Casein kinase 2 phosphorylates and induces the SALL2 tumor suppressor degradation in colon cancer cells.

Spalt-like proteins are Zinc finger transcription factors from Caenorhabditis elegans to vertebrates, with critical roles in development. In vertebrates, four paralogues have been identified (SALL1-4), and SALL2 is the family's most dissimilar member. SALL2 is required during brain and eye development. It is downregulated in cancer and acts as a tumor suppressor, promoting cell cycle arrest and cell death. Despite its critical functions, information about SALL2 regulation is scarce. Public data indicate that SALL2 is ubiquitinated and phosphorylated in several residues along the protein, but the mechanisms, biological consequences, and enzymes responsible for these modifications remain unknown. Bioinformatic analyses identified several putative phosphorylation sites for Casein Kinase II (CK2) located within a highly conserved C-terminal PEST degradation motif of SALL2. CK2 is a serine/threonine kinase that promotes cell proliferation and survival and is often hyperactivated in cancer. We demonstrated that CK2 phosphorylates SALL2 residues S763, T778, S802, and S806 and promotes SALL2 degradation by the proteasome. Accordingly, pharmacological inhibition of CK2 with Silmitasertib (CX-4945) restored endogenous SALL2 protein levels in SALL2-deficient breast MDA-MB-231, lung H1299, and colon SW480 cancer cells. Silmitasertib induced a methuosis-like phenotype and cell death in SW480 cells. However, the phenotype was significantly attenuated in CRISPr/Cas9-mediated SALL2 knockout SW480 cells. Similarly, Sall2-deficient tumor organoids were more resistant to Silmitasertib-induced cell death, confirming that SALL2 sensitizes cancer cells to CK2 inhibition. We identified a novel CK2-dependent mechanism for SALL2 regulation and provided new insights into the interplay between these two proteins and their role in cell survival and proliferation.

Influence of CK2 protein kinase activity on the interaction between Trypanosoma cruzi and its vertebrate and invertebrate hosts.

Chagas disease, endemic from Latin America, is caused by Trypanosoma cruzi and is transmitted by triatomine feces. This parasite undergoes complex morphological changes through its life cycle, promoted by significant changes in signal transduction pathways. The activity of protein kinase CK2 has been described in trypanosomatids. Using a specific peptide and radioactive ATP, we identified CK2 activity on the cellular surface and the cytoplasmic content in Trypanosoma cruzi, apart from the secreted form. Dephosphorylated casein promoted an increase of 48% in the secreted CK2 activity. Total extract of peritoneal macrophages from BALB/c and inactivated human serum promoted an increase of 67% and 36%, respectively, in this activity. The protein secreted by parasites was purified by HPLC and had shown compatibility with the catalytic subunit of mammalian CK2. Incubation of the parasites with CK2 inhibitors, added to the culture medium, prevented their growth. The opposite was observed when CK2 activators were used. Results of interaction between Trypanosoma cruzi and the gut of the vector have revealed that, in the presence of CK2 inhibitors, there is a reduction in the association rate. A similar inhibition profile was seen in the Trypanosoma cruzi-macrophages interaction, confirming the importance of this enzyme in the life cycle of this protozoan.

Identification of potential inhibitors of casein kinase 2 alpha of Plasmodium falciparum with potent in vitro activity.

Drug resistance to commercially available antimalarials is a major obstacle in malaria control and elimination, creating the need to find new antiparasitic compounds with novel mechanisms of action. The success of kinase inhibitors for oncological treatments has paved the way for the exploitation of protein kinases as drug targets in various diseases, including malaria. Casein kinases are ubiquitous serine/threonine kinases involved in a wide range of cellular processes such as mitotic checkpoint signaling, DNA damage response, and circadian rhythm. In Plasmodium, it is suggested that these protein kinases are essential for both asexual and sexual blood-stage parasites, reinforcing their potential as targets for multi-stage antimalarials. To identify new putative PfCK2α inhibitors, we utilized an in silico chemogenomic strategy involving virtual screening with docking simulations and quantitative structure-activity relationship predictions. Our investigation resulted in the discovery of a new quinazoline molecule (542), which exhibited potent activity against asexual blood stages and a high selectivity index (>100). Subsequently, we conducted chemical-genetic interaction analysis on yeasts with mutations in casein kinases. Our chemical-genetic interaction results are consistent with the hypothesis that 542 inhibits yeast Cka1, which has a hinge region with high similarity to PfCK2α. This finding is in agreement with our in silico results suggesting that 542 inhibits PfCK2α via hinge region interaction.

