Preclinical Evaluation of the Copper-64 Labeled GRPR-Antagonist RM26 in Comparison with the Cobalt-55 Labeled Counterpart for PET-Imaging of Prostate Cancer.

Gastrin-releasing peptide receptor (GRPR) is overexpressed in the majority of prostate cancers. This study aimed to investigate the potential of 64Cu (radionuclide for late time-point PET-imaging) for imaging of GRPR expression using NOTA-PEG2-RM26 and NODAGA-PEG2-RM26. Methods: NOTA/NODAGA-PEG2-RM26 were labeled with 64Cu and evaluated in GRPR-expressing PC-3 cells. Biodistribution of [64Cu]Cu-NOTA/NODAGA-PEG2-RM26 was studied in PC-3 xenografted mice and compared to the biodistribution of [57Co]Co-NOTA/NODAGA-PEG2-RM26 at 3 and 24 h p.i. Preclinical PET/CT imaging was performed in tumor-bearing mice. NOTA/NODAGA-PEG2-RM26 were stably labeled with 64Cu with quantitative yields. In vitro, binding of [64Cu]Cu-NOTA/NODAGA-PEG2-RM26 was rapid and GRPR-specific with slow internalization. In vivo, [64Cu]Cu-NOTA/NODAGA-PEG2-RM26 bound specifically to GRPR-expressing tumors with fast clearance from blood and normal organs and displayed generally comparable biodistribution profiles to [57Co]Co-NOTA/NODAGA-PEG2-RM26; tumor uptake exceeded normal tissue uptake 3 h p.i.. Tumor-to-organ ratios did not increase significantly with time. [64Cu]Cu-NOTA-PEG2-RM26 had a significantly higher liver and pancreas uptake compared to other agents. 57Co-labeled radioconjugates showed overall higher tumor-to-non-tumor ratios, compared to the 64Cu-labeled counterparts. [64Cu]Cu-NOTA/NODAGA-PEG2-RM26 was able to visualize GRPR-expression in a murine PC model using PET. However, [55/57Co]Co-NOTA/NODAGA-PEG2-RM26 provided better in vivo stability and overall higher tumor-to-non-tumor ratios compared with the 64Cu-labeled conjugates.

Protein kinase CK2 localizes to sites of DNA double-strand break regulating the cellular response to DNA damage.

BACKGROUND: The DNA-dependent protein kinase (DNA-PK) is a nuclear complex composed of a large catalytic subunit (DNA-PKcs) and a heterodimeric DNA-targeting subunit Ku. DNA-PK is a major component of the non-homologous end-joining (NHEJ) repair mechanism, which is activated in the presence of DNA double-strand breaks induced by ionizing radiation, reactive oxygen species and radiomimetic drugs. We have recently reported that down-regulation of protein kinase CK2 by siRNA interference results in enhanced cell death specifically in DNA-PKcs-proficient human glioblastoma cells, and this event is accompanied by decreased autophosphorylation of DNA-PKcs at S2056 and delayed repair of DNA double-strand breaks. RESULTS: In the present study, we show that CK2 co-localizes with phosphorylated histone H2AX to sites of DNA damage and while CK2 gene knockdown is associated with delayed DNA damage repair, its overexpression accelerates this process. We report for the first time evidence that lack of CK2 destabilizes the interaction of DNA-PKcs with DNA and with Ku80 at sites of genetic lesions. Furthermore, we show that CK2 regulates the phosphorylation levels of DNA-PKcs only in response to direct induction of DNA double-strand breaks. CONCLUSIONS: Taken together, these results strongly indicate that CK2 plays a prominent role in NHEJ by facilitating and/or stabilizing the binding of DNA-PKcs and, possibly other repair proteins, to the DNA ends contributing to efficient DNA damage repair in mammalian cells.

Interaction between CK2α and CK2ß, 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 (CK2ß). 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 CK2ß 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 CK2ß. 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 CK2ß-bound CK2α. Identifying residues on CK2α that play a key role in CK2α/CK2ß interactions is important for the future generation of small molecule drug design.

