Casein kinase 2, the versatile regulator of cell survival

Casein kinase 2 (CK2), a highly conservative, multifunctional serine/threonine protein kinase, is critically important for the regulation of a plethora of processes in eukaryotes, such as cell proliferation, differentiation and death. CK2 is expressed in all tissues; in particular, its amount and activity are elevated in tumor cells. Unlike many regulatory proteins CK2 permanently adopts an active conformation. Of the utmost importance are the anti-apoptotic functions of CK2. This protein kinase is capable of regulating cell survival at multiple levels including DNA repair, NF-kappaB, Wnt, PI3K/Akt and JAK-STAT signaling cascades, chaperones, activation of anti-apoptotic proteins and down-regulation of pro-apoptotic counterparts, in particular, caspases. The versatility of CK2-mediated phosphorylation ensures the survival of tumor cells exposed to stimuli that differ in the origin and mechanisms of cytotoxicity. This manifold mode of CK2-dependent survival makes this enzyme an important target for antitumor therapy.     

Regulatory role of CK2 during the progression of cell cycle

The protein kinase casein kinase 2 (CK2) is a ubiquitous eukaryotic serine/threonine protein kinase that plays an important role in cell cycle progression. We find that (1) CK2 interacts with a tumor suppressor protein, adenomatous polyposis coli (APC) that occurs at the highest level in G₂/M, and (2) the C-terminal region of APC, between amino acid residues 2086–2394, has the strongest activity to suppress CK2. Over-expression of this fragment in HEK293 cells or colorectal carcinoma cells that have truncated mutant APC proteins down-regulates cell proliferation rates as well as colony formation on soft agar. These results indicate that the complex formation between CK2 and full-length APC regulates CK2 activity that, in turn, regulates cell cycle progression, whereas truncated APC in colorectal carcinomas are unable to regulate the cell cycle. In the process to look for the downstream target for CK2, we found that eukaryotic translation initiation factor 5 (eIF5) is phosphorylated by CK2 in vivo as well as in vitro These results suggest an important role of CK2 on promotion of cell growth through eIF5.     

Induction of apoptosis by antisense CK2 in human prostate cancer xenograft model

Protein serine/threonine kinase CK2 (formerly casein kinase 2) is a ubiquitous protein kinase that plays key roles in cell growth, proliferation, and survival. We have shown previously that its molecular down-regulation induces apoptosis in cancer cells in culture. Here, we have employed a xenograft model of prostate cancer to extend these studies to determine whether antisense CK2alpha evokes a similar response in vivo. A single dose of antisense CK2alpha oligodeoxynucleotide given directly into the PC3-LN4 xenograft tumor in nude mouse induced a dose- and time-dependent tumor cell death in vivo. The tumor was completely resolved at the higher tested dose of the antisense. Cell death was due to apoptosis and correlated with a potent down-regulation of the CK2alpha message and loss of CK2 from the nuclear matrix in the xenograft tissue as well as in cancer cells in culture. These observations accorded with several of the earlier studies indicating that loss of CK2 from the nuclear matrix is associated with induction of apoptosis. Comparison of the effects of antisense CK2alpha oligodeoxynucleotide on cancer versus normal or noncancer cells showed that the concentration of antisense CK2alpha that elicited extensive apoptosis in tumor cells in culture or xenograft tumors in vivo had a relatively small or minimal effect on noncancer cells in culture or on normal prostate gland subjected to orthotopic injection of antisense oligodeoxynucleotide in vivo. The basis for the difference in sensitivity of cancer versus noncancer cells to antisense CK2alpha is unknown at this time; however, this differential response under similar conditions of treatment may be significant in considering the potential feasibility of targeting the CK2 signal for induction of apoptosis in cancer cells in vivo. Although much further work will be needed to establish the feasibility of targeting CK2 for cancer therapy, to our knowledge, this is the first report to provide important new evidence as an initial "proof of principle" for the potential application of antisense CK2alpha in cancer therapy, paving the way for future detailed studies of approaches to targeting CK2 in vivo to induce cancer cell death.     

