Inducible expression of the regulatory protein kinase CK2beta subunit: incorporation into complexes with catalytic CK2 subunits and re-examination of the effects of CK2beta on cell proliferation.

The regulatory subunit of protein kinase CK2, designated CK2beta, exists both free in cells and in complexes with the CK2 catalytic subunits. Growing evidence suggests that CK2beta has functions dependent and independent of the CK2 catalytic subunits. There have been indications that CK2beta has functions associated with DNA damage responses and in the control of cell proliferation. For example, transient and stable constitutive overexpression of CK2beta in mammalian cells was previously shown to perturb cell cycle progression and to attenuate proliferation. To systematically investigate the molecular mechanisms responsible for these effects of CK2beta on cell proliferation, we generated human osteosarcoma U2OS cell lines with tetracycline-regulated expression of CK2beta. Increased expression of CK2beta results in increases in total cellular CK2 activity, but no changes in cell cycle profiles or proliferation. Furthermore, following exposure to ultraviolet radiation, p53 induction was identical regardless of the levels of CK2beta in cells. Mouse 3T3-L1 cells stably transfected with CK2beta also showed no alterations in cell proliferation. The differences between these results and those previously reported emphasize the complex nature of CK2beta and its cellular functions. Furthermore, these results indicate that increased expression of CK2beta is not by itself sufficient to effect alterations in cell proliferation.     

The emerging CK2 interactome: insights into the regulation and functions of CK2

Protein kinase CK2 represents a small family of protein serine/threonine kinases implicated in a variety of biological processes including events relating to cell proliferation and survival. Notably, CK2 displays oncogenic activity in mice and exhibits altered expression in several types of cancer. Accordingly, a detailed understanding of the cellular functions of CK2 and elucidation of the mechanisms by which CK2 is regulated in cells is expected to contribute to understanding its role in tumorigenesis with the prospect of novel approaches to therapy. While CK2 has traditionally been viewed as a tetrameric complex composed of two catalytic and two regulatory subunits, mounting evidence suggests that its subunits may have functions independent of tetrameric CK2 complexes. In mammals, as is the case in the budding yeast Saccharomyces cerevisiae, there are two isozymic forms of CK2, adding additional heterogeneity to the CK2 family. Studies in yeast and in human cells demonstrate that the different forms of CK2 interact with a large number of cellular proteins. To reveal new insights regarding the regulation and functions of different forms of CK2, we have examined the emerging interactomes for each of the CK2 subunits. Analysis of these interactomes for both yeast and human CK2 reinforces the view that this family of enzymes participates in a broad spectrum of cellular events. Furthermore, while there is considerable overlap between the interactomes of the individual CK2 subunits, notable differences in each of the individual interactomes provides additional evidence for functional specialization for the individual forms of CK2.     

The multiple personalities of the regulatory subunit of protein kinase CK2: CK2 dependent and CK2 independent roles reveal a secret identity for CK2beta

Protein kinase CK2 (formerly casein kinase II), an enzyme that participates in a wide variety of cellular processes, has traditionally been classified as a stable tetrameric complex consisting of two catalytic CK2alpha or CK2alpha' subunits and two regulatory CK2beta subunits. While consideration of CK2 as a tetrameric complex remains relevant, significant evidence has emerged to challenge the view that its individual subunits exist exclusively within these complexes. This review will summarize biochemical and genetic evidence indicating that the regulatory CK2beta subunit exists and performs functions independently of CK2 tetramers. For example, unbalanced expression of catalytic and regulatory CK2 subunits has been observed in a variety of tissues and tumors. Furthermore, localization studies including live cell imaging have demonstrated that while the catalytic and regulatory subunits of CK2 exhibit extensive co-localization, independent mobility of the individual CK2 subunits can also be observed within cells. Identification of proteins that interact with CK2beta in the absence of catalytic CK2 subunits reinforces the notion that CK2beta has functions distinct from CK2 and begins to offer insights into these CK2-independent functions. In this respect, the discovery that CK2beta can interact with and modulate the activity of a number of other serine/threonine protein kinases including A-Raf, c-Mos and Chk1 is particularly striking. This review will discuss the interactions between CK2beta and these protein kinases with special emphasis on the properties of CK2beta that mediate these interactions and on the implications of these interactions in yielding new prospects for elucidation of the cellular functions of CK2beta.     

