Multitarget Anticancer Agents Based on Histone Deacetylase and Protein Kinase CK2 inhibitors.

The design of multitarget drugs (MTDs) has become an innovative approach for the search of effective treatments in complex diseases such as cancer. In this work, we communicate our efforts in the design of multi-targeting histone deacetylase (HDAC) and protein kinase CK2 inhibitors as a novel therapeutic strategy against cancer. Using tetrabromobenzotriazole (TBB) and 2-dimethylamino-4,5,6,7-tetrabromo-benzimidazole (DMAT) as scaffolds for CK2 inhibition, and a hydroxamate to coordinate the zinc atom present in the active site of HDAC (zinc binding group, ZBG), new multitarget inhibitors have been designed and synthesized. According to the in vitro assays, N-Hydroxy-6-(4,5,6,7-tetrabromo-2-(dimethylamino)-1H-benzo[d]imidazol-1-yl)hexanamide (11b) is the most interesting compound, with IC50 values of 0.66; 1.46 and 3.67 µM. for HDAC6; HDAC1 and CK2; respectively. Cellular assays on different cancer cell lines rendered promising results for N-Hydroxy-8-(4,5,6,7-tetrabromo-2-(dimethylamino)-1H-benzo[d]imidazol-1-yl)octanamide (11d). This inhibitor presented the highest cytotoxic activity, proapoptotic capability, and the best mitochondria-targeting and multidrug-circumventing properties, thus being the most promising drug candidate for further in vivo studies.

Simultaneous Determination of Protein Kinase A and Casein Kinase II by Dual-Color Peptide Biomineralized Metal Nanoclusters.

We design two artificial substrate peptides to synthesize blue-emissive Cu nanoclusters and red-emissive Au nanoclusters, respectively. In addition to the biomineralization function, these two peptides retain the biological activity to be phosphorylated by protein kinase and digested by carboxypeptidase Y. In the absence of protein kinase, the peptides capped on the nanoclusters suffer consecutive exocleavage by carboxypeptidase Y, resulting in oxidation and thus fluorescence quenching of the nanoclusters due to the losing of peptide protection. In the presence of protein kinase A and casein kinase II, the phosphorylation modification on corresponding substrate peptides protects the peptides against carboxypeptidase Y digestion and then the fluorescence of the nanoclusters can be retained. Since a single excitation wavelength can excite the both nanoclusters, blue and red emissive signals can be collected at the same time and then the quantitative determination of protein kinase A and casein kinase II can be achieved simultaneously.

Electrogenerated Chemiluminescence Bioassay of Two Protein Kinases Incorporating Peptide Phosphorylation and Versatile Probe.

A sensitive electrogenerated chemiluminescence (ECL) bioassay was developed for the detection of two protein kinases incorporating the peptide phosphorylation and a versatile ECL probe. Cyclic adenosine monophosphate-dependent protein kinase (PKA) and casein kinase II (CK2) were used as proof-of-concept targets while a PKA-specific peptide (CLRRASLG) and a CK2-specific peptide (CRRRADDSDDDDD) were used as the recognition substrates. Taking advantage of the ability of protein A binding with the Fc region of a variety of antibodies with high affinity, a ruthenium derivative-labeled protein A was utilized as a versatile ECL probe for bioassay of multiple protein kinases. A specific peptide substrate toward target protein kinase was first self-assembled on the surface of gold electrode and then serine in the specific peptide on the electrode was phosphorylated by target protein kinase in the presence of adenosine-5'-triphosphate. After recognition of the phosphorylated peptide by monoclonal antiphosphoserine antibody, the versatile ECL probe was specifically bound to the antiphosphoserine antibody on the electrode surface. The ECL bioassay was developed successfully in the individual detection of PKA and CK2 with detection limit of 0.005 U/mL and 0.004 U/mL, respectively. In addition, the ECL bioassay was applied to quantitative analysis of the kinase inhibitors and monitoring drug-triggered kinase activation in cell lysates. Moreover, an ECL imaging bioassay using electron-multiplying charged coupled device as detector on the gold electrode array was developed for the simultaneous detection of PKA and CK2 activity from 0.01 U/mL to 0.4 U/mL, respectively, at one time. This work demonstrates that the ingenious design and use of a versatile ECL probe are promising to simultaneous detection of multiple protein kinases and screening of kinase inhibitor.