Trypanosoma cruzi DNA Polymerase ß Is Phosphorylated In Vivo and In Vitro by Protein Kinase C (PKC) and Casein Kinase 2 (CK2).

DNA polymerase ß plays a fundamental role in the life cycle of Trypanosoma cruzi since it participates in the kinetoplast DNA repair and replication. This enzyme can be found in two forms in cell extracts of T. cruzi epimastigotes form. The H form is a phosphorylated form of DNA polymerase ß, while the L form is not phosphorylated. The protein kinases which are able to in vivo phosphorylate DNA polymerase ß have not been identified yet. In this work, we purified the H form of this DNA polymerase and identified the phosphorylation sites. DNA polymerase ß is in vivo phosphorylated at several amino acid residues including Tyr35, Thr123, Thr137 and Ser286. Thr123 is phosphorylated by casein kinase 2 and Thr137 and Ser286 are phosphorylated by protein kinase C-like enzymes. Protein kinase C encoding genes were identified in T. cruzi, and those genes were cloned, expressed in bacteria and the recombinant protein was purified. It was found that T. cruzi possesses three different protein kinase C-like enzymes named TcPKC1, TcPKC2, and TcPKC3. Both TcPKC1 and TcPKC2 were able to in vitro phosphorylate recombinant DNA polymerase ß, and in addition, TcPKC1 gets auto phosphorylated. Those proteins contain several regulatory domains at the N-terminus, which are predicted to bind phosphoinositols, and TcPKC1 contains a lipocalin domain at the C-terminus that might be able to bind free fatty acids. Tyr35 is phosphorylated by an unidentified protein kinase and considering that the T. cruzi genome does not contain Tyr kinase encoding genes, it is probable that Tyr35 could be phosphorylated by a dual protein kinase. Wee1 is a eukaryotic dual protein kinase involved in cell cycle regulation. We identified a Wee1 homolog in T. cruzi and the recombinant kinase was assayed using DNA polymerase ß as a substrate. T. cruzi Wee1 was able to in vitro phosphorylate recombinant DNA polymerase ß, although we were not able to demonstrate specific phosphorylation on Tyr35. Those results indicate that there exists a cell signaling pathway involving PKC-like kinases in T. cruzi.

The Catalytic Subunit of Schizosaccharomyces pombe CK2 (Cka1) Negatively Regulates RNA Polymerase II Transcription through Phosphorylation of Positive Cofactor 4 (PC4).

Positive cofactor 4 (PC4) is a transcriptional coactivator that plays important roles in transcription and DNA replication. In mammals, PC4 is phosphorylated by CK2, and this event downregulates its RNA polymerase II (RNAPII) coactivator function. This work describes the effect of fission yeast PC4 phosphorylation on RNAPII transcription in a cell extract, which closely resembles the cellular context. We found that fission yeast PC4 is strongly phosphorylated by the catalytic subunit of CK2 (Cka1), while the regulatory subunit (Ckb1) downregulates the PC4 phosphorylation. The addition of Cka1 to an in vitro transcription assay can diminish the basal transcription from the Ad-MLP promoter; however, the addition of recombinant fission yeast PC4 or Ckb1 can stimulate the basal transcription in a cell extract. Fission yeast PC4 is phosphorylated in a domain which has consensus phosphorylation sites for CK2, and two serine residues were identified as critical for CK2 phosphorylation. Mutation of one of the serine residues in PC4 does not completely abolish the phosphorylation; however, when the two serine residues are mutated, CK2 is no longer able to phosphorylate PC4. The mutant which is not phosphorylated is able to stimulate transcription even though it is previously phosphorylated by Cka1, while the wild type and the point mutant are inactivated by Cka1 phosphorylation, and they cannot stimulate transcription by RNAPII in cell extracts. Those results demonstrate that CK2 can regulate the coactivator function of fission yeast PC4 and suggests that this event could be important in vivo as well.