Inducible expression of protein kinase CK2 in mammalian cells. Evidence for functional specialization of CK2 isoforms.

Protein kinase CK2 (formerly casein kinase II) exhibits elevated expression in a variety of cancers, induces lymphocyte transformation in transgenic mice, and collaborates with Ha-Ras in fibroblast transformation. To systematically examine the cellular functions of CK2, human osteosarcoma U2-OS cells constitutively expressing a tetracycline-regulated transactivator were stably transfected with a bidirectional plasmid encoding either catalytic isoform of CK2 (i.e. CK2alpha or CK2alpha') together with the regulatory CK2beta subunit in order to increase the cellular levels of either CK2 isoform. To interfere with either CK2 isoform, cells were also transfected with kinase-inactive CK2alpha or CK2alpha' (i. e. GK2alpha (K68M) or CK2alpha'(K69M)) together with CK2beta. In these cells, removal of tetracycline from the growth medium stimulated coordinate expression of catalytic and regulatory CK2 subunits. Increased expression of active forms of CK2alpha or CK2alpha' resulted in modest decreases in cell proliferation, suggesting that optimal levels of CK2 are required for optimal proliferation. By comparison, the effects of induced expression of kinase-inactive CK2alpha differed significantly from the effects of induced expression of kinase-inactive CK2alpha'. Of particular interest is the dramatic attenuation of proliferation that is observed following induction of CK2alpha'(K69M), but not following induction of CK2alpha(K68M). These results provide evidence for functional specialization of CK2 isoforms in mammalian cells. Moreover, cell lines exhibiting regulatable expression of CK2 will facilitate efforts to systematically elucidate its cellular functions.     

Expression of estrogen receptor β increases integrin α1 and integrin β1 levels and enhances adhesion of breast cancer cells

Estrogen effects on mammary gland development and differentiation are mediated by two receptors (ERα and ERβ). Estrogen‐bound ERα induces proliferation of mammary epithelial and cancer cells, while ERβ is important for maintenance of the differentiated epithelium and inhibits proliferation in different cell systems. In addition, the normal breast contains higher ERβ levels compared to the early stage breast cancers, suggesting that loss of ERβ could be important in cancer development. Analysis of ERβ?/? mice has consistently revealed reduced expression of cell adhesion proteins. As such, ERβ is a candidate modulator of epithelial homeostasis and metastasis. Consequently, the aim of this study was to analyze estrogenic effects on adhesion of breast cancer cells expressing ERα and ERβ. As ERβ is widely found in breast cancer but not in cell lines, we used ERα positive T47‐D and MCF‐7 human breast cancer cells to generate cells with inducible ERβ expression. Furthermore, the colon cancer cell lines SW480 and HT‐29 were also used. Integrin α1 mRNA and protein levels increased following ERβ expression. Integrin β1—the unique partner for integrin α1—increased only at the protein level. ERβ expression enhanced the formation of vinculin containing focal complexes and actin filaments, indicating a more adhesive potential. This was confirmed by adhesion assays where ERβ increased adhesion to different extracellular matrix proteins, mostly laminin. In addition, ERβ expression was associated to less cell migration. These results indicate that ERβ affects integrin expression and clustering and consequently modulates adhesion and migration of breast cancer cells. J. Cell. Physiol. 222:156–167, 2010. © 2009 Wiley‐Liss, Inc.     

Co-expression of transcription factor AP-2beta (TFAP2B) and GATA3 in human mammary epithelial cells with intense,apicobasal immunoreactivity for CK8/18