Casein kinase II regulates N-methyl-D-aspartate receptor activity in spinal cords and pain hypersensitivity induced by nerve injury.

Increased N-methyl-d-aspartate receptor (NMDAR) activity and phosphorylation in the spinal cord are critically involved in the synaptic plasticity and central sensitization associated with neuropathic pain. However, the mechanisms underlying increased NMDAR activity in neuropathic pain conditions remain poorly understood. Here we show that peripheral nerve injury induces a large GluN2A-mediated increase in NMDAR activity in spinal lamina II, but not lamina I, neurons. However, NMDAR currents in spinal dorsal horn neurons are not significantly altered in rat models of diabetic neuropathic pain and resiniferatoxin-induced painful neuropathy (postherpedic neuralgia). Inhibition of protein tyrosine kinases or protein kinase C has little effect on NMDAR currents potentiated by nerve injury. Strikingly, casein kinase II (CK2) inhibitors normalize increased NMDAR currents of dorsal horn neurons in nerve-injured rats. In addition, inhibition of calcineurin, but not protein phosphatase 1/2A, augments NMDAR currents only in control rats. CK2 inhibition blocks the increase in spinal NMDAR activity by the calcineurin inhibitor in control rats. Furthermore, nerve injury significantly increases CK2α and CK2ß protein levels in the spinal cord. In addition, inhibition of CK2 or CK2ß knockdown at the spinal level substantially reverses pain hypersensitivity induced by nerve injury. Our study indicates that neuropathic pain conditions with different etiologies do not share the same mechanisms, and increased spinal NMDAR activity is distinctly associated with traumatic nerve injury. CK2 plays a prominent role in the potentiation of NMDAR activity in the spinal dorsal horn and may represent a new target for treatments of chronic pain caused by nerve injury.

CK2 involvement in ESCRT-III complex phosphorylation.

The multivesicular body (MVB) sorting pathway is a mechanism for delivering transmembrane proteins into the lumen of the lysosome for degradation. ESCRT-III is the final complex in the pathway that assembles on endosomes and executes membrane scission of intraluminal vesicles. In addition, proteins of this complex are involved in other topologically similar processes such as cytokinesis, virus egress and autophagy. Here we show that protein kinase CK2α is involved in the phosphorylation of the ESCRT-III subunits CHMP3 and CHMP2B, as well as of VPS4B/SKD1, an ATPase that mediates ESCRT-III disassembly. This phosphorylation is observed both in vitro and in cells. While we do not observe recruitment of CK2α to endosomes, we demonstrate the localization of CK2α to midbodies during cytokinesis. Phosphomimetic and non-phosphorylatable mutants of ESCRT-III proteins can still bind endosomes and localize to midbodies, indicating that CK2α does not regulate ESCRT-III localization. Finally, we analyzed two cellular functions where CHMP3, CHMP2B and VPS4 are known to be involved, epidermal growth factor degradation and cytokinetic abscission. We demonstrate that the former is impaired by CK2α downregulation whereas the latter is not affected. Taken together, our results indicate that CK2α regulates the function of ESCRT-III proteins in MVB sorting.

Using graphene quantum dots as photoluminescent probes for protein kinase sensing.

