Effects of hyperactive Janus kinase 2 signaling in mammary epithelial cells

Ornithine decarboxylase (ODC), the first rate-limiting enzyme in the polyamine biosynthesis is one of the most rapidly degraded proteins in eukaryotic cells. Mammalian ODC is a notable exception to the widely accepted dogma that ubiquitination is always required for targeting a protein to degradation by the 26S proteasome. However, while it is well established that in mammalian cells degradation of ODC is ubiquitin independent, the requirement of ubiquitination for degradation of ODC in yeast cells remained undetermined. We have investigated ODC degradation in three mutant strains of Saccharomyces cerevisiae in which ubiquitin-dependent protein degradation activity is severely compromised. While yeast ODC was rapidly degraded in all these mutant strains the degradation of N-end rule substrates was inhibited. A mutant mouse ODC that fails to interact with Az was rapidly degraded in yeast cells but was stable in mammalian cells suggesting that interaction with a mammalian Az like yeast protein is not necessary for the degradation of ODC in yeast cells. Deletion analysis revealed that sequences from its unique N-terminus are involved in targeting yeast ODC to rapid degradation in yeast cells.     

Protein kinase CK2 interacts with adiponectin receptor 1 and participates in adiponectin signaling

Adiponectin is an adipokine with anti-atherogenic, anti-diabetic and insulin sensitizing properties. Its effects on energy homeostasis, glucose and lipid metabolism are mediated by two ubiquitously expressed seven-transmembrane receptors, AdipoR1 and -R2. With the exception of APPL1 and RACK1, no intracellular binding partners of adiponectin receptors are reported and thus signaling pathways downstream of these receptors remain largely unknown. To incorporate adiponectins protective potential in drug development it is essential to understand adiponectin signaling cascades in detail. A yeast two-hybrid approach employing AdipoR1s cytoplasmatic N-terminus led to the identification of the regulatory subunit of protein kinase CK2. We confirmed the interaction in co-immunoprecipitation, ELISA experiments and co-localization analysis in mammalian cells. Furthermore we could localize the interaction site in an N-terminal basic region close to the transmembrane domain. In adiponectin stimulation experiments of C2C12 mouse myotubes and MCF7 cells incorporating CK2 inhibitor 2-dimethylamino-4,5,6,7-tetrabromo-1H-benz-imidazole (DMAT) we found a modulator role of CK2 in adiponectin signaling. Accordingly we identified the regulatory subunit of protein kinase CK2 as a novel intracellular partner of AdipoR1 and have strong evidence of CK2 as an effector molecule in adiponectin signaling. Since CK2 is involved in signaling cascades of other adipokines and hormones, e.g. leptin and insulin, our findings suggest a possible key function in crosstalk between adiponectin and insulin signaling pathways and could provide further insight into the anti-diabetic effects of adiponectin.     

Highlighting protein kinase CK2 movement in living cells

Protein kinase CK2 has traditionally been described as a stable heterotetrameric complex (α < eqid1 > β₂) but new approaches that effectively capture the dynamic behavior of proteins, are bringing a new picture of this complex into focus. To track the spatio-temporal dynamics of CK2 in living cells, we fused its catalytic α and regulatory β subunits with GFP and analog proteins. Beside the mostly nuclear localization of both subunits, and the identification of specific domains on each subunit that triggers their localization, the most significant finding was that the association of both CK2 subunits in a stable tetrameric holoenzyme eliminates their nuclear import (Mol Cell Biol {23}: 975–987, 2003). Molecular movements of both subunits in the cytoplasm and in the nucleus were analyzed using different new and updated fluorescence imaging methods such as: fluorescence recovery after photo bleaching (FRAP), fluorescence loss in photo bleaching (FLIP), fluorescence correlation spectroscopy (FCS), and photoactivation using a biphoton microscope. These fluorescence-imaging techniques provide unprecedented ways to visualize and quantify the mobility of each individual CK2 subunit with high spatial and temporal resolution. Visualization of CK2 heterotetrameric complex formation could also be recorded using the fluorescence resonance energy transfer (FRET) technique. FRET imaging revealed that the assembling of this molecular complex can take place both in the cytoplasmic and nuclear compartments. The spatio–temporal organization of individual CK2 subunits and their dynamic behavior remain now to be correlated with the functioning of this kinase in the complex environment of the cell.     

