Myeloid Cell CK2 Regulates Inflammation and Resistance to Bacterial Infection.

Kinase activity plays an essential role in the regulation of immune cell defenses against pathogens. The protein kinase CK2 (formerly casein kinase II) is an evolutionarily conserved kinase with hundreds of identified substrates. CK2 is ubiquitously expressed in somatic and immune cells, but the roles of CK2 in regulation of immune cell function remain largely elusive. This reflects the essential role of CK2 in organismal development and limited prior work with conditional CK2 mutant murine models. Here, we generated mice with a conditional (floxed) allele of Csnk2a, which encodes the catalytic CK2α subunit of CK2. When crossed to Lyz2-cre mice, excision of Csnk2a sequence impaired CK2α expression in myeloid cells but failed to detectably alter myeloid cell development. By contrast, deficiency for CK2α increased inflammatory myeloid cell recruitment, activation, and resistance following systemic Listeria monocytogenes (Lm) infection. Results from mixed chimera experiments indicated that CK2α deficiency in only a subset of myeloid cells was not sufficient to reduce bacterial burdens. Nor did cell-intrinsic deficiency for CK2α suffice to alter accumulation or activation of monocytes and neutrophils in infected tissues. These data suggest that CK2α expression by Lyz2-expressing cells promotes inflammatory and anti-bacterial responses through effects in trans. Our results highlight previously undescribed suppressive effects of CK2 activity on inflammatory myeloid cell responses and illustrate that cell-extrinsic effects of CK2 can shape inflammatory and protective innate immune responses.

Comparative analysis of two brine shrimps revealed differential expression pattern and functional characterization of CK2α under bacterial stimulation from different geographical distribution.

Understanding how the brine shrimp responds to different geographical populations can provide novel insights on response to bacterial stimulation. In the paper, Artemia sinica from lower altitudes and Artemia parthenogenetica from higher altitudes of the Tibetan Plateau, were used to illustrate different defense against bacteria mechanisms that these organisms used to adapt to different geographical environments. Protein kinase CK2 is a serine/threonine kinase with a multitude of protein substrates. It is a ubiquitous enzyme essential for the viability of eukaryotic cells, where its functions in a variety of cellular processes, including cell cycle progression, apoptosis, transcription, and viral infection. The gene encodes the same mRNA sequence in A. sinica and A. parthenogenetica, named AsCK2α and ApCK2α, respectively. The open reading frame was obtained, a 1047-bp sequence encoding a predicted protein of 349 amino acids. To systematically analyze the expression of AsCK2α and ApCK2α during embryonic development and bacterial challenge, real-time PCR, Western blotting and immunohistochemistry were performed. The results showed that AsCK2α was higher than ApCK2α at different developmental stages. Under bacterial challenge, the expression of ApCK2α was significantly higher than AsCK2α. Protein localization analysis showed that AsCK2α and ApCK2α were mainly distributed in the head and chest. Our research revealed that CK2α plays a vital role in the growth, development and bacterial stimulation of the brine shrimp.

Litopenaeus vannamei CK2 is involved in shrimp innate immunity by modulating hemocytes apoptosis.

Protein kinase CK2 (CK2) is a ubiquitous serine/threonine kinase with multiple cellular functions in vertebrates including apoptosis, differentiation, proliferation, survival, tumorigenesis, signal transduction, immune regulation and inflammation. In the current study, the catalytic and regulatory subunit homologs of Litopenaeus vannamei protein kinase CK2 (LvCK2α and LvCK2ß) were cloned and characterized. LvCK2α has a full-length cDNA sequence of 1764 bp with a 1053 bp open reading frame (ORF) encoding a putative protein of 351 amino acids, which contains a typical serine/threonine kinase domain. On the other hand, LvCK2ß has a 1394 bp full-length cDNA with an ORF of 663 bp encoding a protein with 221 amino acids, which contains a Casein kinase II regulatory subunit domain. Sequence and phylogenetic analysis revealed that LvCK2 was evolutionary related with the CK2 of invertebrates. Quantitative reverse transcription PCR (RT-qPCR) analysis showed that LvCK2α and LvCK2ß transcripts were widely expressed in all shrimp tissues tested, and were both induced in hemocytes and hepatopancreas upon challenge with Vibrio parahaemolyticus, Streptoccocus iniae, lipopolysaccharide (LPS), and white spot syndrome virus (WSSV), suggesting their involvement in shrimp immune response. Moreover, RNA interference (RNAi) of LvCK2α resulted in increased hemocytes apoptosis, shown by high caspase 3/7 activity, increased number of apoptotic cells, coupled with an elevation in transcript levels of pro-apoptotic LvCaspase3 and LvCytochrome C, and a reduction in mRNA levels of pro-survival LvBcl2, LvIAP1, and LvIAP2. In addition, LvCK2α knockdown followed by V. parahaemolyticus challenge resulted in higher cumulative mortality of shrimp. Taken together, our current findings suggest that LvCK2 modulates shrimp hemocytes apoptosis as part of the innate immune response to pathogens.