A simple and sensitive photoluminescence (PL) assay for the activity of a protein kinase based on the selective aggregation of phosphorylated peptide-graphene quantum dot (GQD) conjugates triggered by Zr(4+) ion coordination has been established. With more sophisticated design of the peptide substrate sequences, detecting other enzymes could also be possible. Under optimal conditions, a linear relationship between the decreased PL intensity of peptide-GQD conjugates and the concentration of casein kinase II (CK2) in the range from 0.1 to 1.0 unit mL(-1) with a detection limit of 0.03 unit mL(-1) (3σ) was obtained. The EC50 value (i.e., the enzyme concentration producing 50% substrate conversion) for CK2 was evaluated to be 0.34 unit mL(-1). The proposed method showed potential applications in kinase inhibitor screening. To demonstrate the potential of this GQD-based platform for screening of kinase inhibitors in real biological systems, the inhibition of CK2 phosphorylation activity by four different inhibitors (ellagic acid, 5,6-dichlorobenzimidazole-l-ß-d-ribofuranoside, emodin, and quercetin) was tested in human serum by comparing signals from samples incubated with the inhibitors against that without any inhibitor. As expected, in the presence of inhibitors, the PL intensity increased with increasing inhibitor efficiency. The IC50 value (inhibitor concentration producing 50% inhibition) for ellagic acid was estimated to be 0.041 µM. The developed protocol provides a new and promising tool for the analysis of both the enzyme and its inhibitors with low cost and excellent performance.

Detecting host factors involved in virus infection by observing the clustering of infected cells in siRNA screening images.

MOTIVATION: Detecting human proteins that are involved in virus entry and replication is facilitated by modern high-throughput RNAi screening technology. However, hit lists from different laboratories have shown only little consistency. This may be caused by not only experimental discrepancies, but also not fully explored possibilities of the data analysis. We wanted to improve reliability of such screens by combining a population analysis of infected cells with an established dye intensity readout. RESULTS: Viral infection is mainly spread by cell-cell contacts and clustering of infected cells can be observed during spreading of the infection in situ and in vivo. We employed this clustering feature to define knockdowns which harm viral infection efficiency of human Hepatitis C Virus. Images of knocked down cells for 719 human kinase genes were analyzed with an established point pattern analysis method (Ripley's K-function) to detect knockdowns in which virally infected cells did not show any clustering and therefore were hindered to spread their infection to their neighboring cells. The results were compared with a statistical analysis using a common intensity readout of the GFP-expressing viruses and a luciferase-based secondary screen yielding five promising host factors which may suit as potential targets for drug therapy. CONCLUSION: We report of an alternative method for high-throughput imaging methods to detect host factors being relevant for the infection efficiency of viruses. The method is generic and has the potential to be used for a large variety of different viruses and treatments being screened by imaging techniques.

Biotinylated quercetin as an intrinsic photoaffinity proteomics probe for the identification of quercetin target proteins.

Quercetin is a flavonoid natural product, that is, found in many foods and has been found to have a wide range of medicinal effects. Though a number of quercetin binding proteins have been identified, there has been no systematic approach to identifying all potential targets of quercetin. We describe an O7-biotinylated derivative of quercetin (BioQ) that can act as a photoaffinity proteomics reagent for capturing quercetin binding proteins, which can then be identified by LC-MS/MS. BioQ was shown to inhibit heat induction of HSP70 with almost the same efficiency as quercetin, and to both inhibit and photocrosslink to CK2 kinase, a known target of quercetin involved in activation of the heat shock transcription factor. BioQ was also able to pull down a number of proteins from unheated and heated Jurkat cells following UV irradiation that could be detected by both silver staining and Western blot analysis with an anti-biotin antibody. Analysis of the protein bands by trypsinization and LC-MS/MS led to the identification of heat shock proteins HSP70 and HSP90 as possible quercetin target proteins, along with ubiquitin-activating enzyme, a spliceosomal protein, RuvB-like 2 ATPases, and eukaryotic translation initiation factor 3. In addition, a mitochondrial ATPase was identified that has been previously shown to be a target of quercetin. Most of the proteins identified have also been previously suggested to be potential anticancer targets, suggesting that quercetin's antitumor activity may be due to its ability to inhibit multiple target proteins.