Syk tyrosine kinase participates in beta1-integrin signaling and inflammatory responses in airway epithelial cells

The protein tyrosine kinase Syk is critically involved in immunoreceptor signaling in hematopoietic cells. Recent studies demonstrate Syk expression in nonhematopoietic cells, including fibroblasts, endothelial cells, hepatocytes, and breast epithelium. However, the role of Syk in these cells is uncertain. We hypothesized that Syk is expressed in respiratory epithelial cells (EC) and that it functions as a signaling molecule involved in inflammatory responses in the epithelium. With the use of immunohistochemistry, Western blot, PCR, and laser scanning confocal microscopy, Syk was detected in human, rat, and mouse bronchial epithelium in situ and in cultured human bronchial EC in primary cells and the cell lines HS-24 and BEAS-2B. Syk-dependent signaling pathways in EC were initiated by engagement of beta1-integrin receptors. Stimulation of beta1-integrin receptors by fibronectin or antibody cross-linking caused redistribution of Syk from a cytoplasmic to plasma membrane localization. In stimulated cells, Syk and beta1-integrin colocalized. In addition, following beta1-integrin receptor engagement, tyrosine phosphorylation of Syk was observed. Expression of the intercellular adhesion molecule-1 (ICAM-1) and production of IL-6, both important molecules in lung inflammation, was downregulated in EC treated with Syk small interfering RNA or Syk inhibitor piceatannol. We propose that Syk is involved in signaling pathways induced by integrin engagement in airway EC. Syk-mediated signaling regulates IL-6 and ICAM-1 expression and may be important in the pathophysiology of lung inflammation.     

蛋白激酶CK2的研究进展

蛋白激酶CK2是一种真核细胞中普遍存在的信使非依赖性丝／苏氨酸蛋白激酶。近年来,对蛋白激酶CK2的研究也取得了一些重要进展,尤其是蛋白激酶CK2的结构及其作用底物,蛋白激酶CK2与肿瘤及细胞凋亡的关系,越来越引起人们的关注。     

Regulation of the epithelial Na+ channel by the protein kinase CK2

CK2 is a ubiquitous, pleiotropic, and constitutively active Ser/Thr protein kinase that controls protein expression, cell signaling, and ion channel activity. Phosphorylation sites for CK2 are located in the C terminus of both beta- and gamma-subunits of the epithelial Na(+) channel (ENaC). We examined the role of CK2 on the regulation of both endogenous ENaC in native murine epithelia and in Xenopus oocytes expressing rENaC. In Ussing chamber experiments with mouse airways, colon, and cultured M1-collecting duct cells, amiloride-sensitive Na(+) transport was inhibited dose-dependently by the selective CK2 inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB). In oocytes, ENaC currents were also inhibited by TBB and by the structurally unrelated inhibitors heparin and poly(E:Y). Expression of a trimeric channel lacking both CK2 sites (alphabeta(S631A)gamma(T599A)) produced a largely attenuated amiloride-sensitive whole cell conductance and rendered the mutant channel insensitive to CK2. In Xenopus oocytes, CK2 was translocated to the cell membrane upon expression of wt-ENaC but not of alphabeta(S631A)gamma(T599A)-ENaC. Phosphorylation by CK2 is essential for ENaC activation, and to a lesser degree, it also controls membrane expression of alphabetagamma-ENaC. Channels lacking the Nedd4-2 binding motif in beta-ENaC (R561X, Y618A) no longer required the CK2 site for channel activity and siRNA-knockdown of Nedd4-2 eliminated the effects of TBB. This implies a role for CK2 in inhibiting the Nedd4-2 pathway. We propose that the C terminus of beta-ENaC is targeted by this essential, conserved pleiotropic kinase that directs its constitutive activity toward many cellular protein complexes.     

