Effects of CK2β subunit down-regulation on Akt signalling in HK-2 renal cells.

The PI3K/Akt pathway is interconnected to protein kinase CK2, which directly phosphorylates Akt1 at S129. We have previously found that, in HK-2 renal cells, downregulation of the CK2 regulatory subunit β (shCK2β cells) reduces S129 Akt phosphorylation. Here, we investigated in more details how the different CK2 isoforms impact on Akt and other signaling pathways.

Up-Regulation of the Alpha Prime Subunit of Protein Kinase CK2 as a Marker of Fast Proliferation in GL261 Cultured Cells.

Glioblastoma (GB) is the most prevalent malignant primary brain tumor in adults. The preclinical glioblastoma model GL261 is widely used for investigating new therapeutic strategies. GL261 cultured cells are used for assessing preliminary in vitro data for this model although very little is known about the molecular characteristics of this cell line.

Under-expression of CK2β subunit in ccRCC represents a complementary biomarker of p-STAT3 Ser727 that correlates with patient survival.

Clear cell renal cell carcinoma (ccRCC) is the most common and aggressive subtype of renal cancer. STAT3 pathway is altered in these tumors and p-STAT3 Ser727 is an independent prognostic factor for ccRCC. Protein kinase CK2 is altered in different types of tumors and overexpression of CK2α is considered predictive of bad prognosis and metastatic risk.

Targeting Protein Kinase CK2: Evaluating CX-4945 Potential for GL261 Glioblastoma Therapy in Immunocompetent Mice.

Glioblastoma (GBM) causes poor survival in patients even with aggressive treatment. Temozolomide (TMZ) is the standard chemotherapeutic choice for GBM treatment but resistance always ensues. Protein kinase CK2 (CK2) contributes to tumour development and proliferation in cancer, and it is overexpressed in human GBM.

Protein Kinase CK2 Content in GL261 Mouse Glioblastoma.

Glioblastoma (GBM) is the most prevalent and aggressive human glial tumour with a median survival of 14-15 months. Temozolomide (TMZ) is the standard chemotherapeutic choice for GBM treatment. Unfortunately, chemoresistence always ensues with concomitant tumour regrowth.

HAVCR/KIM-1 activates the IL-6/STAT-3 pathway in clear cell renal cell carcinoma and determines tumor progression and patient outcome.

Renal cell carcinoma (RCC), the third most prevalent urological cancer, claims more than 100,000 lives/year worldwide. The clear cell variant (ccRCC) is the most common and aggressive subtype of this disease. While commonly asymptomatic, more than 30% of ccRCC are diagnosed when already metastatic, resulting in a 95% mortality rate.

Protein kinase CK2-dependent phosphorylation of the human Regulators of Calcineurin reveals a novel mechanism regulating the calcineurin-NFATc signaling pathway.

Cyclosporine A and FK506 produce immunosuppression by blocking calcineurin phosphatase activity and consequently activation of cytosolic Nuclear Factor of Activated T-cell (NFATc) transcription factor. Due to the chronic toxicity associated with their administration, the development of more specific immunosuppressants is currently an important unmet medical need. In this context, an immunosuppressant peptide derived from the CIC motif of the human Regulators of Calcineurin (RCAN) proteins has been shown to inhibit NFATc signaling without affecting general phosphatase activity of calcineurin.

Hepatitis A virus cellular receptor 1/kidney injury molecule-1 is a susceptibility gene for clear cell renal cell carcinoma and hepatitis A virus cellular receptor/kidney injury molecule-1 ectodomain shedding a predictive biomarker of tumour progression.

Aim Of The Study: To correlate hepatitis A virus cellular receptor (HAVCR)/kidney injury molecule-1 (KIM-1) expression in clear cell renal cell carcinoma (ccRCC) tumours with patient outcome and study the consequences of HAVCR/KIM-1 ectodomain shedding.

Methods: HAVCR/KIM-1 expression in ccRCC, oncocytomes, papillary carcinomas and unaffected tissue counterparts was evaluated. Minimal change disease and pre-clamping normal and ccRCC tissue biopsies were included.

A pharmacologically-based array to identify targets of cyclosporine A-induced toxicity in cultured renal proximal tubule cells.

Mechanisms of cyclosporine A (CsA)-induced nephrotoxicity were generally thought to be hemodynamic in origin; however, there is now accumulating evidence of a direct tubular effect. Although genomic and proteomic experiments by our group and others provided overall information on genes and proteins up- or down-regulated by CsA in proximal tubule cells (PTC), a comprehensive view of events occurring after CsA exposure remains to be described. For this purpose, we applied a pharmacologic approach based on the use of known activities of a large panel of potentially protective compounds and evaluated their efficacy in preventing CsA toxicity in cultured mouse PTC.