Protein kinase CK2 and new binding partners during spermatogenesis.

Protein kinase CK2 is an ubiquitously expressed enzyme that is absolutely necessary for the survival of cells. Besides the holoenzyme consisting of the regulatory ß-subunit and the catalytic α- or α'-subunit, the subunits exist in separate forms. The subunits bind to a number of other cellular proteins. We show the expression of individual subunits as well as interaction with the transitional nuclear protein TNP1 and with the motor neuron protein KIF5C during spermatogenesis. TNP1 is a newly identified binding partner of the α-subunit of CK2. CK2α and KIF5C were found in late spermatogenesis, whereas CK2ß and TNP1 were found in early spermatogenesis. CK2α, CK2α', TNP1, and KIF5C were detected in the acrosome of spermatozoa, while CK2ß was detectable in the mid-piece. Combinations of CK2 subunits might determine interactions with other proteins during spermatogenesis. KIF5C as a kinesin motor neuron protein is probably involved in the redistribution of proteins during spermatogenesis.

A FRET-based microplate assay for human protein kinase CK2, a target in neoplastic disease.

Besides cardiovascular diseases, cancer represents the major cause of death in developed countries. In many different human tumors, increased activity of serine/threonine protein kinase CK2 has been detected, and recent in vivo studies support a direct involvement of CK2 in tumor progression. Therefore, potent compounds to decrease CK2 activity to a non-pathogenic level would be a promising effort toward an antineoplastic therapy. In this study, an alternative to the established radiometric phosphorylation assay for quantification of CK2 activity was developed. For this purpose, the substrate peptide RRRDDDSDDD was coupled at the C-terminus to the fluorophore EDANS (5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid) and at the N-terminus to the quencher DABCYL (4-(4-dimethylaminophenylazo)benzoic acid). This resulted in quenched fluorescence of EDANS due to a FRET-based effect. After proteolytic cleavage of the peptide by elastase, the quenching effect was reduced and, as a consequence, fluorescence was increased. Because elastase is supposed to cleave at the S/D site of the peptide, phosphorylation of serine by CK2 hampered substrate binding of elastase and blocked the increase in fluorescence by proteolytic cleavage. This means that the new assay to quantify human CK2 activity is based on the differential accessibility of the proteolytic cleavage site, which is dependent on kinase phosphorylation. It could be used to measure inhibition of the human target in neoplastic diseases by the compounds TBB (4,5,6,7-tetrabromobenzotriazole) and Emodin.

Inhibition of protein kinase CK2 closes the CFTR Cl channel, but has no effect on the cystic fibrosis mutant deltaF508-CFTR.

BACKGROUND: Deletion of phenylalanine-508 (DeltaF508) from the first nucleotide-binding domain (NBD1) in the wild-type cystic fibrosis (CF) transmembrane-conductance regulator (wtCFTR) causes CF. However, the mechanistic relationship between DeltaF508-CFTR and the diversity of CF disease is unexplained. The surface location of F508 on NBD1 creates the potential for protein-protein interactions and nearby, lies a consensus sequence (SYDE) reported to control the pleiotropic protein kinase CK2. METHODS: Electrophysiology, immunofluorescence and biochemistry applied to CFTR-expressing cells, Xenopus oocytes, pancreatic ducts and patient biopsies. RESULTS: Irrespective of PKA activation, CK2 inhibition (ducts, oocytes, cells) attenuates CFTR-dependent Cl(-) transport, closing wtCFTR in cell-attached membrane patches. CK2 and wtCFTR co-precipitate and CK2 co-localized with wtCFTR (but not DeltaF508-CFTR) in apical membranes of human airway biopsies. Comparing wild-type and DeltaF508CFTR expressing oocytes, only DeltaF508-CFTR Cl(-) currents were insensitive to two CK2 inhibitors. Furthermore, wtCFTR was inhibited by injecting a peptide mimicking the F508 region, whereas the DeltaF508-equivalent peptide had no effect. CONCLUSIONS: CK2 controls wtCFTR, but not DeltaF508-CFTR. Others find that peptides from the F508 region of NBD1 allosterically control CK2, acting through F508. Hence, disruption of CK2-CFTR interaction by DeltaF508-CFTR might disrupt multiple, membrane-associated, CK2-dependent pathways, creating a new molecular disease paradigm for deleted F508 in CFTR.