Protein Kinase CK2: An Emerging Regulator of Immunity.

Although it has historically been studied in the context of cancer, recent literature has highlighted the importance of the highly conserved serine/threonine kinase casein kinase II (CK2) in inflammatory disorders. Most strikingly, CK2 is a major regulator of the Th17-Treg axis relevant to many T cell-driven autoimmune disorders including multiple sclerosis (MS).

Regulation of cellular proliferation in acute lymphoblastic leukemia by Casein Kinase II (CK2) and Ikaros.

The IKZF1 gene encodes the Ikaros protein, a zinc finger transcriptional factor that acts as a master regulator of hematopoiesis and a tumor suppressor in leukemia. Impaired activity of Ikaros is associated with the development of high-risk acute lymphoblastic leukemia (ALL) with a poor prognosis. The molecular mechanisms that regulate Ikaros' function as a tumor suppressor and regulator of cellular proliferation are not well understood. We demonstrated that Ikaros is a substrate for Casein Kinase II (CK2), an oncogenic kinase that is overexpressed in ALL. Phosphorylation of Ikaros by CK2 impairs Ikaros' DNA-binding ability, as well as Ikaros' ability to regulate gene expression and function as a tumor suppressor in leukemia. Targeting CK2 with specific inhibitors restores Ikaros' function as a transcriptional regulator and tumor suppressor resulting in a therapeutic, anti-leukemia effect in a preclinical model of ALL. Here, we review the genes and pathways that are regulated by Ikaros and the molecular mechanisms through which Ikaros and CK2 regulate cellular proliferation in leukemia.

Protein kinase CK2 governs the molecular decision between encephalitogenic TH17 cell and Treg cell development.

T helper 17 (TH17) cells represent a discrete TH cell subset instrumental in the immune response to extracellular bacteria and fungi. However, TH17 cells are considered to be detrimentally involved in autoimmune diseases like multiple sclerosis (MS). In contrast to TH17 cells, regulatory T (Treg) cells were shown to be pivotal in the maintenance of peripheral tolerance. Thus, the balance between Treg cells and TH17 cells determines the severity of a TH17 cell-driven disease and therefore is a promising target for treating autoimmune diseases. However, the molecular mechanisms controlling this balance are still unclear. Here, we report that pharmacological inhibition as well as genetic ablation of the protein kinase CK2 (CK2) ameliorates experimental autoimmune encephalomyelitis (EAE) severity and relapse incidence. Furthermore, CK2 inhibition or genetic ablation prevents TH17 cell development and promotes the generation of Treg cells. Molecularly, inhibition of CK2 leads to reduced STAT3 phosphorylation and strongly attenuated expression of the IL-23 receptor, IL-17, and GM-CSF. Thus, these results identify CK2 as a nodal point in TH17 cell development and suggest this kinase as a potential therapeutic target to treat TH17 cell-driven autoimmune responses.

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.

Protein kinase CK2 enables regulatory T cells to suppress excessive TH2 responses in vivo.

The quality of the adaptive immune response depends on the differentiation of distinct CD4(+) helper T cell subsets, and the magnitude of an immune response is controlled by CD4(+)Foxp3(+) regulatory T cells (Treg cells). However, how a tissue- and cell type-specific suppressor program of Treg cells is mechanistically orchestrated has remained largely unexplored. Through the use of Treg cell-specific gene targeting, we found that the suppression of allergic immune responses in the lungs mediated by T helper type 2 (TH2) cells was dependent on the activity of the protein kinase CK2. Genetic ablation of the ß-subunit of CK2 specifically in Treg cells resulted in the proliferation of a hitherto-unexplored ILT3(+) Treg cell subpopulation that was unable to control the maturation of IRF4(+)PD-L2(+) dendritic cells required for the development of TH2 responses in vivo.