AP-2β is a new mammary epithelial differentiation marker and its expression is preferentially retained and enhanced in lobular carcinoma in situ and invasive lobular breast cancer. In normal breast epithelium AP-2β is expressed in a scattered subpopulation of luminal cells. So far, these cells have not been further characterized. Co-expression of AP-2β protein and luminal epithelium markers (GATA3, CK8/18), hormone receptors [estrogen receptor (ER), androgen receptor (AR)] and candidate stem cells markers (CK5/14, CD44) were assessed by double-immunofluorescence staining in normal mammary gland epithelium. The subpopulation of AP-2β-positive mammary epithelial cells showed an almost complete, superimposable co-expression with GATA3 and a peculiar intense, ring-like appearing immunoreactivity for CK8/18. Confocal immunofluorescence microscopy revealed an apicobasal staining for CK8/18 in AP-2β-positive cells, which was not seen in in AP-2β-negative cells. Furthermore, AP-2β-positive displayed a partial co-expression with ER and AR, but lacked expression of candidate stem cell markers CK5/14 and CD44. In summary, AP-2β is a new luminal mammary epithelial differentiation marker, which is expressed in the GATA3-positive subpopulation of luminal epithelial cells. These AP-2β-positive/GATA3-positive cells also show a peculiar CK8/18-expression which may indicate a previously unknown functionally specialized mammary epithelial cell population.

     

Predicting CK2 beta-dependent substrates using linear patterns

CK2 is a constitutively active Ser/Thr protein kinase deregulated in cancer and other pathologies, responsible for about the 20% of the human phosphoproteome. The holoenzyme is a complex composed of two catalytic (α or α´) and two regulatory (β) subunits, with individual subunits also coexisting in the cell. In the holoenzyme, CK2β is a substrate-dependent modulator of kinase activity. Therefore, a comprehensive characterization of CK2 cellular function should firstly address which substrates are phosphorylated exclusively when CK2β is present (class-III or beta-dependent substrates). However, current experimental constrains limit this classification to a few substrates. Here, we took advantage of motif-based prediction and designed four linear patterns for predicting class-III behavior in sets of experimentally determined CK2 substrates. Integrating high-throughput substrate prediction, functional classification and network analysis, our results suggest that beta-dependent phosphorylation might exert particular regulatory roles in viral infection and biological processes/pathways like apoptosis, DNA repair and RNA metabolism. It also pointed, that human beta-dependent substrates are mainly nuclear, a few of them shuttling between nuclear and cytoplasmic compartments.     

The effects of cucurbitacin E on GADD45β‐trigger G2/M arrest and JNK‐independent pathway in brain cancer cells

Cucurbitacin E (CuE), an active compound of the cucurbitacin family, possesses a variety of pharmacological functions and chemotherapy potential. Cucurbitacin E exhibits inhibitory effects in several types of cancer; however, its anticancer effects on brain cancer remain obscure and require further interpretation. In this study, efforts were initiated to inspect whether CuE can contribute to anti‐proliferation in human brain malignant glioma GBM 8401 cells and glioblastoma‐astrocytoma U‐87‐MG cells. An MTT assay measured CuE's inhibitory effect on the growth of glioblastomas (GBMs). A flow cytometry approach was used for the assessment of DNA content and cell cycle analysis. DNA damage 45β (GADD45β) gene expression and CDC2/cyclin‐B1 disassociation were investigated by quantitative real‐time PCR and Western blot analysis. Based on our results, CuE showed growth‐inhibiting effects on GBM 8401 and U‐87‐MG cells. Moreover, GADD45β caused the accumulation of CuE‐treated G2/M‐phase cells. The disassociation of the CDC2/cyclin‐B1 complex demonstrated the known effects of CuE against GBM 8401 and U‐87‐MG cancer cells. Additionally, CuE may also exert antitumour activities in established brain cancer cells. In conclusion, CuE inhibited cell proliferation and induced mitosis delay in cancer cells, suggesting its potential applicability as an antitumour agent.     