Role of polyamines in determining the cellular response to chemotherapeutic agents: modulation of protein kinase CK2 expression and activity.

Numerous studies have shown that platinum compounds stimulate the expression of the polyamine catabolic enzyme spermidine/spermine N(1)-acetyltransferase resulting in anti-proliferative activity and apoptosis. As many cancer cell types including pancreatic cancer cells express high levels of polyamines, the possibility to develop anti-tumor strategies to deplete polyamine pools has drawn considerable attention in recent years. This has been effectively accomplished by treating cells with platinum drugs in combination with polyamine analogs such as N(1),N(11)-diethylnorspermine (DENSPM). The present study, examined the cytotoxic effects of oxaliplatin in combination with stimulators of polyamine catabolism in human pancreatic cancer cells, that are notoriously resistant to chemotherapeutic treatment, and colorectal cancer cells. Additionally, as protein kinase CK2 has been shown to be an anti-apoptotic and pro-survival enzyme regulated by the intracellular polyamine pools, we aimed to investigate the effect of combined DENSPM and oxaliplatin treatment on CK2-mRNA and -protein levels. Results reported here show that treatment with oxaliplatin and DENSPM in combination impairs cell viability particularly in the case of colorectal cancer cells. The analysis of CK2 expression and activity indicates that the response to a specific treatment may depend on the impact that individual compounds exert on pro-survival and pro-death proteins at the transcription and translation levels that should be carefully evaluated in view of subsequent clinical studies.

Lack of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is accompanied by increased CK2α' levels.

DNA-PKcs is the catalytic subunit of DNA-dependent protein kinase, an enzyme necessary for non-homologous end-joining (NHEJ) and hence repair of DNA double strand breaks. Characterization of two isogenic cell lines, M059K and M059J, which are DNA-PKcs-proficient and -deficient, respectively, revealed that lack of DNA-PKcs is accompanied by an increase in the protein level of one of the catalytic isozymes of protein kinase CK2, i.e., CK2α' and a concomitant increase in CK2 activity. The increase was also detectable at the mRNA level as measured by quantitative real time PCR. However, no increase at the DNA level was observed either by comparative PCR or fluorescent in situ hybridization indicating that gene amplification is not involved. Interestingly, only CK2α' was increased and not the other two subunits of CK2, i.e., CK2ß or CK2α. In addition, the increase in CK2α' protein level was also observed in a DNA-PKcs-deficient mouse cell line.

Improving Contrast and Detectability: Imaging with [55Co]Co-DOTATATE in Comparison with [64Cu]Cu-DOTATATE and [68Ga]Ga-DOTATATE.