Phosphorylation by protein kinase CK2: a signaling switch for the caspase-inhibiting protein ARC

Caspases play a central role in apoptosis, but their activity is under the control of caspase-inhibiting proteins. A characteristic of caspase-inhibiting proteins is direct caspase binding. It is yet unknown how the localization of caspase-inhibiting proteins is regulated and whether there are upstream signals controlling their function. Here we report that the function of ARC is regulated by protein kinase CK2. ARC at threonine 149 is phosphorylated by CK2. This phosphorylation targets ARC to mitochondria. ARC is able to bind to caspase-8 only when it is localized to mitochondria but not to the cytoplasm. Our results reveal a molecular mechanism by which a caspase-inhibiting protein requires phosphorylation in order to prevent apoptosis.     

Differential transformation of mammary epithelial cells by Wnt genes.

The mouse Wnt family includes at least 10 genes that encode structurally related secreted glycoproteins. Wnt-1 and Wnt-3 were originally identified as oncogenes activated by the insertion of mouse mammary tumor virus in virus-induced mammary adenocarcinomas, although they are not expressed in the normal mammary gland. However, five other Wnt genes are differentially expressed during development of adult mammary tissue, suggesting that they may play distinct roles in various phases of mammary gland growth and development. Induction of transformation by Wnt-1 and Wnt-3 may be due to interference with these normal regulatory events; however, there is no direct evidence for this hypothesis. We have tested Wnt family members for the ability to induce transformation of cultured mammary cells. The results demonstrate that the Wnt gene family can be divided into three groups depending on their ability to induce morphological transformation and altered growth characteristics of the C57MG mammary epithelial cell line. Wnt-1, Wnt-3A, and Wnt-7A were highly transforming and induced colonies which formed and shed balls of cells. Wnt-2, Wnt-5B, and Wnt-7B also induced transformation but with a lower frequency and an apparent decrease in saturation density. In contrast, Wnt-6 and two other family members which are normally expressed in C57MG cells, Wnt-4 and Wnt-5A, failed to induce transformation. These data demonstrate that the Wnt genes have distinct effects on cell growth and should not be regarded as functionally equivalent.     

The cytosolic protein kinase CK2 phosphorylates cardiac calsequestrin in intact cells

The luminal SR protein CSQ2 contains phosphate on roughly half of the serines found in its C-terminus. The sequence around phosphorylation sites in CSQ2 suggest that the in vivo kinase is protein kinase CK2, even though this enzyme is thought to be present only in the cytoplasm and nucleus. To test whether CSQ2 kinase is CK2, we combined approaches that reduced CK2 activity and CSQ2 phosphorylation in intact cells. Tetrabromocinnamic acid, a specific inhibitor of CK2, inhibited both the CSQ2 kinase and CK2 in parallel across a range of concentrations. In intact primary adult rat cardiomyocytes and COS cells, 24 h of drug treatment reduced phosphorylation of overexpressed CSQ2 by 75%. Down-regulation of CK2α subunits in COS cells using siRNA, produced a 90% decrease in CK2α protein levels, and CK2-silenced COS cells exhibited a twofold reduction in CSQ2 kinase activity. Phosphorylation of CSQ2 overexpressed in CK2-silenced cells was also reduced by a factor of two. These data suggested that CSQ2 in intact cells is phosphorylated by CK2, a cytosolic kinase. When phosphorylation site mutants were analyzed in COS cells, the characteristic rough endoplasmic reticulum form of the CSQ2 glycan (GlcNAc2Man9,8) underwent phosphorylation site dependent processing such that CSQ2-nonPP (Ser to Ala mutant) and CSQ2-mimPP (Ser to Glu mutant) produced apparent lower and greater levels of ER retention, respectively. Taken together, these data suggest CK2 can phosphorylate CSQ2 co-translationally at biosynthetic sites in rough ER, a process that may result in changes in its subsequent trafficking through the secretory pathway.     