Targeting of Protein Kinase CK2 Elicits Antiviral Activity on Bovine Coronavirus Infection.

Coronaviruses constitute a global threat to the human population; therefore, effective pan-coronavirus antiviral drugs are required to tackle future re-emerging virus outbreaks. Protein kinase CK2 has been suggested as a promising therapeutic target in COVID-19 owing to the in vitro antiviral activity observed after both pharmacologic and genetic inhibition of the enzyme. Here, we explored the putative antiviral effect of the anti-CK2 peptide CIGB-325 on bovine coronavirus (BCoV) infection using different in vitro viral infected cell-based assays. The impact of the peptide on viral mRNA and protein levels was determined by qRT-PCR and Western blot, respectively. Finally, pull-down experiments followed by Western blot and/or mass spectrometry analysis were performed to identify CIGB-325-interacting proteins. We found that CIGB-325 inhibited both the cytopathic effect and the number of plaque-forming units. Accordingly, intracellular viral protein levels were clearly reduced after treatment of BCoV-infected cells, with CIGB-325 determined by immunocytochemistry. Pull-down assay data revealed the physical interaction of CIGB-325 with viral nucleocapsid (N) protein and a group of bona fide CK2 cellular substrates. Our findings evidence in vitro antiviral activity of CIGB-325 against bovine coronavirus as well as some molecular clues that might support such effect. Altogether, data provided here strengthen the rationale of inhibiting CK2 to treat betacoronavirus infections.

Pro-Oxidant Effect of Resveratrol on Human Breast Cancer MCF-7 Cells is Associated with CK2 Inhibition.

Casein kinase 2 (CK2) plays a critical role in the proliferation and apoptosis of cancer cells. Resveratrol is a bioactive compound with anticancer and anti-inflammatory effects. This study investigated the pro-oxidant cytotoxic effects of resveratrol in association with the inhibition of CK2 activity on human breast carcinoma cells MCF-7. We showed that resveratrol and TBB, an inhibitor of CK2, decreased cell viability in a concentration dependent manner with an IC50 value of 238 µM and 106 µM after 24 h, of treatment, respectively. Resveratrol and TBB decreased CK2 activity by 1.6 and 1.4-fold, respectively, and both significantly decreased mitochondrial membrane potential. However, only resveratrol increased reactive oxygen species (ROS) levels by 1.7-fold as opposed to TBB, which did not affect ROS levels. Indeed, incubating MCF-7 cells with the antioxidant polyethylene glycol-catalase (PEG-CAT) preserved cell viability from the cytotoxic effects of resveratrol, but not from TBB toxicity. This effect seemed to be related to PEG-CAT ability to prevent CK2 inhibition induced by resveratrol incubation. In conclusion, this study demonstrated that the cytotoxic effect of resveratrol on MCF-7 cells might be associated with its pro-oxidant action, which inhibited CK2 activity, affecting cell viability and mitochondrial function.

Phosphoproteomic Landscape of AML Cells Treated with the ATP-Competitive CK2 Inhibitor CX-4945.

