Subcellular localization of casein kinase I

An anti-yeast CKI antiserum was shown to cross-react with CKI isolated from Krebs II mouse ascites tumour cells. The mammalian CKI showed virtually the same molecular mass (app. 45 kDa) as the yeast enzyme. By immunofluorescence it could be shown that CKI is preferably located in the nucleolus.     

Development of a high-throughput screening-compatible assay to identify inhibitors of the CK2α/CK2β interaction

Increased activity of protein kinase CK2 is associated with various types of cancer, neurodegenerative diseases, and chronic inflammation. In the search for CK2 inhibitors, attention has expanded toward compounds disturbing the interaction between CK2α and CK2β in addition to established active site-directed approaches. The current article describes the development of a fluorescence anisotropy-based assay that mimics the principle of CK2 subunit interaction by using CK2α^1–335 and the fluorescent probe CF-Ahx-Pc as a CK2β analog. In addition, we identified new inhibitors able to displace the fluorescent probe from the subunit interface on CK2α^1–335. Both CF-Ahx-Pc and the inhibitors I-Pc and Cl-Pc were derived from the cyclic peptide Pc, a mimetic of the C-terminal CK2α-binding motif of CK2β. The design of the two inhibitors was based on docking studies using the known crystal structure of the Pc/CK2α^1–335 complex. The dissociation constants obtained in the fluorescence anisotropy assay for binding of all compounds to human CK2α^1–335 were validated by isothermal titration calorimetry. I-Pc was identified as the tightest binding ligand with a K_D value of 240 nM and was shown to inhibit the CK2 holoenzyme-dependent phosphorylation of PDX-1, a substrate requiring the presence of CK2β, with an IC₅₀ value of 92 μM.     

The C-terminal domain of human grp94 protects the catalytic subunit of protein kinase CK2 (CK2alpha) against thermal aggregation. Role of disulfide bonds.

The C-terminal domain (residues 518-803) of the 94 kDa glucose regulated protein (grp94) was expressed in Escherichia coli as a fusion protein with a His6-N-terminal tag (grp94-CT). This truncated form of grp94 formed dimers and oligomers that could be dissociated into monomers by treatment with dithiothreitol. Grp94-CT conferred protection against aggregation on the catalytic subunit of protein kinase CK2 (CK2alpha), although it did not protect against thermal inactivation. This anti-aggregation effect of grp94-CT was concentration dependent, with full protection achieved at grp94-CT/CK2alpha molar ratios of 4 : 1. The presence of dithiothreitol markedly reduced the anti-aggregation effects of grp94-CT on CK2alpha without altering the solubility of the chaperone. It is concluded that the chaperone activity of the C-terminal domain of human grp94 requires the maintenance of its quaternary structure (dimers and oligomers), which seems to be stabilised by disulphide bonds.     

ATP sensitive bi-quinoline activator of the AMP-activated protein kinase

The AMP-activated protein kinase (AMPK) regulates cellular and whole-body energy balance in response to changes in adenylate charge and hormonal signals. Activation of AMPK in tissues such as skeletal muscle and liver reverses many of the metabolic defects associated with obesity and Type 2 diabetes. Here we report a bi-quinoline (JJO-1) that allosterically activates all AMPK αβγ isoforms in vitro except complexes containing the γ3 subunit. JJO-1 does not directly activate the autoinhibited α subunit kinase domain and differs among other known direct activators of AMPK in that allosteric activation occurs only at low ATP concentrations, and is not influenced by either mutation of the γ subunit adenylate-nucleotide binding sites or deletion of the β subunit carbohydrate-binding module. Our findings indicate that AMPK has multiple modes of allosteric activation that may be exploited to design isoform-specific activators as potential therapeutics for metabolic diseases.     

The Protein Kinase CK2 Holoenzyme Structure Supports Proposed Models of Autoregulation and Trans-Autophosphorylation

Eukaryotic protein kinases are typically strictly controlled by second messenger binding, protein/protein interactions, dephosphorylations or similar processes. None of these regulatory mechanisms is known to work for protein kinase CK2 (former name “casein kinase 2”), an acidophilic and constitutively active eukaryotic protein kinase. CK2 predominantly exists as a heterotetrameric holoenzyme composed of two catalytic subunits (CK2α) complexed to a dimer of non-catalytic subunits (CK2β). One model of CK2 regulation was proposed several times independently by theoretical docking of the first CK2 holoenzyme structure. According to this model, the CK2 holoenzyme forms autoinhibitory aggregates correlated with trans-autophosphorylation and driven by the down-regulatory affinity between an acidic loop of CK2β and the positively charged substrate binding region of CK2α from a neighboring CK2 heterotetramer. Circular trimeric aggregates in which one-half of the CK2α chains show the predicted inhibitory proximity between those regions were detected within the crystal packing of the human CK2 holoenzyme. Here, we present further in vitro support of the “regulation-by-aggregation” model by an alternative crystal form in which CK2 tetramers are arranged as approximately linear aggregates coinciding essentially with the early predictions. In this assembly, the substrate binding region of every CK2α chain is blocked by a CK2β acidic loop from a neighboring tetramer. We found these crystals with CK2^Andante that contains a CK2β variant mutated in a CK2α-contact helix and described to be responsible for a prolonged circadian rhythm in Drosophila. The increased propensity of CK2^Andante to form aggregates with completely blocked active sites may contribute to this phenotype.     