Protein kinase CK2 signal in neoplasia

Protein kinase CK2 (previously known as casein kinase II) is a protein serine/threonine kinase that has been implicated in cell growth and proliferation. The focus of this review is on the apparent role of CK2 in cancer. Studies from several laboratories have shown a dysregulated expression of the kinase in tumors. Nuclear matrix and chromatin appear to be key sites for signaling of the CK2 activity in relation to cell growth. Several types of growth stimuli produce a common downstream response in CK2 by enhancing its nuclear shuttling. The neoplastic change is also associated with changes in intracellular localization of the kinase so that a higher nuclear localization is observed in tumor cells compared with normal cells. Experimental studies suggest that dysregulated expression of the alpha subunit of CK2 imparts an oncogenic potential in the cells such that in cooperation with certain oncogenes it produces a profound enhancement of the tumor phenotype. Recent studies have provided evidence that overexpression of CK2 in tumor cells is not simply a reflection of tumor cell proliferation alone but additionally may reflect the pathobiological characteristics of the tumor. Of considerable interest is the possibility that CK2 dysregulation in tumors may influence the apoptotic activity in those cells. Approaches to interfering with the CK2 signal may provide a useful means for inducing tumor cell death.     

Validation of protein kinase CK2 as oncological target

Protein kinase CK2 is a highly conserved enzyme composed of two catalytic subunits α and/or α′ and two regulatory subunits β whose activity is elevated in diverse tumour types as well as in highly proliferating tissues. Several results suggest that the overexpression of either CK2 catalytic subunits or the CK2 holoenzyme contributes to cellular transformation. In a similar vein, experiments performed compromising the intracellular expression of CK2 has led to somehow contradictory results with respect to the ability of this enzyme to control survival and apoptosis. To better elucidate the role of CK2 in programmed cell death, we have depleted cells of CK2 catalytic subunits by the application of antisense oligodeoxynucleotides and siRNAs techniques, respectively. Our results indicate that protein kinase CK2 is characterized by an extremely high stability that might be due to its association with other intracellular proteins, enhanced half-life or lower vulnerability towards proteolytic degradation. In addition, we show that despite the effectiveness of the methods applied in lowering CK2 kinase activity in all cells investigated, CK2 might not by itself be sufficient to trigger enhanced drug-induced apoptosis in cells.     

CK2 inhibition induces apoptosis via the ER stress response

Protein kinase CK2 is a ubiquitously expressed serine/threonine kinase consisting of two catalytic α/α′ and two regulatory β subunits. Expression of CK2 is highly elevated in tumor cells where it protects cells from apoptosis. Accordingly inhibition of CK2 is known to induce programmed cell death, making it a promising target for cancer therapy. In the present study we investigated apoptosis induction by the CK2 inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB) in prostate tumor cells. In contrast to PC-3 cells LNCaP cells respond to CK2 inhibition with apoptosis. Most interestingly we found the mitochondrial pathway induced in LNCaP as well as in PC-3 cells as monitored by down-regulation of bcl-2 and subsequent cytochrome c release. In both cell lines activation of caspase 9 was not detected. Instead, an activation of the endoplasmic reticulum (ER) stress response in LNCaP cells after treatment with the CK2 inhibitor TBB was found. We show that this ER stress response led to an up-regulation of the death receptor DR5 and subsequent apoptosis in LNCaP cells.     