Casein kinase 2 (CK2) regulates a plethora of proteins with pivotal roles in solid and hematological neoplasia. Particularly, in acute myeloid leukemia (AML) CK2 has been pointed as an attractive therapeutic target and prognostic marker. Here, we explored the impact of CK2 inhibition over the phosphoproteome of two cell lines representing major AML subtypes. Quantitative phosphoproteomic analysis was conducted to evaluate changes in phosphorylation levels after incubation with the ATP-competitive CK2 inhibitor CX-4945. Functional enrichment, network analysis, and database mining were performed to identify biological processes, signaling pathways, and CK2 substrates that are responsive to CX-4945. A total of 273 and 1310 phosphopeptides were found differentially modulated in HL-60 and OCI-AML3 cells, respectively. Despite regulated phosphopeptides belong to proteins involved in multiple biological processes and signaling pathways, most of these perturbations can be explain by direct CK2 inhibition rather than off-target effects. Furthermore, CK2 substrates regulated by CX-4945 are mainly related to mRNA processing, translation, DNA repair, and cell cycle. Overall, we evidenced that CK2 inhibitor CX-4945 impinge on mediators of signaling pathways and biological processes essential for primary AML cells survival and chemosensitivity, reinforcing the rationale behind the pharmacologic blockade of protein kinase CK2 for AML targeted therapy.

Preclinical efficacy of CIGB-300, an anti-CK2 peptide, on breast cancer metastasic colonization.

CK2 is a serine/threonine kinase that is overexpressed in breast cancer and its inhibition is associated to reduced tumor growth and disease progression. CIGB-300 is an antitumor peptide with a novel mechanism of action, since it binds to protein kinase CK2 catalytic subunit alpha and to CK2 substrates thus preventing the enzyme activity. Our aim was to evaluate the potential therapeutic benefits of CIGB-300 on breast cancer disease using experimental models with translational relevance. We demonstrated that CIGB-300 reduces breast cancer cell growth in MDA-MB-231, MCF-7 and F3II cells, exerting a pro-apoptotic action and cell cycle arrest. We also found that CIGB-300 decreased cell adhesion, migration and clonogenic capacity of malignant cells. Effect on experimental breast cancer lung metastasis was evaluated after surgical removal of primary F3II tumors or after tail vein injection of tumor cells, also we evaluated CIGB-300 effect on spontaneous lung metastasis in an orthotopic model. Systemic CIGB-300 treatment inhibited breast cancer colonization of the lung, reducing the size and number of metastatic lesions. The present preclinical study establishes for the first time the efficacy of CIGB-300 on breast cancer. These encouraging results suggest that CIGB-300 could be used for the management of breast cancer as an adjuvant therapy after surgery, limiting tumor metastatic spread and thus protecting the patient from distant recurrence.

IKAROS and CK2 regulate expression of BCL-XL and chemosensitivity in high-risk B-cell acute lymphoblastic leukemia.

High-risk B-cell acute lymphoblastic leukemia (B-ALL) is an aggressive disease, often characterized by resistance to chemotherapy. A frequent feature of high-risk B-ALL is loss of function of the IKAROS (encoded by the IKZF1 gene) tumor suppressor. Here, we report that IKAROS regulates expression of the BCL2L1 gene (encodes the BCL-XL protein) in human B-ALL. Gain-of-function and loss-of-function experiments demonstrate that IKAROS binds to the BCL2L1 promoter, recruits histone deacetylase HDAC1, and represses BCL2L1 expression via chromatin remodeling. In leukemia, IKAROS' function is impaired by oncogenic casein kinase II (CK2), which is overexpressed in B-ALL. Phosphorylation by CK2 reduces IKAROS binding and recruitment of HDAC1 to the BCL2L1 promoter. This results in a loss of IKAROS-mediated repression of BCL2L1 and increased expression of BCL-XL. Increased expression of BCL-XL and/or CK2, as well as reduced IKAROS expression, are associated with resistance to doxorubicin treatment. Molecular and pharmacological inhibition of CK2 with a specific inhibitor CX-4945, increases binding of IKAROS to the BCL2L1 promoter and enhances IKAROS-mediated repression of BCL2L1 in B-ALL. Treatment with CX-4945 increases sensitivity to doxorubicin in B-ALL, and reverses resistance to doxorubicin in multidrug-resistant B-ALL. Combination treatment with CX-4945 and doxorubicin show synergistic therapeutic effects in vitro and in preclinical models of high-risk B-ALL. Results reveal a novel signaling network that regulates chemoresistance in leukemia. These data lay the groundwork for clinical testing of a rationally designed, targeted therapy that combines the CK2 inhibitor, CX-4945, with doxorubicin for the treatment of hematopoietic malignancies.