PET imaging at late time points after injection may allow tracer clearance from normal tissue and hence improve image contrast and detectability. 55Co is a promising isotope with high positron yield and a long half-life suitable for imaging at delayed time points. Here, we compared the 3 radioconjugates [68Ga]Ga-DOTATATE, [64Cu]Cu-DOTATATE, and [55Co]Co-DOTATATE by PET/CT imaging in NOD-SCID mice bearing subcutaneous somatostatin receptor-expressing AR42J tumors. Methods:55Co and 64Cu were produced by the 54Fe(d,n)55Co and 64Ni(p,n)64Cu nuclear reactions, whereas 68Ga was obtained from a 68Ge/68Ga generator. 55Co and 64Cu were labeled with DOTATATE by heating in a sodium acetate buffer and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, respectively. AR42J tumor-bearing mice were dynamically scanned 0-1 h after injection. For 64Cu and 55Co, additional imaging was also performed at late time points after 4 and 24 h. Dose calculations were based on a known biodistribution. The cumulated disintegrations in each organ were calculated by integration of a fitted exponential function to the biodistribution of each respective organ. Equivalent doses were calculated by OLINDA/EXM using the MIRD formalism. Results: Tumor uptake was rapid from 0 to 1 h after injection for all 3 radioconjugates. Normal-tissue ratios as represented by tumor-to-liver, tumor-to-kidney, and tumor-to-muscle ratios increased significantly over time, with [55Co]Co-DOTATATE reaching the highest ratio of all radioconjugates. For [55Co]Co-DOTATATE, the tumor-to-liver ratio increased to 65 ± 16 at 4 h and 50 ± 6 at 24 h, which were 15 (P < 0.001) and 30 (P < 0.001) times higher, respectively, than the corresponding ratios for [64Cu]Cu-DOTATATE and 5 (P < 0.001) times higher than that of [68Ga]Ga-DOTATATE at 1 h. Correspondingly, tumor-to-kidney and tumor-to-muscle ratios for [55Co]Co-DOTATATE were 4 (P < 0.001) and 11 (P < 0.001) times higher than that of [64Cu]Cu-DOTATATE at 24 h. An equivalent dose was calculated as 9.6E-02 mSv/MBq for [55Co]Co-DOTATATE. Conclusion: [55Co]Co-DOTATATE demonstrated superior image contrast compared with [64Cu]Cu-DOTATATE and [68Ga]Ga-DOTATATE for PET imaging of somatostatin receptor-expressing tumors, warranting translation into clinical trials. Dosimetry calculations found that effective doses for [55Co]Co-DOTATATE were comparable to those for both [64Cu]Cu-DOTATATE and [68Ga]Ga-DOTATATE.

Exploring the intramolecular phosphorylation sites in human Chk2.

A comparative biochemical analysis was performed using recombinant human protein kinase Chk2 (checkpoint kinase 2) expressed in bacteria and insect cells. Dephosphorylated, inactive, recombinant human Chk2 could be reactivated in a concentration-dependent manner. Despite distinct time-dependent autophosphorylation kinetics by monitoring the phosphorylation of amino acid residues T68, S19, S33/35, T432, in Chk2 wildtype and Chk2 mutants (T68A, T68D and Q69E) they gave identical specific activities. However, upon gel filtration of Chk2 wildtype and the mutants, only Chk2 wildtype and the T68D mutant led to the formation of a 'pure' dimer; dephosphorylated wildtype Chk2 eluted as a monomer. Transfection of HEK293 cells with Chk2 wildtype and Chk2 mutants in the absence or presence of DNA damage showed significant T68 phosphorylation already in the absence of DNA damaging reagents. Upon DNA damage, phosphorylation of additional Chk2 sites was observed (S19, S33/35). A comparison of ATM+/+ and ATM-/- cells with respect to phosphorylation of residues T68, S19, S33/35 in the absence and presence of DNA damage showed in all cases phosphorylation of T68, although signal intensity was increased ca. three-fold after DNA damage. Mass spectrometric analyses of human recombinant Chk2 isolated from bacteria and insect cells showed distinct differences. The number of phosphorylated residues in human recombinant Chk2 isolated from bacteria was 16, whereas in the case of the recombinant human Chk2 from insect cells it was 8. Except for phosphorylated amino acid T378 which was not found in the Chk2 isolated from bacteria, all other phosphorylated residues identified in human Chk2 from insect cells were present also in Chk2 from bacteria.

Emodin negatively affects the phosphoinositide 3-kinase/AKT signalling pathway: a study on its mechanism of action.