Intermolecular contact sites in protein kinase CK2

Chemical crosslinking and analysis of CNBr-digested fusion products by immunoblotting with sequence-specific antibodies identifies an interaction between positions 55-70 of subunit (55-70) and 65-80 of subunit (65-80). This has been supported by crosslinking of subunits with peptides 65-80 and 55-70, by binding of subunits to immobilized peptides, and by the hindrance of coprecipitation with peptide-raised antibodies (anti-65-80; anti-55-70). Functionally, 55-70 is a negative regulatory region for the kinase activity of subunit . The opposite, stimulatory property of subunit has been assigned to its C-terminal part. Subdivision of peptide 155-181, that has stimulatory effect, into overlapping peptides and assaying for a binding and binding competition revealed a tight physical contact at 162-175. This region, however, is non-stimulatory indicating binding a necessary but not sufficient quality for stimulation. A contact might exist to regions surrounding C147 and/or C220 at subunit a as indicated by crosslinking and peptide competition. The crosslinking data also confirm a - contact in CK2 holoenzyme. Effects by non-ionic detergents show hydrophobic interactions to play an important role in catalytic activity adjustment.     

Interactions of protein kinase CK2 subunits

Several approaches have been used to study the interactions of the subunits of protein kinase CK2. The inactive mutant of CK2 that has Asp 156 mutated to Ala (CK2A156) is able to bind the CK2 subunit and to compete effectively in this binding with wild-type subunits and . The interaction between CK2A156 and CK2 was also demonstrated by transfection of epitope-tagged cDNA constructs into COS-7 cells. Immunoprecipitation of epitope-tagged CK2A156 coprecipitated the subunit and vice-versa. The assay of the CK2 activity of the extracts obtained from cells transiently transfected with these different subunits yielded some surprising results: The CK2 specific phosphorylating activity of these cells transfected with the inactive CK2A156 was considerably higher than the control cells transfected with vectors alone. Assays of the immunoprecipitated CK2A156 expressed in these cells, however, demonstrated that the mutant was indeed inactive. It can be concluded that transfection of the inactive CK2A156 affects the endogenous activity of CK2. Transfection experiments with CK2 and subunits and CK2A156 were also used to confirm the interaction of CK2 with the general CDK inhibitor p21WAF1/CIP1 co-transfected into these cells. Finally a search in the SwissProt databank for proteins with properties similar to those derived from the amino acid composition of CK2 indicated that CK2 is related to protein phosphatase 2A and to other phosphatases as well as to a subunit of some ion-transport ATPases.     

Cellular regulators of protein kinase CK2

Protein phosphorylation is a key regulatory post-translational modification and is involved in the control of many cellular processes. Protein kinase CK2, formerly known as casein kinase II, which is a ubiquitous and highly conserved protein serine/threonine kinase, plays a central role in the control of a variety of pathways in cell proliferation, transformation, apoptosis and senescence. An understanding of the regulation of such a central protein kinase would greatly help our comprehension of the regulation of many pathways in cellular regulation. A number of reviews have addressed the detection, the development, and the characterization of inhibitors of CK2. The present review focuses on possible natural regulators of CK2, i.e. proteins and other cellular factors that bind to CK2 and thereby regulate its activity.     