Lysophosphatidylcholine triggers cell differentiation in the protozoan parasite Herpetomonas samuelpessoai through the CK2 pathway.

BACKGROUND: Protozoa are distantly related to vertebrates but present some features of higher eukaryotes, making them good model systems for studying the evolution of basic processes such as the cell cycle. Herpetomonas samuelpessoai is a trypanosomatid parasite isolated from the hemipteran insect Zelus leucogrammus. Lysophosphatidylcholine (LPC) is implicated in the transmission and establishment of Chagas disease, whose etiological agent is Trypanosoma cruzi. LPC is synthesized by T. cruzi and its vectors, the hemipteran Rhodnius prolixus and Triatoma infestans. Platelet-activating factor (PAF), a phospholipid with potent and diverse physiological and pathophysiological actions, is a powerful inducer of cell differentiation in Herpetomonas muscarum muscarum and T. cruzi. The enzyme phospholipase A2 (PLA2) catalyzes the hydrolysis of the 2-ester bond of 3-sn-phosphoglyceride, transforming phosphatidylcholine (PC) into LPC. METHODS: In this study, we evaluated cellular differentiation, PLA2 activity and protein kinase CK2 activity of H. samuelpessoai in the absence and in the presence of LPC and PAF. RESULTS: We demonstrate that both PC and LPC promoted a twofold increase in the cellular differentiation of H. samuelpessoai, through CK2, with a concomitant inhibition of its cell growth. Intrinsic PLA2 most likely directs this process by converting PC into LPC. CONCLUSIONS: Our results suggest that the actions of LPC on H. samuelpessoai occur upon binding to a putative PAF receptor and that the protein kinase CK2 plays a major role in this process. Cartoon depicting a model for the synthesis and functions of LPC in Herpetomonas samuelpessoai, based upon our results regarding the role of LPC on the cell biology of Trypanosoma cruzi [28-32]. N nucleus, k kinetoplast, PC phosphatidylcholine, LPC lysophosphatidylcholine, PLA2 phospholipase A2, PAFR putative PAF receptor in trypanosomatids [65], CK2 protein kinase CK2 [16].

Endothelin-converting enzyme-1c promotes stem cell traits and aggressiveness in colorectal cancer cells.

Endothelin-1 is a mitogenic peptide that activates several proliferation, survival, and invasiveness pathways. The effects of endothelin-1 rely on its activation by endothelin-converting enzyme-1 (ECE1), which is expressed as four isoforms with different cytoplasmic N termini. Recently, isoform ECE1c has been suggested to have a role in cancer aggressiveness. The N terminus of ECE1c is phosphorylated by protein kinase CK2 (also known as casein kinase 2), and this enhances its stability and promotes invasiveness in colorectal cancer cells. However, it is not known how phosphorylation improves stability and why this is correlated with increased aggressiveness. We hypothesized that CK2 phosphorylation protects ECE1c from N-terminal ubiquitination and, consequently, from proteasomal degradation. Here, we show that lysine 6 is the bona fide residue involved in ubiquitination of ECE1c and its mutation to arginine (ECE1cK6R ) significantly impairs proteasomal degradation, thereby augmenting ECE1c stability, even in the presence of the CK2 inhibitor silmitasertib. Furthermore, colorectal cancer cells overexpressing ECE1cK6R displayed enhanced cancer stem cell (CSC) traits, including increased stemness gene expression, chemoresistance, self-renewal, and colony formation and spheroid formation in vitro, as well as enhanced tumor growth and metastasis in vivo. These findings suggest that CK2-dependent phosphorylation enhances ECE1c stability, promoting an increase in CSC-like traits. Therefore, phospho-ECE1c may be a biomarker of poor prognosis and a potential therapeutic target for colorectal cancer.