CD5 enhances Th17-cell differentiation by regulating IFN-γ response and RORγt localization.

Mechanisms that modulate the generation of Th17 cells are incompletely understood. We report that the activation of casein kinase 2 (CK2) by CD5 is essential for the efficient generation of Th17 cells in vitro and in vivo. In our study, the CD5-CK2 signaling pathway enhanced TCR-induced activation of AKT and promoted the differentiation of Th17 cells by two independent mechanisms: inhibition of glycogen synthase kinase 3 (GSK3) and activation of mTOR. Genetic ablation of the CD5-CK2 signaling pathway attenuated TCR-induced AKT activation and consequently increased activity of GSK3 in Th17 cells. This resulted in increased sensitivity of Th17 cells to IFN-γ-mediated inhibition. In the absence of CD5-CK2 signaling, we observed decreased activity of S6K and attenuated nuclear translocation of RORγt (ROR is retinoic acid receptor related orphan receptor). These results reveal a novel and essential function of the CD5-CK2 signaling pathway and GSK3-IFN-γ axis in regulating Th-cell differentiation and provide a possible means to dampen Th17-type responses in autoimmune diseases.

Computer-aided analysis of structural properties and epitopes of Iranian HPV-16 E7 oncoprotein.

Infection by human papillomavirus type 16 (HPV-16) is the cause of 50% or more of cervical cancers in women. The E7 oncoprotein of HPV-16 has long been known as a potent immortalizing and transforming agent. We used different servers like PseAAC, MHC_binding, MHC_II_binding and Expasy for the present computational prediction. The results for T cell epitopes showed that B1501, A0203, A0201, A0202, A6801 and DRB0405 alleles had lower IC50 than other alleles. We also predicted several peptides with the best binding affinities for alleles of the most frequent MHC class I and II alleles of the various ethnic groups living in the different region of Iran. Two peptides (26-35) and (44-52) were predicted as B-cell epitopes. According to this analysis 1 N-glycosylation site, 2 PKC sites, 4 CK2 sites and 3 disulfide sites were predicted. Our computational study predicted that B cell epitope 1 was Casein kinase II phosphorylated (site No. 31) and glycosylated (site No. 29). Putative MHC-I epitopes 3 and 5 and MHC-II epitopes 19, 21 and 26 were predicted to be casein kinase II phosphorylated. MHC-II epitopes 19 and 21 was predicted to be glycosylated. T cell epitopes 1, 13, 16 and 24 were demonstrated to be kinase C phosphorylated. The result of this analysis for Iranian HPV-16 E7 also indicated that 21.43%, 18.37% and 60.20% of the protein were in the α-helix, extended strand and random coil respectively.

The interferon-gamma-mediated inhibition of lipoprotein lipase gene transcription in macrophages involves casein kinase 2- and phosphoinositide-3-kinase-mediated regulation of transcription factors Sp1 and Sp3.

The mechanisms underlying transcriptional inhibition by interferon-gamma (IFN-gamma) are poorly understood despite the existence of a large number of genes that are regulated in this manner and the key role of this cytokine in inflammatory disorders such as atherosclerosis. We have previously identified a novel mechanism for transcriptional inhibition by IFN-gamma that involves a reduction in the binding of transcription factors Sp1 and Sp3 to regulatory sequences in the lipoprotein lipase (LPL) gene. In the present study, we have investigated the signalling pathways that impact on the IFN-gamma-mediated regulation of Sp1/Sp3 binding and LPL gene transcription in macrophages. The IFN-gamma-mediated inhibition of LPL promoter activity was prevented by expression of dominant negative forms of casein kinase 2 (CK2) and protein kinase B (PKB), a key downstream component of the phosphoinositide-3-kinase (PI3K) pathway. IFN-gamma activated both the catalytic subunits of CK2 without affecting their expression. CK2 interacted with both Sp1 and Sp3 and this association was increased by IFN-gamma. Electrophoretic mobility shift assays showed that a CK2-mediated phosphorylation of either cellular extracts or recombinant Sp1 reduced binding to the regulatory region in the LPL gene. The action of PKB was potentially mediated through mammalian target for rapamycin proteins. Taken together, these results suggest a key role for CK2 and PI3K signalling pathways in the IFN-gamma-mediated inhibition of macrophage LPL gene transcription through the regulation of Sp1/Sp3 binding.