Structural and functional determinants of protein kinase CK2��: facts and open questions

Ser/Thr protein kinase CK2 is involved in several fundamental processes that regulate the cell life, such as cell cycle progression, gene expression, cell growth, and differentiation and embryogenesis. In various cancers, CK2 shows a markedly elevated activity that has been associated with conditions that favor the onset of the tumor phenotype. This prompts to numerous studies aimed at the identification of compounds that are able to inhibit the catalytic activity of this oncogenic kinase, in particular, of ATP-competitive inhibitors. The many available crystal structures indicate that this enzyme owns some regions of remarkable flexibility which were associated to important functional properties. Of particular relevance is the flexibility, unique among protein kinases, of the hinge region and the following helix αD. This study attempts to unveil the structural bases of this characteristic of CK2. We also analyze some controversial issues concerning the functional interpretation of structural data on maize and human CK2 and try to recognize what is reasonably established and what is still unclear about this enzyme. This analysis can be useful also to outline some principles at the basis of the development of effective ATP-competitive CK2 inhibitors.     

蛋白质酪氨酸磷酸化在抗失巢凋亡的癌细胞中的失调变化

失巢凋亡是细胞与细胞外基质脱离发生的一种特定的凋亡方式 . 癌细胞抗失巢凋亡或失巢生存能力可以使之在转移过程中生存 . 业已发现癌细胞失巢生存与 PI3K-PKB/Akt 、 MAPK 这两条重要信号途径有关,但是 PI3K-PKB/Akt 、 MAPK 通路的上游酪氨酸激酶途径还不甚清楚 . 为此设计了一种基于 SH2-pTyr 特异性结合特性的功能性筛选方法,以期发现癌细胞失巢生存相关的酪氨酸磷酸化蛋白质,为最终明确酪氨酸激酶途径提供有力的实验依据 . 实验发现, MDCK 细胞悬浮培养后失巢凋亡,但癌细胞可以失巢生存 . 与这一现象相一致的是,悬浮培养后, MDCK 细胞中一系列 SH2 结合的酪氨酸磷酸化蛋白质水平急剧下降,而癌细胞中蛋白质酪氨酸磷酸化水平并不呈锚着依赖性 . 细胞悬浮培养后,随着培养时间的延长, MDCK 细胞中 Abl S SH2 结合的靶蛋白酪氨酸磷酸化水平逐渐降低,在 H460 肺癌细胞中经过短暂下降后升高, H1792 肺癌细胞随着培养时间的延长, Abl SH2 结合的靶蛋白酪氨酸磷酸化水平逐渐增加 . Fyn SH2 和 Crk SH2 结合的蛋白质分别为 FAK 和 p130Cas ,后者是重要的失巢生存信号 . 这些结果提示,酪氨酸磷酸化蛋白质可能赋予肺癌细胞失巢生存能力 . 结果也表明,功能性 SH2 筛查方法可以有效地发现肿瘤细胞中失巢生存相关的酪氨酸磷酸化蛋白质 .     

In CK2 inactivated cells the cyclin dependent kinase inhibitor Sic1 is involved in cell-cycle arrest before the onset of S phase

Protein kinase CK2 is a heterotetramer composed of two catalytic and two regulatory subunits. In Saccharomyces cerevisiae the catalytic subunits (alpha and alpha') are encoded by the CKA1, CKA2 genes. cka1Deltacka2(ts) mutants arrest cell cycle in both G1 and G2/M at 37 degrees C. Hence, it has been proposed that CK2 plays an important role in cell-cycle progression and several cell-cycle proteins have been reported to be CK2 substrates. We have previously shown that Sic1, the inhibitor of Clb5-Cdc28 complexes required for the G1/S transition, is a physiologically relevant CK2 substrate. Here we show that CK2 inactivation up-regulates Sic1 level resulting in severe down-regulation of Clb5-Cdc28 kinase activity. Concurrent inactivation of Sic1 and CK2 leads to accumulation of cells with a post-synthetic DNA content and short/elongated spindles, typical of cells arrested in mitosis. These findings indicate that Sic1 plays a major role during G1 arrest of CK2-inactivated cells.     