The development of selective cell-permeable inhibitors of protein kinases whose aberrant activation contributes to cell transformation is a promising approach in cancer treatment. Emodin is a natural anthraquinone derivative that exhibits anti-proliferative effects in various cancer cell lines by efficient induction of apoptosis. The phosphoinositide 3-kinase (PI3K)/AKT pathway has been shown to be central in the promotion of cell survival since the alteration of this signalling cascade is a frequent event in human malignancies. Previous published results indicated that treatment of cells with inhibitors of protein kinase CK2, such as emodin, induces apoptosis and that the anti-apoptotic effect of CK2 is partially mediated by target phosphorylation and up-regulation of AKT by CK2. In the present study, a screening with selected CK2 inhibitors induced a variable response with respect to AKT down-regulation, emodin being the most effective, suggesting that other mechanisms other than the inhibition of CK2 were responsible for the emodin-mediated modulation of AKT. We found that emodin does not directly affect AKT kinase. Furthermore, we show that the down-regulation of AKT is due to the emodin-mediated target inhibition of components of the PI3K pathway, which directly or indirectly affect AKT activity, i.e. the mammalian target of rapamycin and the phosphatase and tensin homolog deleted on chromosome 10, but not the phosphoinositide-dependent kinase 1. Taken together, our results highlight a new mechanism by which emodin exerts anti-cancer activity and suggest the further investigation of plant polyphenols, such as emodin, as therapeutic and preventive agents for cancer therapy.

Disruption of the regulatory beta subunit of protein kinase CK2 in mice leads to a cell-autonomous defect and early embryonic lethality.

Protein kinase CK2 is a ubiquitous protein kinase implicated in proliferation and cell survival. Its regulatory beta subunit, CK2beta, which is encoded by a single gene in mammals, has been suspected of regulating other protein kinases. In this work, we show that knockout of the CK2beta gene in mice leads to postimplantation lethality. Mutant embryos were reduced in size at embryonic day 6.5 (E6.5). They did not exhibit signs of apoptosis but did show reduced cell proliferation. Mutant embryos were resorbed at E7.5. In vitro, CK2beta(-/-) morula development stopped after the blastocyst stage. Attempts to generate homozygous embryonic stem (ES) cells failed. By using a conditional knockout approach, we show that lack of CK2beta is deleterious for mouse ES cells and primary embryonic fibroblasts. This is in contrast to what occurs with yeast cells, which can survive without functional CK2beta. Thus, our study demonstrates that in mammals, CK2beta is essential for viability at the cellular level, possibly because it acquired new functions during evolution.

The use of radiocobalt as a label improves imaging of EGFR using DOTA-conjugated Affibody molecule.

Several anti-cancer therapies target the epidermal growth factor receptor (EGFR). Radionuclide imaging of EGFR expression in tumours may aid in selection of optimal cancer therapy. The 111In-labelled DOTA-conjugated ZEGFR:2377 Affibody molecule was successfully used for imaging of EGFR-expressing xenografts in mice. An optimal combination of radionuclide, chelator and targeting protein may further improve the contrast of radionuclide imaging. The aim of this study was to evaluate the targeting properties of radiocobalt-labelled DOTA-ZEGFR:2377. DOTA-ZEGFR:2377 was labelled with 57Co (T1/2 = 271.8 d), 55Co (T1/2 = 17.5 h), and, for comparison, with the positron-emitting radionuclide 68Ga (T1/2 = 67.6 min) with preserved specificity of binding to EGFR-expressing A431 cells. The long-lived cobalt radioisotope 57Co was used in animal studies. Both 57Co-DOTA-ZEGFR:2377 and 68Ga-DOTA-ZEGFR:2377 demonstrated EGFR-specific accumulation in A431 xenografts and EGFR-expressing tissues in mice. Tumour-to-organ ratios for the radiocobalt-labelled DOTA-ZEGFR:2377 were significantly higher than for the gallium-labelled counterpart already at 3 h after injection. Importantly, 57Co-DOTA-ZEGFR:2377 demonstrated a tumour-to-liver ratio of 3, which is 7-fold higher than the tumour-to-liver ratio for 68Ga-DOTA-ZEGFR:2377. The results of this study suggest that the positron-emitting cobalt isotope 55Co would be an optimal label for DOTA-ZEGFR:2377 and further development should concentrate on this radionuclide as a label.

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.

Characterization of ATM and DNA-PK wild-type and mutant cell lines upon DSB induction in the presence and absence of CK2 inhibitors.

