Structural and functional analysis of the flexible regions of the catalytic α-subunit of protein kinase CK2

CK2 is a Ser/Thr protein kinase essential for cell viability. Its activity is anomalously high in several solid (prostate, mammary gland, lung, kidney and head and neck) and haematological tumours (AML, CML and PML), creating conditions favouring the onset of cancer. Cancer cells become addicted to high levels of CK2 activity and therefore this kinase is a remarkable example of "non-oncogene addiction". CK2 is a validated target for cancer therapy with one inhibitor in phase I clinical trials. Several crystal structures of CK2 are available, many in complex with ATP-competitive inhibitors, showing the presence of regions with remarkable flexibility. We present the structural characterisation of these regions by means of seven new crystal structures, in the apo form and in complex with inhibitors. We confirm previous findings about the unique flexibility of the CK2α catalytic subunit in the hinge/αD region, the p-loop and the β4β5 loop, and show here that there is no clear-cut correlation between the conformations of these flexible zones. Our findings challenge some of the current interpretations on the functional role of these regions and dispute the hypothesis that small ligands stabilize an inactive state. The mobility of the hinge/αD region in the human enzyme is unique among protein kinases, and this can be exploited for the development of more selective ATP-competitive inhibitors. The identification of different ligand binding modes to a secondary site can provide hints for the design of non-ATP-competitive inhibitors targeting the interaction between the α catalytic and the β regulatory subunits.     

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.     

CK2α/CK1α chimeras are sensitive to regulation by the CK2β subunit

The purpose of this study was to analyse the frequency and type of mutations in the coding region of androgen receptor (AR) and to determine the role of polymorphisms in the intron 1 of ERalpha, exon 5 of ERbeta, intron 7 of progesterone, exon 7 of the aromatase (CYP19) and exon 9 of VDR genes in the risk of prostate cancer. PCR-RFLP analysis of all above the genes was on 100 prostate cancer patients and an equal number of matching controls. The study also included PCR-SSCP analyses of exons 2-8 of AR gene. The genotype containing -/- allele of ERalpha gene was statistically significant for the risk of prostate cancer pose (OR, 2.70; 95% CI, 1.08-6.70, P = 0.032) Rr genotype of ERbeta gene also have a higher risk (OR, 1.65; 95% CI, 0.52-5.23) for prostate cancer. The Cys allele of CYP19 gene was also associated with statistically significant increased risk of prostate cancer (OR; 2.28, 95% CI, 1.20-4.35, P = 0.012). tt genotype of codon 352 of VDR gene showed an OR of 0.43 for (95% CI, 0.13-1.39) and an OR for Tt genotype was 0.65 (95% CI, 0.36-1.16). Taken together, the results showed that in North Indian population, ERalpha and CYP19 genes may be playing a role in the risk of prostate cancer.     

Predominance of CK2α over CK2α′ in the mammalian brain

CK2 is a heterotetrameric ubiquitous kinase consisting of two catalytic subunits and two regulatory subunits. The two catalytic subunits, α and α', are highly homologous but differ in their C-terminal regions. It is not known whether CK2α and α' have distinctive substrate specificity, since no α- or α'-specific substrate has been identified. Thus, it is assumed that the two kinase isoforms overlap in their substrate specificity. CK2 protein levels and activity were found to be elevated in the brain when compared to other organs. Here we have studied the protein levels of CK2α and α' isoforms in nine major brain regions. We found that both, CK2α and α', are expressed in all brain regions tested. Whereas CK2α levels do not vary strongly across the regions, CK2α' levels are slightly higher in the cortex and hippocampus than in other regions. Furthermore, we show that CK2α protein levels in the striatum are relatively high when compared to CK2α'. The approximate stoichiometry ratio of CK2α:CK2α' is 8:1. Therefore, one can consider that CK2α levels are predominant in comparison to CK2α' levels throughout the mammalian brain.     