NKX3.1 is regulated by protein kinase CK2 in prostate tumor cells.

Diminished expression of NKX3.1 is associated with prostate cancer progression in humans, and in mice, loss of nkx3.1 leads to epithelial cell proliferation and altered gene expression patterns. The NKX3.1 amino acid sequence includes multiple potential phosphoacceptor sites for protein kinase CK2. To investigate posttranslational regulation of NKX3.1, phosphorylation of NKX3.1 by CK2 was studied. In vitro kinase assays followed by mass spectrometric analyses demonstrated that CK2 phosphorylated recombinant NKX3.1 on Thr89 and Thr93. Blocking CK2 activity in LNCaP cells with apigenin or 5,6-dichlorobenzimidazole riboside led to a rapid decrease in NKX3.1 accumulation that was rescued by proteasome inhibition. Replacing Thr89 and Thr93 with alanines decreased NKX3.1 stability in vivo. Small interfering RNA knockdown of CK2alpha' but not CK2alpha also led to a decrease in NKX3.1 steady-state level. In-gel kinase assays and Western blot analyses using fractionated extracts of LNCaP cells demonstrated that free CK2alpha' could phosphorylate recombinant human and mouse NKX3.1, whereas CK2alpha' liberated from the holoenzyme could not. These data establish CK2 as a regulator of NKX3.1 in prostate tumor cells and provide evidence for functionally distinct pools of CK2alpha' in LNCaP cells.

Detection of casein kinase II by aggregation-induced emission.

A novel aggregation-induced emission (AIE) probe comprised of a hydrophilic protein kinase specific peptide and a hydrophobic tetraphenylethene (TPE) unit was synthesized through click reaction. The prepared TPE-peptide probe could be completely degraded by carboxypeptidase Y (CPY) to release hydrophobic TPE part, which aggregated in buffer solution and showed strong TPE emission. In the presence of casein kinase (CKII), the phosphorylation of peptide prevented the complete degradation by CPY producing the nonemissive probe. Thus, the developed probe can be used to detect CKII homogeneously and conveniently. This detection process can be finished within 1.5 h with high sensitivity (0.05 mU/µL) and good selectivity. The developed method can also be used to screen protein kinase inhibitor even in a complex biological system.

Obesity-Linked Phosphorylation of SIRT1 by Casein Kinase 2 Inhibits Its Nuclear Localization and Promotes Fatty Liver.

Sirtuin1 (SIRT1) deacetylase delays and improves many obesity-related diseases, including nonalcoholic fatty liver disease (NAFLD) and diabetes, and has received great attention as a drug target. SIRT1 function is aberrantly low in obesity, so understanding the underlying mechanisms is important for drug development. Here, we show that obesity-linked phosphorylation of SIRT1 inhibits its function and promotes pathological symptoms of NAFLD. In proteomic analysis, Ser-164 was identified as a major serine phosphorylation site in SIRT1 in obese, but not lean, mice, and this phosphorylation was catalyzed by casein kinase 2 (CK2), the levels of which were dramatically elevated in obesity. Mechanistically, phosphorylation of SIRT1 at Ser-164 substantially inhibited its nuclear localization and modestly affected its deacetylase activity. Adenovirus-mediated liver-specific expression of SIRT1 or a phosphor-defective S164A-SIRT1 mutant promoted fatty acid oxidation and ameliorated liver steatosis and glucose intolerance in diet-induced obese mice, but these beneficial effects were not observed in mice expressing a phosphor-mimic S164D-SIRT1 mutant. Remarkably, phosphorylated S164-SIRT1 and CK2 levels were also highly elevated in liver samples of NAFLD patients and correlated with disease severity. Thus, inhibition of phosphorylation of SIRT1 by CK2 may serve as a new therapeutic approach for treatment of NAFLD and other obesity-related diseases.