Cross-talk between IGF-I and TGF-beta signaling pathways

Insulin-like growth factor-I (IGF-I) has gained broad recognition as an important survival factor for epithelial cells in numerous tissues. The IGF-I receptor signaling pathway is deregulated in the majority of carcinomas, and such deregulation has also been reported to be tightly associated with enhanced tumor progression and metastasis. One of the key proteins that transduces IGF-I signals and is phospho-activated downstream of the IGF-I receptor, is the non-receptor serine/threonine kinase proto-oncogene protein kinase B (PKB, also known as Akt). This kinase serves as a major molecular node to control the function of many cell survival and death proteins through phosphorylation-mediated protein modification. The end result of the activation of Akt is enhanced cell survival and proliferation, pre-requisites for malignant transformation. Recent studies show that IGF-I signals cross-talk at multiple levels with various components of the TGF-beta signaling pathway, which depending on context may function either as tumor suppressor or as tumor promoter. Thus, a better understanding of how the IGF-I and TGF-beta signaling pathways are mutually interconnected is likely to unveil novel targets for the therapeutic intervention of many cancers.     

Subcellular immunolocalization of protein kinase CK2 in normal and carcinoma cells.

CK2 is a messenger-independent protein serine/threonine kinase that has been implicated in cell growth and proliferation. Our recent analysis of squamous cell carcinomas of the head and neck (SCCHN) revealed a significant elevation in CK2 activity in these tumor cells relative to normal mucosa of the upper aerodigestive tract and suggested a correlation with aggressive tumor behavior and poor clinical outcome. In order to further define the distribution of CK2 in these tissues, we have examined the immunohistochemical staining pattern of surgical specimens of both SCCHN tumors and normal upper aerodigestive tract mucosa using a monoclonal antibody directed against the catalytic subunit CK2-alpha of the kinase, and have compared these data with the subcellular distribution of CK2 activity in these same tissues. These measurements showed that CK2 is predominantly localized to the nuclei of the tumor cells, which agreed closely with the immunohistochemical staining pattern of CK2-alpha in tumor cells. The chiefly nuclear distribution of CK2-alpha immunostaining found consistently in SCCHN tumor cells and tumor-infiltrating lymphocytes contrasted with a relatively more predominant cytosolic staining pattern exhibited by various cellular constituents of normal oropharyngeal mucosa. The immunostaining pattern of CK2-alpha revealed that staining was observed in the cells stained for the proliferation-marker Ki-67; however, strong distinct immunostaining for CK2-alpha was also observed in large numbers of other cells in these same tumors, suggesting that CK2 elevation in these tumors is not a reflection of proliferative activity alone, but may also relate to the pathobiological behavior of the tumor.     

Regulation of caspase pathways by protein kinase CK2: identification of proteins with overlapping CK2 and caspase consensus motifs

Apoptosis, or programmed cell death, is a vital cellular process often impaired in diseases such as cancer. Aspartic acid-directed proteases known as caspases cleave a broad spectrum of cellular proteins and are central constituents of the apoptotic machinery. Caspases are regulated by a variety of mechanisms including protein phosphorylation. One intriguing mechanism by which protein kinases can modulate caspase pathways is by blocking substrate cleavage through phosphorylation of residues adjacent to caspase cleavage sites. To explore this mechanism in detail, we recently undertook a systematic investigation using a combination of bioinformatics, peptide arrays, and peptide cleavage assays to identify proteins with overlapping protein kinase and caspase recognition motifs (Duncan et al., Sci Signal 4:ra30, 2011). These studies implicated protein kinase CK2 as a global regulator of apoptotic pathways. In this article, we extend the analysis of proteins with overlapping CK2 and caspase consensus motifs to examine the convergence of CK2 with specific caspases and to identify CK2/caspase substrates known to be phosphorylated or cleaved in cells. Given its constitutive activity and elevated expression in cancer, these observations suggest that the ability of CK2 to modulate caspase pathways may contribute to a role in promoting cancer cell survival and raise interesting prospects for therapeutic targeting of CK2.     