Kinetics of renaturation and self-assembly of intermediates on the pathway of folding of the β2-subunit of Escherichia coli tryptophan-synthetase

The kinetics of renaturation of the β₂-subunit of Escherichia coli tryptophan-synthetase (l-serine hydrolyase (adding indole) E.C. 4.2.1.20) and those of its two proteolytic fragments F₁ and F₂ are studied and compared. Steps corresponding to the refolding of F₁, to the association of the folded F₁ and F₂ fragments, and to an isomerization of the associated protein are identified. These steps are ordered on the pathway of renaturation and some of their kinetic parameters are determined. This leads to a tentative kinetic model for the renaturation of nicked-β₂ starting from the denatured F₁ and F₂ fragments.The step corresponding to the refolding of the F₁ domain, as well as that corresponding to the last rate-limiting isomerization leading to the native protein, is shown to be the same in the refolding of the entire, uncleaved β₂-protein. It is concluded that the refolded F₁ fragment corresponds to a folding intermediate on the pathway of renaturation of the β₂-subunit.     

Enzyme enhancement activity of N-octyl-β-valienamine on β-glucosidase mutants associated with Gaucher disease

Gaucher disease (GD), caused by a defect of β-glucosidase (β-Glu), is the most common form of sphingolipidosis. We have previously shown that a carbohydrate mimic N-octyl-β-valienamine (NOV), an inhibitor of β-Glu, could increase the protein level and enzyme activity of F213I mutant β-Glu in cultured GD fibroblasts, suggesting that NOV acted as a pharmacological chaperone to accelerate transport and maturation of this mutant enzyme. In the current study, NOV effects were evaluated in GD fibroblasts with various β-Glu mutations and in COS cells transiently expressing recombinant mutant proteins. In addition to F213I, NOV was effective on N188S, G202R and N370S mutant forms of β-Glu, whereas it was ineffective on G193W, D409H and L444P mutants. When expressed in COS cells, the mutant proteins as well as the wild-type protein were localized predominantly in the endoplasmic reticulum and were sensitive to Endo-H treatment. NOV did not alter this localization or Endo-H sensitivity, suggesting that it acted in the endoplasmic reticulum. Profiling of N-alkyl-β-valienamines with various lengths of the acyl chain showed that N-dodecyl-β-valienamine was as effective as NOV. These results suggest a potential therapeutic value of NOV and related compounds for GD with a broad range of β-Glu mutations.     

Expression of the β-subunit of β-conglycinin in seeds of transgenic plants

Soybean (Glycine max (L.) Merr.) seeds contain the storage protein -conglycinin, encoded by a multigene family. -Conglycinin consists of three subunits; , , and . A genomic clone for a -subunit of -conglycinin has been characterized by restriction-enzyme mapping and hybrid selected in-vitro translation followed by immunoprecipitation. In order to determine the developmental regulation of this -subunit gene, its expression was studied in seeds of transgenic petunia (Petunia hybrida) and tobacco (Nicotiana tabacum L.) plants. The -subunit expressed in seeds of petunia and tobacco was recognized by anti--conglycinin serum at a relative molecular mass of 53 000, equivalent to that of the native protein. Separation of the petunia-seed proteins by isoelectric focusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis showed that multiple isoelectric forms of the -subunit were produced. There was approximately a twofold variation in the accumulation of the -subunit protein in the mature seeds of transgenic petunia plants, each containing a single -subunit gene. However, the level of protein accumulation in mature seeds and the amount of -subunit mRNA in developing seeds was not correlated. Accumulation of the -subunit protein in transgenic seeds was less than the -subunit protein that accumulated in transgenic petunia seeds containing a single -subunit gene and less than the amount of the -subunit in mature soybean seeds which contain 8–13 -subunit genes. In transgenic tobacco plants, the accumulation of the -subunit protein in seeds was generally well correlated with the number of genes that were incorporated in the different transformants.Abbreviations kb kilobase - kDa kilodalton - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