Identification and validation of novel prognostic markers in Renal Cell Carcinoma.

Kidney cancer (Renal Cell Carcinoma (RCC)) is one of the most deadly malignancies due to frequent late diagnosis and poor treatment options. Histologically, RCC embraces a wide variety of different subtypes with the clear cell variant (ccRCC) being the most common, accounting for 75-90% of all RCCs. At present, the surveillance protocols for follow-up of RCC patients after radical nephrectomy are based on the American Joint Committee on Cancers (AJCC) pathological tumor-node-metastasis (TNM) classification system. Other comprehensive staging modalities have emerged and have been implemented in an attempt to improve prognostication by combining other pathological and clinical variables, including Fuhrman nuclear grade and Leibovich score. However, even early stage tumors remain at risk of metastatic progression after surgical resection and 20-40% of patients undergoing nephrectomy for clinically localized RCC will develop a recurrence. Identifying this high-risk group of RCC patients remains a challenge. Hence, novel molecular prognostic biomarkers are needed to better predict clinical outcomes. An intensive search within this field has been ongoing in the past few years, and the three main predictive and prognostic markers validated in RCC are Von Hippel Lindau (VHL), vascular endothelial growth factor (VEGF) and carbonic anhydrase IX (CAIX). Nonetheless, the use of these is still debated and none of them have yet been implemented in clinical routine. RCC is resistant to conventional oncological therapies, such as chemotherapy and radiation. The availability of novel targeted therapies directed against tumorigenic and angiogenic pathways have increased over the last years, and the outcome of patients with advanced RCC has significantly improved as a consequence. Unfortunately, all patients eventually become resistant. Thus, the development of novel targeted therapies is of great importance. The aim of this thesis was therefore to contribute in the search for novel prognostic molecular markers in RCC and to identify novel targeted therapies by in-vitro studies. This was specifically conducted by investigating; 1) The impact of symptom presentation of RCC on prognosis, 2) The expression of Calcium-activated potassium channels in RCC, the correlation of KCa3.1 to prognosis in ccRCC and the ability of TRAM-34, RA-2 and Paxilline to inhibit the proliferation of ccRCC cell lines in-vitro, 3) The gene expression and prognostic value of 19 selected genes in ccRCC and 4) The expression of the protein kinase CK subunits in subtypes of RCC, the prognostic impact of high protein expression of the CK2α subunit in ccRCC and the ability of CX-4945 and E9 to inhibit ccRCC growth in-vitro. Our molecular study cohort consisted of 155 patients with different subtypes of RCC and the benign renal neoplasm, oncocytoma. They were diagnosed in Region of Southern Denmark in 2001-2013. Frozen tissue from tumor and normal renal cortex parenchyma, together with paraffin-embedded tissue was available for every patient. We performed gene expression analysis by qRT-PCR, immunohistochemical staining of Tissue Micro Arrays, protein kinase activity analysis and functional studies. Study I was performed as a descriptive observational study focusing on the prognostic impact of symptom presentation in RCC. We included 204 patients with renal neoplasms diagnosed in 2011-2012. Incidentally discovered RCC without symptomatic presentation had overall a better prognosis, and presented with smaller tumors, a lower T-stage, lower Fuhrman grade and lower Leibovich score. In addition, the non-symptomatic patient group experienced metastatic disease less frequently. In study II we focused on the expression of two calcium-activated potassium channels in ccRCC and oncocytoma. Both KCa3.1 and KCa1.1 were higher expressed in ccRCC compared to oncocytoma. High expression of KCa3.1 was moreover correlated with poor progression free survival of ccRCC. Functional studies provided new insights since we could detect currents compatible with KCa3.1 and KCa1.1 in the cell membrane of primary and commercial ccRCC cell lines. Nonetheless, we were not able to show any significant inhibition of cell growth by the selective inhibitors of KCa3.1 and KCa1.1, TRAM-34, RA-2 and Paxilline. In study III our aim was to investigate the prognostic role of 19 genes selected on the basis of an earlier study done by the group. We used Taqman® Low Density Array to perform a quantitative real-time PCR analysis. By selecting an optimal cut-point and correct for overestimation of the p-value, we could identify three genes with impact on prognosis of ccRCC in both univariate and multivariate analysis. High expression of the genes SPP1 and CSNK2A1 (encoding Osteopontin and CK2α respectively) correlated with poor prognosis while high expression of DEFB1 (encoding ß-Defensin) correlated with better prognosis. Study IV focused on validating the results obtained in Paper III by investigating the protein expression of CK2α (Protein kinase 2, alpha subunit) in the different subtypes of RCC and oncocytoma. Furthermore, we investigated whether protein expression of CK2α in ccRCC correlated with prognosis. Here we could show, that a positive nuclear staining was a marker of poor prognosis in high-stage ccRCC. Moreover, enzyme activity analysis revealed a higher activity of the protein kinase in tumor tissue of ccRCC than in normal renal cortex. Novel insights were provided in a proliferation study where we investigated the selective inhibitors of CK2α, CX-4945 and E9. CX-4945 was able to inhibit ccRCC cell growth by nearly 50%. All together the studies presented in this thesis add additional information to the ongoing research within identification of novel prognostic markers in ccRCC. We have discovered four new molecular markers, which reliably can predict prognosis at the time of diagnosis. Additionally, we identified CK2α as a novel therapeutic target of ccRCC. The studies suggest further research to validate the findings on larger cohorts and thereby obtain more insight into the involved pathways. Future research initiatives based on the results presented in this thesis could clarify the potential role of CX-4945 as a novel targeted treatment of ccRCC patients.