Subcellular localization of protein kinase CK2

More than 46 years ago, Burnett and Kennedy first described protein kinase CK2 (formerly known as casein kinase 2) in liver extracts. Since then, protein kinase CK2 has been investigated in many organisms from yeast to man. It is now well established that protein kinase CK2 is a pleiotropic and ubiquitous serine or threonine kinase, which is highly conserved during evolution. A great number of studies deal with substrates of CK2, but the fact that over 160 substrates exist is more confusing than elucidatory. The holoenzyme is composed of two regulatory beta-subunits and two catalytic alpha- or alpha'-subunits. There is now increasing evidence for individual functions of the subunits that are different from their functions in the holoenzyme. Furthermore, more and more studies describe interacting partners of the kinase that may be decisive in the regulation of this enzyme. A big step forward has been the determination of the crystal structure of the two subunits of protein kinase CK2. Now the interactions of the catalytic subunit of CK2 with ATP as well as GTP and the interaction between the regulatory subunits can be explained. However, cellular functions of protein kinase CK2 still remain unclear. In the present review we will focus our interest on the subcellular localization of protein kinase CK2. Protein kinase CK2 is found in many organisms and tissues and nearly every subcellular compartment. There is ample evidence that protein kinase CK2 has different functions in these compartments and that the subcellular localization of protein kinase CK2 is tightly regulated. Therefore studying the subcellular localization of protein kinase CK2 may be a key to its function.     

Cell cycle dependent regulation of protein kinase CK2 signaling to the nuclear matrix

Protein kinase CK2 is a ubiquitous protein serine/threonine kinase that is involved in cell growth and proliferation as well as suppression of apoptosis. Several studies have suggested that the kinase plays a role in cell cycle progression; however, changes in enzyme activity during phases of cell cycle have not been detected. Nuclear matrix is a key locus for CK2 signaling in the nucleus. We therefore examined CK2 signaling to the nuclear matrix in distinct phases of cell cycle by employing synchronized ALVA-41 prostate cancer cells. Removal of serum from the culture medium resulted in G0/G1 arrest, and a reduction in the nuclear matrix-associated CK2 activity which was rapidly reversed on addition of serum. Arresting the cells in G(0)/G(1) phase with hydroxyurea and subsequent release to S phase by serum gave similar results. Cells arrested in the G(2)/M phase by treatment with nocodazole demonstrated an extensive reduction in the nuclear matrix-associated CK2 which was reversed rapidly on addition of serum. Changes in the immunoreactive CK2 protein were concordant with the activity data reflecting a dynamic trafficking of the kinase in distinct phases of cell cycle. Under the same conditions, CK2 activity in total cellular lysate remained essentially unaltered. These results provide the first direct evidence of discrete modulations of CK2 in the nuclear matrix during the cell cycle progression. Inducible overexpression of CK2 in CHO cells yielded only a modest increase in CK2 activity even though a significant increase in expression was apparent at the level of CK2 alpha-specific message. Stably transfected ALVA-41 cells, however, did not show a significant change in CK2 levels despite increased expression at the message level. Not surprisingly, both types of the stably transfected cells failed to show any alteration in cell cycle progression. Distribution of the CK2 activity in the cytosolic versus nuclear matrix fractions in normal cells appears to be different from that in the cancer cells such that the ratio of nuclear matrix to cytosolic activity is much higher in the latter. Considering that nuclear matrix is central to several nuclear functions, this pattern of intracellular distribution of CK2 may have implications for its role in the oncogenic process. Published 2003 Wiley-Liss, Inc.     

Regulation of taurine transport systems by protein kinase CK2 in mammalian cells

Maintaining cell volume is critical for cellular function yet shift in cell volume is a prerequisite for mitosis and apoptosis. The ubiquitously and evolutionary conserved serine/threonine kinase CK2 promotes cell survival and suppresses apoptosis. The present review describes how mammalian cells regulate the cellular content of the major cellular organic osmolyte, taurine with emphasis on CK2 mediated regulation of active taurine uptake and volume-sensitive taurine release. Furthermore, we discuss how CK2-mediated regulation of taurine homeostasis is potentially involved in cellular functions such as proliferation and survival.     

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.     