Protein kinase CK2 catalyzes tyrosine phosphorylation in mammalian cells

Protein kinase CK2 exhibits oncogenic activity in mice and is over-expressed in a number of tumors or leukemic cells. On the basis of its amino acid sequence and a wealth of experimental information, CK2 has traditionally been classified as a protein serine/threonine kinase. In contrast to this traditional view of CK2, recent evidence has shown that CK2 can also phosphorylate tyrosine residues under some circumstances in vitro and in yeast. In this study, we provide definitive evidence demonstrating that CK2 also exhibits tyrosine kinase activity in mammalian cells. Tyrosine phosphorylation of CK2 in cells and in CK2 immunoprecipitates is dependent on CK2 activity and is inhibited by the CK2 selective inhibitor 4,5,6,7-tetrabromobenzotriazole. Examination of phosphotyrosine profiles in cells reveals a number of proteins, including CK2 itself, which exhibit increased tyrosine phosphorylation when CK2 levels are increased. Peptide arrays to evaluate the specificity determinants for tyrosine phosphorylation by CK2 reveal that its specificity for tyrosine phosphorylation is distinct from its specificity for serine/threonine phosphorylation. Of particular note is the requirement for an aspartic acid immediately C-terminal to the phosphorylatable tyrosine residue. Collectively, these data provide conclusive evidence that CK2 catalyzes the phosphorylation of tyrosine residues in mammalian cells, a finding that adds a new level of complexity to the challenge of elucidating its cellular functions. Furthermore, these results raise the possibility that increased CK2 levels that frequently accompany transformation may contribute to the increased tyrosine phosphorylation that occurs in transformed cells.     

Comparative analysis of CK2 expression and function in tumor cell lines displaying sensitivity vs. resistance to chemical induced apoptosis

CK2 is a pleiotropic protein kinase, which phosphorylates many substrates and has a global role in promoting cell survival and preventing apoptosis. In this study, we investigated its involvement in the phenomenon of the drug resistance, by which tumor cells frequently become unresponsive to chemical apoptosis. By comparing the expression of CK2 subunits in four different pairs of sensitive (S) and resistant (R) cancer cell lines, we found that in three cases the resistant phenotype is accompanied by the overexpression of the CK2 catalytic alpha subunit, either alone or in combination with the regulatory beta subunit. The degree of CK2 expression correlates with the CK2 catalytic activity, when measured toward endogenous protein substrates. All the tested R cell lines, including the one with no CK2 overexpression, can be induced to undergo death by treatment with CK2 inhibitors. We therefore conclude that, although CK2 overexpression is not an absolute requirement for the resistant phenotype, its activity is essential for cell survival and contributes to a high degree of resistance. We also found that CK2 inhibition increases the accumulation of cytotoxic drugs inside the R cells, presumably by impairing the functionality of the extrusion pump P-gp. We therefore propose that CK2 should be considered a target to counteract the pharmaco-resistant phenotype.     

Inhibition of protein kinase CK2 with the clinical-grade small ATP-competitive compound CX-4945 or by RNA interference unveils its role in acute myeloid leukemia cell survival,p53-dependent apoptosis and daunorubicin-induced cytotoxicity

Background

The involvement of protein kinase CK2 in sustaining cancer cell survival could have implications also in the resistance to conventional and unconventional therapies. Moreover, CK2 role in blood tumors is rapidly emerging and this kinase has been recognized as a potential therapeutic target. Phase I clinical trials with the oral small ATP-competitive CK2 inhibitor CX-4945 are currently ongoing in solid tumors and multiple myeloma.

Methods

We have analyzed the expression of CK2 in acute myeloid leukemia and its function in cell growth and in the response to the chemotherapeutic agent daunorubicin We employed acute myeloid leukemia cell lines and primary blasts from patients grouped according to the European LeukemiaNet risk classification. Cell survival, apoptosis and sensitivity to daunorubicin were assessed by different means. p53-dependent CK2-inhibition-induced apoptosis was investigated in p53 wild-type and mutant cells.