Phospholipase D is activated and phosphorylated by casein kinase-II in human U87 astroglioma cells.

Elevated expression of protein casein kinase II (CKII) stimulated basal phospholipase D (PLD) activity as well as PMA-induced PLD activation in human U87 astroglioma cells. Moreover, CKII-selective inhibitor, emodin and apigenin suppressed PMA-induced PLD activation in a dose-dependent manner as well as basal PLD activity, suggesting the involvement of CKII in the activation of both PLD1 and PLD2. CKII was associated with PLD1 and PLD2 in co-transfection experiments. Furthermore, CKII induced serine/threonine phosphorylation of PLD2 in vivo, and the multiple regions of PLD2 were phosphorylated by CKII in vitro kinase assay using glutathione S-transferase-PLD2 fusion protein fragments. Elevated expression of CKII or PLD increased cell proliferation but pretreatment of cells with 1-butanol suppressed CKII-induced cell proliferation. These results suggest that CKII is involved in proliferation of U87 cells at least in part, through stimulation of PLD activity.

Targeting protein kinase CK2 suppresses bladder cancer cell survival via the glucose metabolic pathway.

Casein kinase 2 (CK2) is a constitutively active serine/threonine kinase that promotes cell proliferation and resists apoptosis. Elevated CK2 expression has been demonstrated in several solid tumors. The expression of CK2α in bladder cancer was elevated in tumor tissues compared with that in adjacent normal tissues. Amplified expression of CK2α was highly correlated with histological grade in bladder cancer(P = 0.024). Knockdown of CK2α in bladder cancer cell lines resulted in a reduction in tumor aerobic glycolysis, accompanied with lower phosphorylated AKT. Moreover, low CK2α levels suppressed cell growth, and similar results could be reproduced after treatment with CX-4945 with a dose-dependent response. CX-4945 inhibited migration and induced apoptosis. Furthermore, knockdown of CK2α decreased the tumorigenicity of bladder cancer cells in vivo. This study is the first to report that CK2 increases glucose metabolism in human bladder cancer. Blocking CK2 function may provide novel diagnostic and potential therapeutic.