Cyclin D1 Gene Silencing Promotes IL‐1β‐Induced Apoptosis in Rat Chondrocytes

This study investigated the effects of cyclin D1 gene silencing on cell proliferation and apoptosis of interleukin‐1β (IL‐1β)‐induced osteoarthritis (OA) chondrocytes. Chondrocytes from healthy sprague‐dawley rats were divided into blank, OA model (chondrocytes underwent IL‐1β inducement), OA trial (chondrocytes underwent IL‐1β inducement with cyclin D1‐shRNA treatment), and negative control (NC; chondrocytes underwent IL‐1β inducement and control‐shRNA treatment) groups. Cell proliferation was assessed by CCK‐8 assay, and cell cycle and apoptosis by flow cytometry. qRT‐PCR and Western blotting were performed to detect cyclin D1 and apoptosis‐related factors expression levels. Chondrocyte proliferation increased after 72–96 h after incubation. The OA trial group exhibited reduced cell proliferation at 48, 72, and 96 h after treatment. The OA model, OA trial, and NC groups all contained more cells arrested in G1 phase and had higher apoptosis rates than the blank group. Additionally, the OA trial group contained more cells arrested in G1 phase, with increased apoptosis rates compared to the OA model and NC groups. The OA model group had lowest expression of cyclin D1 whereas the blank group contained the highest among the four groups. qRT‐PCR also showed that the OA model, OA trial, and NC groups all had increased expression levels of Bax and reduced expression levels of Bcl‐2 and P53 compared to the blank group, whereby by the OA group had the most significant change. The combined evidence in our study shows that cyclin D1 gene silencing suppresses proliferation and induces apoptosis of rat chondrocytes in IL‐1β‐induced OA. J. Cell. Biochem. 119: 290–299, 2018. © 2017 Wiley Periodicals, Inc.     

Kappa opioids promote the proliferation of astrocytes via Gβγ and β‐arrestin 2‐dependent MAPK‐mediated pathways

GTP binding regulatory protein (G protein)‐coupled receptors can activate MAPK pathways via G protein‐dependent and ‐independent mechanisms. However, the physiological outcomes correlated with the cellular signaling events are not as well characterized. In this study, we examine the involvement of G protein and β‐arrestin 2 pathways in kappa opioid receptor‐induced, extracellular signal‐regulated kinase 1/2 (ERK1/2)‐mediated proliferation of both immortalized and primary astrocyte cultures. As different agonists induce different cellular signaling pathways, we tested the prototypic kappa agonist, U69593 as well as the structurally distinct, non‐nitrogenous agonist, C(2)‐methoxymethyl salvinorin B (MOM‐Sal‐B). In immortalized astrocytes, U69593, activated ERK1/2 by a rapid (min) initial stimulation that was sustained over 2 h and increased proliferation. Sequestration of activated Gβγ subunits attenuated U69593 stimulation of ERK1/2 and suppressed proliferation in these cells. Furthermore, small interfering RNA silencing of β‐arrestin 2 diminished sustained ERK activation induced by U69593. In contrast, MOM‐Sal‐B induced only the early phase of ERK1/2 phosphorylation and did not affect proliferation of immortalized astrocytes. In primary astrocytes, U69593 produced the same effects as seen in immortalized astrocytes. MOM‐Sal‐B elicited sustained ERK1/2 activation which was correlated with increased primary astrocyte proliferation. Proliferative actions of both agonists were abolished by either inhibition of ERK1/2, Gβγ subunits or β‐arrestin 2, suggesting that both G protein‐dependent and ‐independent ERK pathways are required for this outcome.     

Structure�Cfunction analysis of the beta regulatory subunit of protein kinase CK2 by targeting embryonic stem cell

Programs that govern stem cell maintenance and pluripotency are dependent on extracellular factors and of intrinsic cell modulators. Embryonic stem (ES) cells with a specific depletion of the gene encoding the regulatory subunit of protein kinase CK2 (CK2β) revealed a viability defect. However, analysis of CK2β functions along the neural lineage established CK2β as a positive regulator for neural stem/progenitor cell (NSC) proliferation and multipotency. By using an in vitro genetic conditional approach, we demonstrate in this work that specific domains of CK2β involved in the regulatory function towards CK2 catalytic subunits are crucial structural determinants for ES cell homeostasis.     

