observation is consistent with the growing evidence that CK2a monomers and CK2b dimers can exist in vivo independently from the CK2 holoenzyme and may possess physiological roles of their own.

The EMBO Journal, 1999

Protein kinase CK2 is a tetramer composed of two α catalytic subunits and two β regulatory subunits. The structure of a C-terminal truncated form of the human β subunit has been determined by X-ray crystallography to 1.7 Å resolution. One dimer is observed in the asymmetric unit of the crystal. The most striking feature of the structure is the presence of a zinc finger mediating the dimerization. The monomer structure consists of two domains, one entirely α-helical and one including the zinc finger. The dimer has a crescent shape holding a highly acidic region at both ends. We propose that this acidic region is involved in the interactions with the polyamines and/or catalytic subunits. Interestingly, conserved amino acid residues among β subunit sequences are clustered along one linear ridge that wraps around the entire dimer. This feature suggests that protein partners may interact with the dimer through a stretch of residues in an extended conformation.

Cet article dresse un premier bilan des recherches sur la confiance dans le contexte du commerce électronique « grand public ». Les spécificités du concept, sa définition et sa dimensionnalité sont d’abord examinées. Une typologie des déterminants de la confiance électronique est ensuite présentée. Des éléments de réflexion sur sa dynamique sont enfin proposés. The aim of this paper is to provide a literature review of research on online consumer trust. First, the concept specificities are identified and the construct definition and dimensionality are examined. Then, a taxonomy of the online trust determinants is suggested. Finally, to gain understanding of the concept evolvement over the time, some guidelines are proposed. oui

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