Involvement of endothelial CK2 in the radiation induced perivascular resistant niche (PVRN) and the induction of radioresistance for non-small cell lung cancer (NSCLC) cells.

BACKGROUND: Tumor microenvironment (TME) plays a vital role in determining the outcomes of radiotherapy. As an important component of TME, vascular endothelial cells are involved in the perivascular resistance niche (PVRN), which is formed by inflammation or cytokine production induced by ionizing radiation (IR). Protein kinase CK2 is a constitutively active serine/threonine kinase which plays a vital role in cell proliferation and inflammation. In this study, we investigated the potential role of CK2 in PVRN after IR exposure. RESULT: Specific CK2 inhibitors, Quinalizarin and CX-4945, were employed to effectively suppressed the kinase activity of CK2 in human umbilical vein endothelial cells (HUVECs) without affecting their viability. Results showing that conditioned medium from IR-exposed HUVECs increased cell viability of A549 and H460 cells, and the pretreatment of CK2 inhibitors slowed down such increment. The secretion of IL-8 and IL-6 in HUVECs was induced after exposure with IR, but significantly inhibited by the addition of CK2 inhibitors. Furthermore, IR exposure elevated the nuclear phosphorylated factor-κB (NF-κB) p65 expression in HUVECs, which was a master factor regulating cytokine production. But when pretreated with CK2 inhibitors, such elevation was significantly suppressed. CONCLUSION: This study indicated that protein kinase CK2 is involved in the key process of the IR induced perivascular resistant niche, namely cytokine production, by endothelial cells, which finally led to radioresistance of non-small cell lung cancer cells. Thus, the inhibition of CK2 may be a promising way to improve the outcomes of radiation in non-small cell lung cancer cells.

CK2 inhibition with silmitasertib promotes methuosis-like cell death associated to catastrophic massive vacuolization of colorectal cancer cells.

Protein kinase CK2 is a highly conserved and constitutively active Ser/Thr-kinase that phosphorylates a large number of substrates, resulting in increased cell proliferation and survival. A known target of CK2 is Akt, a player in the PI3K/Akt/mTORC1 signaling pathway, which is aberrantly activated in 32% of colorectal cancer (CRC) patients. On the other hand, mTORC1 plays an important role in the regulation of protein synthesis, cell growth, and autophagy. Some studies suggest that CK2 regulates mTORC1 in several cancers. The most recently developed CK2 inhibitor, silmitasertib (formerly CX-4945), has been tested in phase I/II trials for cholangiocarcinoma and multiple myeloma. This drug has been shown to induce autophagy and enhance apoptosis in pancreatic cancer cells and to promote apoptosis in non-small cell lung cancer cells. Nevertheless, it has not been tested in studies for CRC patients. We show in this work that inhibition of CK2 with silmitasertib decreases in vitro tumorigenesis of CRC cells in response to G2/M arrest, which correlates with mTORC1 inhibition and formation of large cytoplasmic vacuoles. Notably, molecular markers indicate that these vacuoles derive from massive macropinocytosis. Altogether, these findings suggest that an aberrantly elevated expression/activity of CK2 may play a key role in CRC, promoting cell viability and proliferation in untreated cells, however, its inhibition with silmitasertib promotes methuosis-like cell death associated to massive catastrophic vacuolization, accounting for decreased tumorigenicity at later times. These characteristics of silmitasertib support a potential therapeutic use in CRC patients and probably other CK2-dependent cancers.