Leukemia inhibitory factor induces apoptosis of the mammary epithelial cells and participates in mouse mammary gland involution

Leukemia inhibitory factor (LIF) is a multifunctional glycoprotein that displays multiple biological activities in different cell types, but to date there has been no report on its expression in the normal mammary gland. In this study we found that LIF is expressed at low but detectable levels in postpubertal, adult virgin, and pregnant mouse mammary glands. However, LIF expression drops after parturition to become almost undetectable in lactating glands. Interestingly, LIF expression shows a steep increase shortly after weaning that is maintained for the following 3 days. During this period, known as the first stage of mammary gland involution, the lack of suckling induces local factors that cause extensive epithelial cell death. It has been shown that Stat3 is the main factor in signaling the initiation of apoptosis, but the mechanism of its activation remains unclear. Herein, we show that LIF expression in the gland is induced by milk stasis and not by the decrease of circulating lactogenic hormones after weaning. Implantation of LIF containing pellets in lactating glands results in a significant increase in epithelium apoptosis. In addition, this treatment also induces Stat3 phosphorylation. We conclude that LIF regulated expression in the mouse mammary gland may play a relevant role during the first stage of mammary gland involution. Our results also show that LIF-induced mammary epithelium apoptosis could be mediated, at least partially, by Stat3 activation.     

The orphan receptor tyrosine kinase Ror2 modulates canonical Wnt signaling in osteoblastic cells

Ror2 is an orphan receptor tyrosine kinase that plays crucial roles in developmental morphogenesis, particularly of the skeleton. We have identified human Ror2 as a novel regulator of canonical Wnt signaling in osteoblastic (bone-forming) cells with selective activities, enhancing Wnt1 but antagonizing Wnt3. Immunoprecipitation studies demonstrated physical interactions between human Ror2 and mammalian Wnt1 and Wnt3. Functionally, Ror2 antagonized Wnt1- and Wnt3-mediated stabilization of cytosolic beta-catenin in osteoblastic cells. However, Ror2 had opposing effects on a more distal step of canonical Wnt signaling: it potentiated Wnt1 activity but inhibited Wnt3 function as assessed by changes in Wnt-responsive reporter gene activity. Despite binding to Ror2, neither Wnt1 nor Wnt3 altered receptor activity as assessed by levels of Ror2 autophosphorylation. The ability of Ror2 to regulate canonical Wnt signaling in osteoblastic cells should have physiological consequences in bone, because Wnt signaling is known to modulate osteoblast survival and differentiation. Expression of Ror2 mRNA was highly regulated in a biphasic manner during human osteoblast differentiation, being virtually undetectable in pluripotent stem cells, increasing 300-fold in committed preosteoblasts, and disappearing again in osteocytes. Furthermore, Ror2 expression in osteoblasts was suppressed by the Wnt antagonist, secreted frizzled-related protein 1. The regulated expression of Ror2 during osteoblast differentiation, its inverse expression pattern with secreted frizzled-related protein 1, and its ability to modulate Wnt signaling in osteoblastic cells suggest that Ror2 may regulate bone formation.     

NKX3.1 is regulated by protein kinase CK2 in prostate tumor cells

Diminished expression of NKX3.1 is associated with prostate cancer progression in humans, and in mice, loss of nkx3.1 leads to epithelial cell proliferation and altered gene expression patterns. The NKX3.1 amino acid sequence includes multiple potential phosphoacceptor sites for protein kinase CK2. To investigate posttranslational regulation of NKX3.1, phosphorylation of NKX3.1 by CK2 was studied. In vitro kinase assays followed by mass spectrometric analyses demonstrated that CK2 phosphorylated recombinant NKX3.1 on Thr89 and Thr93. Blocking CK2 activity in LNCaP cells with apigenin or 5,6-dichlorobenzimidazole riboside led to a rapid decrease in NKX3.1 accumulation that was rescued by proteasome inhibition. Replacing Thr89 and Thr93 with alanines decreased NKX3.1 stability in vivo. Small interfering RNA knockdown of CK2alpha' but not CK2alpha also led to a decrease in NKX3.1 steady-state level. In-gel kinase assays and Western blot analyses using fractionated extracts of LNCaP cells demonstrated that free CK2alpha' could phosphorylate recombinant human and mouse NKX3.1, whereas CK2alpha' liberated from the holoenzyme could not. These data establish CK2 as a regulator of NKX3.1 in prostate tumor cells and provide evidence for functionally distinct pools of CK2alpha' in LNCaP cells.