Expression and localization of epitope-tagged protein kinase CK2

Protein kinase CK2, formerly known as casein kinase II, is a ubiquitous protein serine/threonine kinase. The enzyme exists in tetrameric complexes composed of two catalytic (CK2α and/or CK2α′) subunits and two subunits (CK2β) that appear to have a role in modulating the activity of the catalytic subunits. With the exception of their unrelated carboxy-terminal domains, the two isozymic forms of mammalian CK2 display extensive sequence identity. Furthermore, CK2α and CK2α′ exhibit remarkable conservation between species, suggesting that they may have unique functions. In the present study, the cDNAs encoding CK2α and CK2α′ were modified by addition of the hemagglutinin tag of the influenza virus at the amino terminus of the respective proteins. The epitope-tagged proteins were transfected into Cos-7 cells and the localization of the expressed proteins determined by indirect immunofluorescence using monoclonal antibodies specific for the epitope tag. The use of transfection favors the formation of homotetrameric complexes (i.e., α₂β₂, α′₂β₂) instead of heterotetrameric complexes (i.e., αα′β₂) that are present in many cells. Epitope-tagged CK2α and CK2α′ displayed kinase activity and the ability to form complexes with CK2β. The results of these studies also indicate definitively that CK2α and CK2α′ are both localized predominantly within the nucleus. Mutation of conserved lysine residues within the ATP binding domains of CK2α and CK2α′ resulted in loss of kinase activity. However, examination of these mutants indicates that kinase activity is not essential for formation of complexes between subunits of CK2 and is not required for nuclear localization of CK2. J. Cell. Biochem. 64: 525–537. © 1997 Wiley-Liss, Inc.     

β‐arrestin2/miR‐155/GSK3β regulates transition of 5′‐azacytizine‐induced Sca‐1‐positive cells to cardiomyocytes

Stem‐cell antigen 1–positive (Sca‐1+) cardiac stem cells (CSCs), a vital kind of CSCs in humans, promote cardiac repair in vivo and can differentiate to cardiomyocytes with 5′‐azacytizine treatment in vitro. However, the underlying molecular mechanisms are unknown. β‐arrestin2 is an important scaffold protein and highly expressed in the heart. To explore the function of β‐arrestin2 in Sca‐1+ CSC differentiation, we used β‐arrestin2–knockout mice and overexpression strategies. Real‐time PCR revealed that β‐arrestin2 promoted 5′‐azacytizine‐induced Sca‐1+ CSC differentiation in vitro. Because the microRNA 155 (miR‐155) may regulate β‐arrestin2 expression, we detected its role and relationship with β‐arrestin2 and glycogen synthase kinase 3 (GSK3β), another probable target of miR‐155. Real‐time PCR revealed that miR‐155, inhibited by β‐arrestin2, impaired 5′‐azacytizine‐induced Sca‐1+ CSC differentiation. On luciferase report assay, miR‐155 could inhibit the activity of β‐arrestin2 and GSK3β, which suggests a loop pathway between miR‐155 and β‐arrestin2. Furthermore, β‐arrestin2‐knockout inhibited the activity of GSK3β. Akt, the upstream inhibitor of GSK3β, was inhibited in β‐arrestin2‐Knockout mice, so the activity of GSK3β was regulated by β‐arrestin2 not Akt. We transplanted Sca‐1+ CSCs from β‐arrestin2‐knockout mice to mice with myocardial infarction and found similar protective functions as in wild‐type mice but impaired arterial elastance. Furthermore, low level of β‐arrestin2 agreed with decreased phosphorylation of AKT and increased phophorylation of GSK3β, similar to in vitro findings. The β‐arrestin2/miR‐155/GSK3β pathway may be a new mechanism with implications for treatment of heart disease.     

The kinesin I family member KIF5C is a novel substrate for protein kinase CK2

Protein kinase CK2 is ubiquitously expressed. The holoenzyme is composed of two catalytic α- or α′-subunits and two regulatory β-subunits but evidence is accumulating that the subunits can function independently. The composition of the holoenzyme as well as the expression of the individual subunits varies in different tissues, with high expression of CK2α′ in testis and brain. CK2 phosphorylates a number of different substrates which are implicated in basal cellular processes such as proliferation and survival of cells. Here, we report a new substrate, KIF5C, which is a member of the kinesin 1 family of motor neuron proteins. Phosphorylation of KIF5C was demonstrated in vitro and in vivo. Using deletion mutants, a peptide library, and mutation analysis a phosphorylation site for CK2 was mapped to amino acid 338 which is located in the non-motor domain of KIF5C. Interestingly, KIF5C is phosphorylated by holoenzymes composed of CK2α/CK2β and CK2α′/CK2β as well as by CK2α′ alone but not by CK2α alone.