Protein kinase CK2 in human diseases.

Protein kinase CK2 (formerly referred to as casein kinase II) is an evolutionary conserved, ubiquitous protein kinase. There are two paralog catalytic subunits, i.e. alpha (A1) and alpha' (A2). The alpha and alpha' subunits are linked to two beta subunits to produce a heterotetrameric structure. The catalytic alpha subunits are distantly related to the CMGC subfamily of kinases, such as the Cdk kinases. There are some peculiarities associated with protein kinase CK2, which are not found with most other protein kinases: (i) the enzyme is constitutively active, (ii) it can use ATP and GTP and (iii) it is found elevated in most tumors investigated and rapidly proliferating tissues. With the elucidation of the structure of the catalytic subunit, it was possible to explain why the enzyme is constitutively active [1] and why it can bind GTP [2]. Considerable information on the potential roles of CK2 in various disease processes including cancer has been gained in recent years, and the present review may help to further elucidate its aberrant role in many disease states. Its peculiar structural features [3-9] may be advantageous in designing tailor-made compounds with the possibility to specifically target this protein kinase [10]. Since not all the aspects of what has been published on CK2 can be covered in this review, we would like to recommend the following reviews; (i) for general information on CK2 [11-18] and (ii) with a focus on aberrant CK2 [19-22].

Generation and characterization of monoclonal antibodies to protein kinase 2 (CK2) beta subunit.

Protein kinase 2 (CK2) is a ubiquitous and constitutively active serine/threonine protein kinase with various cell functions. It typically forms tetrameric complexes consisting of two catalytic (alpha and/or (alpha') and two regulatory (beta) subunits. The aim of this study was to produce monoclonal antibodies (MAbs) against the CK2beta subunit and to characterize their suitability for Western blotting, immunoprecipitation, and immunohistochemical applications. Bacterially expressed CK2beta-6His-GST recombinant protein has been used as an antigen. Balb/c mice were immunized and given a final boost, and their spleen cells were collected and fused with SP2/0 myeloma cells using PEG 2000. The fused cells were then selected in the HAT-RPMI medium. Anti- CK2beta high-titer antibody-producing hybridoma cell lines were identified by enzyme-linked immunosorbent assay (ELISA) and then subcloned by limiting dilution in HT-RPMI medium supplemented with 20% fetal bovine serum (FBS). A total of 10 IgG-producing cell lines were selected and further tested for their reactivity with the CK2beta subunit using ELISA, Western blots, immunoprecipitation, and immunostaining of formaldehyde-fixed paraffin-embedded tissue sections. The results obtained clearly indicate that several clones produce antibodies that recognize specifically recombinant and endogenous CK2beta subunit in Western blotting and immunoprecipitation, and are suitable for immunohistochemical analysis. In summary, the produced antibodies will be useful for researchers investigating signaling pathways involving CK2 kinase and their deregulation in human pathologies.

Autoimmune uveitis and antigenic mimicry of environmental antigens.

Autoimmunity directed against antigens of immune privileged sites, which are hidden from the immune system by blood-organ-barriers, is difficult to explain: it would require already activated cells to enter the tissue where the respective autoantigens are sequestered. Autoimmune uveitis, a sight-threatening inflammatory disease of the eye, is such an example. To induce disease autoreactive T cells must have been activated outside the eye to pass the blood-retina-barrier and then crossreact with retinal autoantigen. We have described two environmental peptides mimicking a highly pathogenic epitope from retinal S-antigen. One mimicry antigen is from rotavirus, a common pathogen causing gastroenteritis, the other from bovine milk alpha s2casein, a frequent nutritional protein ought to induce oral tolerance. Lewis rats develop uveitis after immunization with both mimicry peptides and casein protein. However, these mimicry antigens failed to induce oral tolerance for protection from uveitis, suspecting that they rather induce immunity than tolerance. Humoral and cellular immune responses to these antigens are enhanced and more frequent in patients with uveitis compared to healthy individuals. Our findings suggest that multiple environmental antigens mimic autoantigens and might cause autoimmune diseases by eliciting defensive immune responses, however, they are not necessarily useful for therapeutic tolerance induction.