Protein kinase CK2 is involved in several cellular processes and has lately also been linked to the DNA damage response through phosphorylations and interactions. Herein, we have analysed two sets of mouse cell lines, one pair, which is proficient and deficient in ATM and the other set expressing or lacking a functional catalytic subunit of the DNA dependent protein kinase (DNA-PKcs). Both kinases are implicated in the downstream phosphorylation of the signaling molecules such as BID and AKT1 in response to DNA damage. BID and AKT1 are also targets of CK2, hence the four cell lines were treated with the three established CK2 inhibitors DMAT, TBB and resorufin in the absence and presence of the radiomimetic drug neocarzinostatin, which induces DNA double-strand breaks. We show that there are differences with respect to the effect of the CK2 inhibitors on the phosphorylation of AKT1 at S473 and its downstream target GSK3ß as well as between the two sets of cell lines. However, no such change was seen with BID phosphorylation. The most notably difference was the higher expression of CK2α' and CK2ß in DNA-PKcs defective cells compared to the DNA-PKcs proficient cells.

Downregulation of protein kinase CK2 induces autophagic cell death through modulation of the mTOR and MAPK signaling pathways in human glioblastoma cells.

Glioblastoma multiforme is the most common primary brain tumor and one of the most aggressive types of cancer in adults. Survival signaling and apoptosis resistance are hallmarks of malignant glioma cells. However, recent studies have shown that other types of cell death such as autophagy can be induced in malignant glioma cells. This suggests that stimulation of this process may be explored in new therapeutic strategies against glioblastoma multiforme. Protein kinase CK2 is a highly conserved and constitutively active enzyme that promotes numerous cellular processes such as survival, proliferation and differentiation. CK2 has been found elevated in several malignancies including brain tumors, and to confer resistance against chemotherapeutic agents and apoptotic stimuli. Recently, we have shown that the siRNA-mediated downregulation of CK2 leads to cell death in DNA-PK-proficient human glioblastoma cells. We show, here, that lack of CK2 results in significant induction of autophagic cell death in two human glioblastoma cell lines, M059K and T98G, as indicated by the positive staining of cells with the acidotropic dye acridine orange, and the specific recruitment of microtubule-associated protein 1 light chain 3 (LC3) to autophagosome membranes. Induction of autophagy is accompanied by CK2-dependent decreased phosphorylation of p70 ribosomal S6 and AKT kinases and significantly reduced expression levels of Raptor. In contrast, phosphorylation and activity levels of ERK1/2 are enhanced suggesting an inhibition of the PI3K/AKT/mTORC1 and activation of the ERK1/2 pathways. Furthermore, siRNA-mediated silencing of CK2 results in increased mitochondrial superoxide production in both glioblastoma cell lines. However, mitochondrial reactive oxygen species release correlates with induction of autophagy only in T98G cells. Taken together, our findings identify CK2 as a novel component of the autophagic machinery and underline the potential of its downregulation to kill glioblastoma cells by overcoming the resistance to multiple anticancer agents.

Structural basis of the constitutive activity of protein kinase CK2.

Protein kinase CK2 (formerly referred to as casein kinase II) is an evolutionary conserved, ubiquitous protein kinase. In mammals, there are two paralog catalytic subunits, that is, CK2α (A1) and CK2α' (A2), and one CK2ß dimer, which together form the heterotetrameric holoenzyme. The presence of full functioning CK2α and CK2ß subunits are absolutely mandatory for embryonic development. Total knockouts are lethal. The CK2α' paralog seems to be an exception inasmuch as a total knockout only leads to sterility in male mice. The catalytic subunits are distantly related to the CMGC subfamily of protein kinases, such as the cyclin-dependent kinases (CDKs). There are some peculiarities associated with protein kinase CK2, which are not found with most of the other protein kinases: the enzyme is constitutively active, it can use ATP and GTP as phosphoryl donors, and it is found elevated in most tumors investigated and rapidly proliferating tissues. In this review, we explain (i) its constitutive activity at the intramolecular level, and (ii) come forward with a model how this protein kinase could be regulated in cells by a mechanism involving intermolecular interactions.

Mapping of the interaction sites between Wee1 kinase and the regulatory beta-subunit of protein kinase CK2.