Involvement of endothelial CK2 in the radiation induced perivascular resistant niche (PVRN) and the induction of radioresistance for non-small cell lung cancer (NSCLC) cells

BACKGROUND: Tumor microenvironment (TME) plays a vital role in determining the outcomes of radiotherapy. As an important component of TME, vascular endothelial cells are involved in the perivascular resistance niche (PVRN), which is formed by inflammation or cytokine production induced by ionizing radiation (IR). Protein kinase CK2 is a constitutively active serine/threonine kinase which plays a vital role in cell proliferation and inflammation. In this study, we investigated the potential role of CK2 in PVRN after IR exposure. RESULT: Specific CK2 inhibitors, Quinalizarin and CX-4945, were employed to effectively suppressed the kinase activity of CK2 in human umbilical vein endothelial cells (HUVECs) without affecting their viability. Results showing that conditioned medium from IR-exposed HUVECs increased cell viability of A549 and H460 cells, and the pretreatment of CK2 inhibitors slowed down such increment. The secretion of IL-8 and IL-6 in HUVECs was induced after exposure with IR, but significantly inhibited by the addition of CK2 inhibitors. Furthermore, IR exposure elevated the nuclear phosphorylated factor-κB (NF-κB) p65 expression in HUVECs, which was a master factor regulating cytokine production. But when pretreated with CK2 inhibitors, such elevation was significantly suppressed. CONCLUSION: This study indicated that protein kinase CK2 is involved in the key process of the IR induced perivascular resistant niche, namely cytokine production, by endothelial cells, which finally led to radioresistance of non-small cell lung cancer cells. Thus, the inhibition of CK2 may be a promising way to improve the outcomes of radiation in nonsmall cell lung cancer cells.

Prion protein inhibits fast axonal transport through a mechanism involving casein kinase 2.

Prion diseases include a number of progressive neuropathies involving conformational changes in cellular prion protein (PrPc) that may be fatal sporadic, familial or infectious. Pathological evidence indicated that neurons affected in prion diseases follow a dying-back pattern of degeneration. However, specific cellular processes affected by PrPc that explain such a pattern have not yet been identified. Results from cell biological and pharmacological experiments in isolated squid axoplasm and primary cultured neurons reveal inhibition of fast axonal transport (FAT) as a novel toxic effect elicited by PrPc. Pharmacological, biochemical and cell biological experiments further indicate this toxic effect involves casein kinase 2 (CK2) activation, providing a molecular basis for the toxic effect of PrPc on FAT. CK2 was found to phosphorylate and inhibit light chain subunits of the major motor protein conventional kinesin. Collectively, these findings suggest CK2 as a novel therapeutic target to prevent the gradual loss of neuronal connectivity that characterizes prion diseases.

Ck2-Dependent Phosphorylation Is Required to Maintain Pax7 Protein Levels in Proliferating Muscle Progenitors.

Skeletal muscle regeneration and long term maintenance is directly link to the balance between self-renewal and differentiation of resident adult stem cells known as satellite cells. In turn, satellite cell fate is influenced by a functional interaction between the transcription factor Pax7 and members of the MyoD family of muscle regulatory factors. Thus, changes in the Pax7-to-MyoD protein ratio may act as a molecular rheostat fine-tuning acquisition of lineage identity while preventing precocious terminal differentiation. Pax7 is expressed in quiescent and proliferating satellite cells, while its levels decrease sharply in differentiating progenitors Pax7 is maintained in cells (re)acquiring quiescence. While the mechanisms regulating Pax7 levels based on differentiation status are not well understood, we have recently described that Pax7 levels are directly regulated by the ubiquitin-ligase Nedd4, thus promoting proteasome-dependent Pax7 degradation in differentiating satellite cells. Here we show that Pax7 levels are maintained in proliferating muscle progenitors by a mechanism involving casein kinase 2-dependent Pax7 phosphorylation at S201. Point mutations preventing S201 phosphorylation or casein kinase 2 inhibition result in decreased Pax7 protein in proliferating muscle progenitors. Accordingly, this correlates directly with increased Pax7 ubiquitination. Finally, Pax7 down regulation induced by casein kinase 2 inhibition results in precocious myogenic induction, indicating early commitment to terminal differentiation. These observations highlight the critical role of post translational regulation of Pax7 as a molecular switch controlling muscle progenitor fate.