KAP degradation by calpain is associated with CK2 phosphorylation and provides a novel mechanism for cyclosporine A-induced proximal tubule injury.

The use of cyclosporine A (CsA) is limited by its severe nephrotoxicity that includes reversible vasoconstrictor effects and proximal tubule cell injury, the latter associated whith chronic kidney disease progression. The mechanisms of CsA-induced tubular injury, mainly on the S3 segment, have not been completely elucidated. Kidney androgen-regulated protein (KAP) is exclusively expressed in kidney proximal tubule cells, interacts with the CsA-binding protein cyclophilin B and its expression diminishes in kidneys of CsA-treated mice.

Protein kinase CK2 associates to lipid rafts and its pharmacological inhibition enhances neurotransmitter release.

In the present work we report the presence of protein kinase CK2 in lipid raft preparations from rat brain synaptosomes, obtained after detergent extraction and subsequent isolation of detergent-resistant membranes using sucrose gradient ultracentrifugation. Moreover, the phosphorylation of syntaxin-1 at Ser14, a specific CK2 target, has been detected in lipid rafts, as assessed by a phospho-specific antibody. Treatment with DMAT, a specific CK2 inhibitor, results in a decrease of syntaxin-1 Ser14 phosphorylation in lipid rafts, while the glutamate release from synaptosomes is enhanced.

The regulatory beta subunit of protein kinase CK2 contributes to the recognition of the substrate consensus sequence. A study with an eIF2 beta-derived peptide.

CK2 is a ubiquitous and pleiotropic Ser/Thr-specific protein kinase that phosphorylates more than 300 protein substrates at sites specified by an acidic consensus sequence in which positions n + 3 and n + 1 are particularly important. Recognition of substrates by CK2 is known to rely on basic residues located in the catalytic site of the alpha subunit which make electrostatic contacts with the negative charges in the substrate consensus sequence, thereby assuring optimal binding; the regulatory beta subunit is believed to play a protective and stabilizing role. We describe a biochemical and structural analysis of CK2-mediated phosphorylation of a 22-mer synthetic peptide corresponding to the N-terminal tail of the eukaryotic translation initiation factor eIF2beta.

Phosphoinositide 3-kinase inhibitors protect mouse kidney cells from cyclosporine-induced cell death.

The use of cyclosporine has been restricted by its nephrotoxic effects mediated, in part, by reactive oxygen species (ROS). Phosphoinositide 3-kinase, protein kinase B, and extracellular regulated kinase (ERK) pathways are related to survival and cell death and are activated after ROS generation. In this study, we evaluated the effects of cyclosporine on these pathways and their contribution to cyclosporine-induced toxicity.

Proteomic analysis of SET-binding proteins.

The protein SET is involved in essential cell processes such as chromatin remodeling, apoptosis and cell cycle progression. It also plays a critical role in cell transformation and tumorogenesis. With the aim to study new SET functions we have developed a system to identify SET-binding proteins by combining affinity chromatography, MS, and functional studies.

Discrimination between the activity of protein kinase CK2 holoenzyme and its catalytic subunits.

The acronym CK2 denotes a highly pleiotropic Ser/Thr protein kinase whose over-expression correlates with neoplastic growth. A vexed question about the enigmatic regulation of CK2 concerns the actual existence in living cells of the catalytic (alpha and/or alpha') and regulatory beta-subunits of CK2 not assembled into the regular heterotetrameric holoenzyme. Here we take advantage of novel reagents, namely a peptide substrate and an inhibitor which discriminate between the holoenzyme and the catalytic subunits, to show that CK2 activity in CHO cells is entirely accounted for by the holoenzyme.

Cross talk between protein kinase CK2 and eukaryotic translation initiation factor eIF2beta subunit.

The beta-subunit of eukaryotic translation initiation factor eIF2 is a substrate and a partner for protein kinase CK2. Surface plasmon resonance analysis shows that the truncated form corresponding to residues 138-333 of eIF2beta (eIF2beta-CT) interacts with CK2beta as efficiently as full length eIF2beta, whereas the form corresponding to residues 1-137, which contains the CK2 phosphorylation sites, (eIF2beta-NT) does not bind. The use of different mutants and truncated forms of CK2alpha allowed us to map the basic segment K74-K83 at the beginning of helix alphaC and residues R191R195K198 in the p + 1 loop as the main determinants for the binding to eIF2beta-CT of either the isolated CK2alpha subunit or the CK2 holoenzyme.

The N-terminal domain of the human eIF2beta subunit and the CK2 phosphorylation sites are required for its function.