CK2α is essential for embryonic morphogenesis

CK2 is a highly conserved serine-threonine kinase involved in biological processes such as embryonic development, circadian rhythms, inflammation, and cancer. Biochemical experiments have implicated CK2 in the control of several cellular processes and in the regulation of signal transduction pathways. Our laboratory is interested in characterizing the cellular, signaling, and molecular mechanisms regulated by CK2 during early embryonic development. For this purpose, animal models, including mice deficient in CK2 genes, are indispensable tools. Using CK2α gene-deficient mice, we have recently shown that CK2α is a critical regulator of mid-gestational morphogenetic processes, as CK2α deficiency results in defects in heart, brain, pharyngeal arch, tail bud, limb bud, and somite formation. Morphogenetic processes depend upon the precise coordination of essential cellular processes in which CK2 has been implicated, such as proliferation and survival. Here, we summarize the overall phenotype found in CK2α (-/- ) mice and describe our initial analysis aimed to identify the cellular processes affected in CK2α mutants.     

Structure of the human protein kinase CK2 catalytic subunit CK2α' and interaction thermodynamics with the regulatory subunit CK2β

Protein kinase CK2 (formerly “casein kinase 2”) is composed of a central dimer of noncatalytic subunits (CK2β) binding two catalytic subunits. In humans, there are two isoforms of the catalytic subunit (and an additional splicing variant), one of which (CK2α) is well characterized. To supplement the limited biochemical knowledge about the second paralog (CK2α′), we developed a well-soluble catalytically active full-length mutant of human CK2α′, characterized it by Michaelis-Menten kinetics and isothermal titration calorimetry, and determined its crystal structure to a resolution of 2 Å. The affinity of CK2α′ for CK2β is about 12 times lower than that of CK2α and is less driven by enthalpy. This result fits the observation that the β4/β5 loop, a key element of the CK2α/CK2β interface, adopts an open conformation in CK2α′, while in CK2α, it opens only after assembly with CK2β. The open β4/β5 loop in CK2α′ is stabilized by two elements that are absent in CK2α: (1) the extension of the N-terminal β-sheet by an additional β-strand, and (2) the filling of a conserved hydrophobic cavity between the β4/β5 loop and helix αC by a tryptophan residue. Moreover, the interdomain hinge region of CK2α′ adopts a fully functional conformation, while unbound CK2α is often found with a nonproductive hinge conformation that is overcome only by CK2β binding. Taken together, CK2α′ exhibits a significantly lower affinity for CK2β than CK2α; moreover, in functionally critical regions, it is less dependent on CK2β to obtain a fully functional conformation.     

Is dimerization required for the catalytic activity of bacterial biotin carboxylase?

Acetyl-coenzyme A carboxylases (ACCs) have crucial roles in fatty acid metabolism. The biotin carboxylase (BC) subunit of Escherichia coli ACC is believed to be active only as a dimer, although the crystal structure shows that the active site of each monomer is 25 A from the dimer interface. We report here biochemical, biophysical, and structural characterizations of BC carrying single-site mutations in the dimer interface. Our studies demonstrate that two of the mutants, R19E and E23R, are monomeric in solution but have only a 3-fold loss in catalytic activity. The crystal structures of the E23R and F363A mutants show that they can still form the correct dimer at high concentrations. Our data suggest that dimerization is not an absolute requirement for the catalytic activity of the E. coli BC subunit, and we propose a new model for the molecular mechanism of action for BC in multisubunit and multidomain ACCs.     

Influence of thyroid hormones on the human ATP synthase β-subunit gene promoter

The action of thyroid hormones on the expression of the mitochondrial ATP synthase -subunit gene (ATPsyn) is controversial. We detected a binding site for the thyroid hormone receptor between-366 and-380 in the human ATPsyn gene by DNase I footprint analysis and band-shift assays. However, expression vectors in which the chloramphenicol acetyl transferase (CAT) reporter gene is driven by the 5 upstream region of ATPsyn gene were unresponsive to T₃ when transiently transfected to HepG2 or GH4C1 cells. CAT constructs driven by the rat phosphoenolpyruvate carboxykinase (PEPCK) or the growth hormone (GH) promoters were stimulated several fold by T₃ in parallel experiments. It is proposed that the biological effects of thyroid hormones on the ATPsyn expression occur through indirect mechanisms.