Colon cancer cell invasion is promoted by protein kinase CK2 through increase of endothelin-converting enzyme-1c protein stability.

Endothelin-converting enzyme-1c (ECE-1c) is a membrane metalloprotease involved in endothelin-1 synthesis, which has been shown in vitro to have a role in breast, ovary and prostate cancer cell invasion. N-terminal end of ECE-1c displays three putative phosphorylation sites for the protein kinase CK2. We studied whether CK2 phosphorylates N-terminal end of ECE-1c as well as whether this has a role in migration and invasion of colon cancer cells. CK2 phosphorylated the N-terminal end of ECE-1c and this was precluded upon inhibition of CK2. Inhibition also led to diminished protein levels of both endogen ECE-1 or GFP-fused N-terminal end of ECE-1c in 293T embryonic and DLD-1 colon cancer cells, which highlighted the importance of this motif on UPS-dependent ECE-1c degradation. Full-length ECE-1c mutants designed either to mimic or abrogate CK2-phosphorylation displayed increased or decreased migration/invasion of colon cancer cells, respectively. Moreover, ECE-1c overexpression or its silencing with a siRNA led to increased or diminished cell migration/invasion, respectively. Altogether, these data show that CK2-increased ECE-1c protein stability is related to augmented migration and invasion of colon cancer cells, shedding light on a novel mechanism by which CK2 may promote malignant progression of this disease.

Phenolic indeno[1,2-b]indoles as ABCG2-selective potent and non-toxic inhibitors stimulating basal ATPase activity.

Ketonic indeno[1,2-b]indole-9,10-dione derivatives, initially designed as human casein kinase II (CK2) inhibitors, were recently shown to be converted into efficient inhibitors of drug efflux by the breast cancer resistance protein ABCG2 upon suited substitutions including a N (5)-phenethyl on C-ring and hydrophobic groups on D-ring. A series of ten phenolic and seven p-quinonic derivatives were synthesized and screened for inhibition of both CK2 and ABCG2 activities. The best phenolic inhibitors were about threefold more potent against ABCG2 than the corresponding ketonic derivatives, and showed low cytotoxicity. They were selective for ABCG2 over both P-glycoprotein and MRP1 (multidrug resistance protein 1), whereas the ketonic derivatives also interacted with MRP1, and they additionally displayed a lower interaction with CK2. Quite interestingly, they strongly stimulated ABCG2 ATPase activity, in contrast to ketonic derivatives, suggesting distinct binding sites. In contrast, the p-quinonic indenoindoles were cytotoxic and poor ABCG2 inhibitors, whereas a partial inhibition recovery could be reached upon hydrophobic substitutions on D-ring, similarly to the ketonic derivatives.

CK2 Secreted by Leishmania braziliensis Mediates Macrophage Association Invasion: A Comparative Study between Virulent and Avirulent Promastigotes.

CK2 is a protein kinase distributed in different compartments of Leishmania braziliensis: an externally oriented ecto-CK2, an intracellular CK2, and a secreted CK2. This latter form is constitutively secreted from the parasite (CsCK2), but such secretion may be highly enhanced by the association of specific molecules, including enzyme substrates, which lead to a higher enzymatic activity, called inductively secreted CK2 (IsCK2). Here, we examined the influence of secreted CK2 (sCK2) activity on the infectivity of a virulent L. braziliensis strain. The virulent strain presented 121-fold higher total CK2 activity than those found in an avirulent strain. The use of specific CK2 inhibitors (TBB, DRB, or heparin) inhibited virulent parasite growth, whereas no effect was observed in the avirulent parasites. When these inhibitors were added to the interaction assays between the virulent L. braziliensis strain and macrophages, association index was drastically inhibited. Polyamines enhanced sCK2 activity and increased the association index between parasites and macrophages. Finally, sCK2 and the supernatant of the virulent strain increased the association index between the avirulent strain and macrophages, which was inhibited by TBB. Thus, the kinase enzyme CK2 seems to be important to invasion mechanisms of L. braziliensis.