Results

CK2α was found highly expressed in the majority of samples across the different acute myeloid leukemia prognostic subgroups as compared to normal CD34⁺ hematopoietic and bone marrow cells. Inhibition of CK2 with CX-4945, K27 or siRNAs caused a p53-dependent acute myeloid leukemia cell apoptosis. CK2 inhibition was associated with a synergistic increase of the cytotoxic effects of daunorubicin. Baseline and daunorubicin-induced STAT3 activation was hampered upon CK2 blockade.

Conclusions

These results suggest that CK2 is over expressed across the different acute myeloid leukemia subsets and acts as an important regulator of acute myeloid leukemia cell survival. CK2 negative regulation of the protein levels of tumor suppressor p53 and activation of the STAT3 anti-apoptotic pathway might antagonize apoptosis and could be involved in acute myeloid leukemia cell resistance to daunorubicin.

     

The tumor suppressor PTEN is phosphorylated by the protein kinase CK2 at its C terminus. Implications for PTEN stability to proteasome-mediated degradation

The tumor suppressor phosphatase PTEN regulates cell migration, growth, and survival by dephosphorylating phosphatidylinositol second messengers and signaling phosphoproteins. PTEN possesses a C-terminal noncatalytic regulatory domain that contains multiple putative phosphorylation sites, which could play an important role in the control of its biological activity. The protein kinase CK2 phosphorylated, in a constitutive manner, a cluster of Ser/Thr residues located at the PTEN C terminus. PTEN-phosphorylated defective mutants showed decreased stability in comparison with wild type PTEN and were more rapidly degraded by the proteasome. Inhibition of PTEN phosphorylation by the CK2 inhibitor 5,6-dichloro-1-beta-d-ribofuranosyl-benzimidazole also diminished the PTEN protein content. Our results support the notion that proper phosphorylation of PTEN by CK2 is important for PTEN protein stability to proteasome-mediated degradation.     

Stem/progenitor and intermediate cell types and the origin of human prostate cancer

Theories of cell lineage in human prostatic epithelium, based on protein expression, propose that basal and luminal cells: 1) are either independently capable of self-renewal or 2) arise from stem cells expressing a full spectrum of proteins (p63, cytokeratins CK5/14, CK8/18, and glutathione-S-transferase-pi [GST-pi]) similar to cells of the embryonic urogenital sinus (UGS). Such embryonic-like stem cells are thought to give rise to mature basal cells and secretory luminal cells. By single cell cloning of an immortalized, normal human prostate-derived, non-tumorigenic RWPE-1 cell line, we isolated and characterized two epithelial cell types, WPE-stem and WPE-int. WPE-stem cells show: i) strong, sixfold greater nuclear expression of p63; ii) nearly twofold greater expression of CK14; iii) threefold less CK18, and iv) low androgen receptor (AR) expression as compared with WPE-int cells. WPE-stem cells are androgen-independent for growth and survival. WPE-int cells express very low p63 and CK5/14, and high CK18. WPE-int cells are androgen-independent for growth and survival but are highly responsive as shown by androgen induction of AR and prostate specific antigen (PSA). Compared with WPE-int cells, WPE-stem cells are smaller and show more rapid growth. WPE-stem cells can grow in an anchorage-independent manner in agar with 4.5-fold greater cloning efficiency and as free floating "prostaspheres" in liquid medium; and express over 40-fold higher matrix metalloproteinase-2 activity. These results indicate that WPE-stem cells express several features characteristic of stem/progenitor cells present in the UGS and in adult prostatic epithelium. In contrast, WPE-int cells have an intermediate, committed phenotype on the pathway to luminal cell differentiation. We propose that in normal prostatic epithelium, cells exist at many stages in a continuum of differentiation progressing from stem cells to definitive basal and luminal cells. Establishment and characterization of clones of human prostatic epithelial cells provide novel models for determining cell lineages, the origin of prostate cancer, and for developing new strategies for tumor prevention and treatment.