Regulation of thyroid hormone receptor alpha2 RNA binding and subcellular localization by phosphorylation.

Thyroid hormone receptor alpha2 (TRalpha2) is an alternative splice product of the TRalpha primary transcript whose unique carboxyl terminus does not bind T3 or activate transcription. The physiological function of TRalpha2 is unknown. We have found that TRalpha2 is a single stranded RNA binding protein and that the RNA binding domain localizes to a 41 amino acid region immediately distal to the second zinc finger. TRalpha2 contains a single protein kinase CK2 phosphorylation site in its amino terminus and potentially nine CK2 sites in its unique carboxyl terminus. In vitro CK2 treatment of TRalpha2 eliminated its RNA binding. Mutational analysis indicated that phosphorylations at the N- and C-terminal sites both contribute to this inhibitory effect. Cellular localization studies demonstrated that phosphorylated TRalpha2 is primarily cytoplasmic, whereas unphosphorylated TRalpha2 is primarily nuclear. Since RNA binding is a property of unphosphorylated TRalpha2, the TRalpha2-RNA interaction likely represents a nuclear function of TRalpha2.

Label-free fluorescence assay for protein kinase based on peptide biomineralized gold nanoclusters as signal sensing probe.

A label-free, sensitive and simple method to detect protein kinase based on the selective aggregation of phosphorylated peptide-gold nanoclusters (peptide-AuNCs) triggered by Zr(4+) ion coordination is developed. The AuNCs were synthesized by peptide without any strong reducing agents, which prevent peptides from being disrupted. Under optimal conditions, a linear relationship between the decreased PL intensity of peptide-AuNCs and the concentration of casein kinase II (CK2) in the range of 0.08-2.0 unit mL(-1) with a detection limit of 0.027 unit mL(-1) (3σ) was obtained. The feasibility of this AuNCs-based sensor was further demonstrated by the assessment of kinase inhibition by ellagic acid, 5,6-dichlorobenzimidazole-1-ß-D-ribofuranoside, emodin, and quercetin in human serum. As expected, the PL intensity increased with increasing inhibitor efficiency in the presence of inhibitors. The IC50 value (inhibitor concentration producing 50% inhibition) for ellagic acid was estimated to be 0.045 µM. With more sophisticated design of the peptide substrate sequences, the detection of other enzymes will be realized. With characteristics of homogeneous, facile, universal, label-free, and applicable for kinase assay, the proposed sensor provides potential application in kinase-related biochemical fundamental research and inhibitor screening.

Electrochemical detection of protein kinase activity based on carboxypeptidase Y digestion triggered signal amplification.

An effective assay method for monitoring protein kinase activity and screening inhibitors is greatly beneficial to kinase-related drug discovery, early diagnosis of diseases, and therapeutic effect evaluation. Herein, we develop a simple electrochemical method for detecting the activity of casein kinase II (CK2) based on phosphorylation against carboxypeptidase Y (CPY) digestion triggered signal amplification, where CK2 catalyzed phosphorylation event protects the substrate peptide from the digestion of CPY, maintains the repulsive force of the substrate peptide towards the redox probe, and results in a weak electrochemical signal. Whereas, without phosphorylation, the substrate peptide is digested by CPY and a strong electrochemical signal is obtained. The detection feasibility is demonstrated for the assay of CK2 activity with low detection limit of 0.047unit/mL. Moreover, the biosensor was used for the analysis of kinase inhibition. Based on the electrochemical signal dependent inhibitor concentration, the IC50 value of ellagic acid was estimated to be 39.77nM. The proposed method is also successfully applied to analyze CK2 activity in cell lysates, proving the applicability in complex biological samples.