Somatic Wee1 is a protein kinase that plays a key role in cell cycle progression at the onset of mitosis by phosphorylating CDK1 at the inhibitory Tyr15 amino acid residue. Wee1 is regulated at multiple levels, i.e., phosphorylation, protein-protein association and proteasome-mediated degradation. We have recently shown that the regulatory beta-subunit of protein kinase CK2 participates in PLK1-Wee1 complex formation interacting directly with Wee1 and thereby contributing to the regulation of G2-M cell cycle transition. Here, we show that Wee1 binds CK2beta via two domains comprising amino acids 59-71 and 232-332. By employing deletion mutants of the CK2beta-subunit we also show that two regions between residues 1-5 and 155-170 are necessary for binding Wee1. Furthermore, we demonstrate that the interaction between CK2beta and Wee1 does not modify the kinase activity of the latter, instead CK2beta directly upregulates CDK1 kinase activity by reversing the inhibitory effect which follows Wee1-mediated phosphorylation. Taken together, our findings reinforce the notion that CK2beta is a modulator of protein kinases implicated in cell cycle regulation and exerts functions that are independent of CK2 tetramers.

Ability of CK2beta to selectively regulate cellular protein kinases.

The Wee1 protein kinase plays a prominent role in keeping cyclin dependent kinase 1 (CDK1) inactive during the G2 phase of the cell cycle. At the onset of mitosis, Wee1 is ubiquitinated by the E3 ubiquitin ligase SCF(beta-TrCP) and subsequently degraded by the proteasome machinery. Previously, it has been reported that although Wee1 lacks the conserved binding motif recognised by beta-TrCP, the CDK-catalysed phosphorylation of Wee1 at Ser123 creates a phosphodegron and primes phosphorylation of two other protein kinases, polo-like kinase 1 (PLK1) and protein kinase CK2, which create two additional phosphodegrons recognised by beta-TrCP. These events contribute to destabilise Wee1 at the onset of mitosis (Watanabe et al. Proc Natl Acad Sci USA 101:4419-4424, 2004). We show here that in addition to the ability of CK2 to phosphorylate Wee1 as reported earlier, the regulatory beta-subunit of protein kinase CK2 can interact with Wee1 in high molecular mass complexes. Indirect immunofluorescence microscopy revealled subcellular co-localisation of CK2beta and Wee1 in the nucleus. Moreover, in vitro phosphorylation assays showed that CK2beta indirectly up-regulates the activity of CDK1 with respect to histone H1 phosphorylation by inhibiting Wee1 kinase. These findings support the view that CK2beta regulates various intracellular processes by modulating the activity of protein kinases that are distinct from CK2 and that protein kinase CK2 plays an important role in events related to the regulation of cell cycle progression as a tetrameric enzyme but also through the individual subunits.

Biochemical characterization of CK2alpha and alpha' paralogues and their derived holoenzymes: evidence for the existence of a heterotrimeric CK2alpha'-holoenzyme forming trimeric complexes.

Altogether 2 holoenzymes and 4 catalytic CK2 constructs were expressed and characterized i.e. CK2alpha(2)1-335 beta2; CK2alpha'-derived holoenzyme; CK2alpha1-335; MBP-CK2alpha'; His-tagged CK2alpha and His-tagged CK2alpha'. The two His-tagged catalytic subunits were expressed in insect cells, all others in Escherichia coli. IC50 studies involving the established CK2 inhibitors DMAT, TBBt, TBBz, apigenin and emodin were carried out and the Ki values calculated. Although the differences in the Ki values found were modest, there was a general tendency showing that the CK2 holoenzymes were more sensitive towards the inhibitors than the free catalytic subunits. Thermal inactivation experiments involving the individual catalytic subunits showed an almost complete loss of activity after only 2 min at 45 degrees C. In the case of the two holoenzymes, the CK2alpha'-derived holoenzyme lost ca. 90% of its activity after 14 min, whereas CK2alpha2(1-335) beta2 only showed a loss of ca. 40% by this time of incubation. Gel filtration analyses were performed at high (500 mM) and low (150 mM) monovalent salt concentrations in the absence or presence of ATP. At 500 mM NaCl the CK2alpha'-derived holoenzyme eluted at a position corresponding to a molecular mass of 105 kDa which is significantly below the elution of the CK2alpha(2)1-335 beta2 holoenzyme (145 kDa). Calmodulin was not phosphorylated by either CK2alpha2(1-335) beta2 or the CK2alpha'-derived holoenzyme. However, in the presence of polylysine only the CK2alpha(2)1-335 beta2 holoenzyme could use calmodulin as a substrate such as the catalytic subunits, in contrast to the CK2alpha'-derived holoenzyme which only phosphorylated calmodulin weakly. This attenuation may be owing to a different structural interaction between the catalytic CK2alpha' subunit and non-catalytic CK2beta subunit.