CK2 (protein kinase CK2) is known to phosphorylate eIF2 (eukaryotic translation initiation factor 2) in vitro; however, its implication in this process in living cells has remained to be confirmed. The combined use of chemical inhibitors (emodin and apigenin) of CK2 together with transfection experiments with the wild-type of the K68A kinase-dead mutant form of CK2alpha evidenced the direct involvement of this protein kinase in eIF2beta phosphorylation in cultured HeLa cells. Transfection of HeLa cells with human wild-type eIF2beta or its phosphorylation site mutants showed Ser2 as the main site for constitutive eIF2beta phosphorylation, whereas phosphorylation at Ser67 seems more restricted.

Unbalanced activation of ERK1/2 and MEK1/2 in apigenin-induced HeLa cell death.

Apigenin, a dietary bioflavonoid with anticarcinogenic properties, was highly cytotoxic for HeLa cells (incubated with 0.5% FBS). This effect was accompanied with a marked increase in ERK1/2 but not MEK1/2 phosphorylation.

Eukaryotic translation-initiation factor eIF2beta binds to protein kinase CK2: effects on CK2alpha activity.

eIF2 (eukaryotic translation-initiation factor 2) is a substrate and an interacting partner for CK2 (protein kinase CK2). Co-immuno-precipitation of CK2 with eIF2beta has now been observed in HeLa cells, overexpressing haemagglutinin-tagged human recombinant eIF2beta. A direct association between His6-tagged human recombinant forms of eIF2beta subunit and both the catalytic (CK2alpha) and the regulatory (CK2beta) subunits of CK2 has also been shown by using different techniques.

Persistent nuclear accumulation of protein kinase CK2 during the G1-phase of the cell cycle does not depend on the ERK1/2 pathway but requires active protein synthesis.

Protein kinase CK2 and phosphorylated ERK1/2 accumulated in nucleus after serum stimulation of quiescent HepG2 cells. Nonetheless, phospho-ERK1/2 accumulated mainly in the nuclease-extracted fraction (NE) whereas the increases in nuclear CK2 (either CK2alpha or CK2beta) occurred initially in the nuclease-resistant fraction (NR). Transient decreases in CK2 were observed in cytoplasm and NE in the first 3h but thereafter they either reverted (cytoplasm) or increased above the control (NE).

PP1/PP2A phosphatases inhibitors okadaic acid and calyculin A block ERK5 activation by growth factors and oxidative stress.

Okadaic acid is an inhibitor of the protein Ser/Thr phosphatases PP1 and PP2A, which blocks the activation of extracellular signal-regulated protein kinase 5 (ERK5), a member of the MAP kinase family activated by growth factors and several types of stressors. The blocking of ERK5 activation by okadaic acid was observed in HeLa cells exposed to epidermal growth factor and H(2)O(2) as well as in PC12 cells stimulated by nerve growth factor and H(2)O(2). Calyculin A, another PP1 and PP2A inhibitor, behaved similarly although these compounds are not structurally related.

Apigenin and LY294002 prolong EGF-stimulated ERK1/2 activation in PC12 cells but are unable to induce full differentiation.

In rat pheochromocytoma cell line (PC12) cells, initial epidermal growth factor (EGF)-stimulated extracellular signal-regulated protein kinases 1/2 (ERK1/2) phosphorylation was similar to that promoted by nerve growth factor (NGF), but declined rapidly. Pre-treatment with apigenin or LY294002 sustained EGF-stimulated ERK1/2 phosphorylation whereas wortmannin partially blocked initial ERK1/2 phosphorylation. Changes in ERK1/2 phosphorylation correlated with alterations in p90 ribosomal S6 kinase activity.

The transcriptional factor Tcf-4 contains different binding sites for beta-catenin and plakoglobin.

beta-Catenin and plakoglobin are two related armadillo proteins necessary for the establishment of adhesion junctions and desmosomes. Moreover, beta-catenin can also act as a transcriptional co-activator through its interaction with the members of Tcf/LEF-1 transcriptional factor family. We show here that Tcf-4 can be phosphorylated in vitro by protein kinase CK2 stoichiometrically in amino acids Ser-58-Ser-59-Ser-60.

The carboxy-terminal domain of Grp94 binds to protein kinase CK2 alpha but not to CK2 holoenzyme.

Surface plasmon resonance analysis shows that the carboxy-terminal domain of Grp94 (Grp94-CT, residues 518-803) physically interacts with the catalytic subunit of protein kinase CK2 (CK2 alpha) under non-stressed conditions. A K(D) of 4 x 10(-7) was determined for this binding. Heparin competed with Grp94-CT for binding to CK2 alpha.