Evolved to be active: sulfate ions define substrate recognition sites of CK2alpha and emphasise its exceptional role within the CMGC family of eukaryotic protein kinases

CK2alpha is the catalytic subunit of protein kinase CK2 and a member of the CMGC family of eukaryotic protein kinases like the cyclin-dependent kinases, the MAP kinases and glycogen-synthase kinase 3. We present here a 1.6 A resolution crystal structure of a fully active C-terminal deletion mutant of human CK2alpha liganded by two sulfate ions, and we compare this structure systematically with representative structures of related CMGC kinases. The two sulfate anions occupy binding pockets at the activation segment and provide the structural basis of the acidic consensus sequence S/T-D/E-X-D/E that governs substrate recognition by CK2. The anion binding sites are conserved among those CMGC kinases. In most cases they are neutralized by phosphorylation of a neighbouring threonine or tyrosine side-chain, which triggers conformational changes for regulatory purposes. CK2alpha, however, lacks both phosphorylation sites at the activation segment and structural plasticity. Here the anion binding sites are functionally changed from regulation to substrate recognition. These findings underline the exceptional role of CK2alpha as a constitutively active enzyme within a family of strictly controlled protein kinases.     

Interaction between CK2α and CK2β, the subunits of protein kinase CK2: thermodynamic contributions of key residues on the CK2α surface

The protein Ser/Thr kinase CK2 (former name: casein kinase II) exists predominantly as a heterotetrameric holoenzyme composed of two catalytic subunits (CK2α) bound to a dimer of noncatalytic subunits (CK2β). We undertook a study to further understand how these subunits interact to form the tetramer. To this end, we used recombinant, C-terminal truncated forms of human CK2 subunits that are able to form the holoenzyme. We analyzed the interaction thermodynamics between the binding of CK2α and CK2β as well as the impact of changes in temperature, pH, and the ionization enthalpy of the buffer using isothermal titration calorimetry (ITC). With structure-guided alanine scanning mutagenesis we truncated individual side chains in the hydrophobic amino acid cluster located within the CK2α interface to identify experimentally the amino acids that dominate affinity. The ITC results indicate that Leu41 or Phe54 single mutations were most disruptive to binding of CK2β. Additionally, these CK2α mutants retained their kinase activity. Furthermore, the substitution of Leu41 in combination with Phe54 showed that the individual mutations were not additive, suggesting that the cooperative action of both residues played a role. Interestingly, the replacement of Ile69, which has a central position in the interaction surface of CK2α, only had modest effects. The differences between Leu41, Phe54, and Ile69 in interaction relevance correlate with solvent accessibility changes during the transition from unbound to CK2β-bound CK2α. Identifying residues on CK2α that play a key role in CK2α/CK2β interactions is important for the future generation of small molecule drug design.     

Enzymatic activity with an incomplete catalytic spine: insights from a comparative structural analysis of human CK2?? and its paralogous isoform CK2????

Eukaryotic protein kinases are fundamental factors for cellular regulation and therefore subject of strict control mechanisms. For full activity a kinase molecule must be penetrated by two stacks of hydrophobic residues, the regulatory and the catalytic spine that are normally well conserved among active protein kinases. We apply this novel spine concept here on CK2α, the catalytic subunit of protein kinase CK2. Homo sapiens disposes of two paralog isoforms of CK2α (hsCK2α and hsCK2α'). We describe two new structures of hsCK2α constructs one of which in complex with the ATP-analog adenylyl imidodiphosphate and the other with the ATP-competitive inhibitor 3-(4,5,6,7-tetrabromo-1H-benzotriazol-1-yl)propan-1-ol. The former is the first hsCK2α structure with a well defined cosubstrate/magnesium complex and the second with an open β4/β5-loop. Comparisons of these structures with existing CK2α/CK2α' and cAMP-dependent protein kinase (PKA) structures reveal: in hsCK2α' an open conformation of the interdomain hinge/helix αD region that is critical for ATP-binding is found corresponding to an incomplete catalytic spine. In contrast hsCK2α often adopts the canonical, PKA-like version of the catalytic spine which correlates with a closed conformation of the hinge region. HsCK2α can switch to the incomplete, non-canonical, hsCK2α'-like state of the catalytic spine, but this transition apparently depends on binding of either ATP or of the regulatory subunit CK2β. Thus, ATP looks like an activator of hsCK2α rather than a pure cosubstrate.     

Protein kinase CK2 phosphorylates the Fas-associated factor FAF1 in vivo and influences its transport into the nucleus

We previously identified the Fas-associated factor FAF1 as an in vitro substrate of protein kinase CK2 and determined Ser289 and Ser291 as phosphorylation sites. Here we demonstrate that these two serine residues are the only sites phosphorylated by CK2 in vitro, and that at least one site is phosphorylated in vivo. Furthermore, we analyzed putative physiological functions of FAF1 phosphorylation. The ability of FAF1 to potentiate Fas-induced apoptosis is not influenced by the FAF1 phosphorylation status; however, the nuclear import of a phosphorylation-deficient FAF1 mutant was delayed in comparison to wild-type FAF1.