BID, an interaction partner of protein kinase CK2alpha.

Recombinant murine BID protein was used as an in vitro substrate for the CK2 holoenzyme and the catalytic CK2alpha subunit. The results obtained show that BID can only serve as a substrate for the catalytic CK2alpha subunit. Phosphorylation of BID using the CK2 holoenzyme was only possible in the presence of polylysine, supporting the notion that BID behaves similarly to calmodulin. Co-immunoprecipitation of BID and CK2 subunits revealed that BID is preferentially associated with the CK2alpha subunit. Enzyme kinetic analyses yielded a Km value for BID that is a level of magnitude lower than that measured for casein and the synthetic peptide, suggesting more specific and tight binding of BID to CK2alpha. In contrast are the Vmax values observed, with a significantly higher phosphorylation rate measured for casein and the synthetic peptide than for BID. When BID was phosphorylated by polylysine-stimulated CK2 holoenzyme prior to caspase-8 cleavage, the formation of tC-BID was reduced in comparison to treatment with caspase-8 in the absence of protein kinase. Mass spectrometric analysis of BID phosphorylated by CK2alpha before and after cleavage with caspase-8 showed phosphorylation of residues Thr58 and Ser76.

Purification and characterization of the CK2alpha'-based holoenzyme, an isozyme of CK2alpha: a comparative analysis.

Protein kinase CK2 (former name: "casein kinase 2") is a pivotal and ubiquitously expressed member of the eukaryotic protein kinase superfamily. It predominantly exists as a heterotetrameric holoenzyme composed of two catalytic subunits (CK2alpha) and two regulatory subunits (CK2beta). In higher animals two paralog catalytic chains-abbreviated CK2alpha and CK2alpha'--exist which can combine with CK2beta to three isoforms of the holoenzyme: CK2alpha(2)beta(2), CK2alpha(2)(')beta(2), and CK2alphaalpha(')beta(2). While CK2alpha and the "normal" holoenzyme CK2alpha(2)beta(2) have been extensively characterized in vitro and in vivo, little is known about the enzymological properties of CK2alpha' and the "alternative" holoenzyme CK2alpha(2)(')beta(2) and about their specific physiological roles. A major reason for this lack of knowledge is the fact that so far CK2alpha' rather than CK2alpha has caused serious stability and solubility problems during standard heterologous expression procedures. To overcome them, we developed a preparation scheme for CK2alpha(2)(')beta(2) from Homo sapiens in catalytically active form based on two critical steps: first expression of human CK2alpha' as a well soluble fusion protein with the maltose binding protein (MBP) and second proteolytic cleavage of CK2alpha'-MBP in the presence of human CK2beta so that CK2alpha' subunits are incorporated into holoenzyme complexes directly after their release from MBP. This successful strategy which may be adopted in comparably difficult cases of protein/protein complex preparation is presented here together with evidence that the CK2alpha'-based and the CK2alpha-based holoenzymes are similar concerning their catalytic activities but are significantly different with respect to some well-known CK2 properties like autophosphorylation and supra-molecular aggregation.