Enhanced casein kinase II activity during mouse embryogenesis. Identification of a 110-kDa phosphoprotein as the major phosphorylation product in mouse embryos and Krebs II mouse ascites tumor cells

Mouse embryos at various stages of development were used to study the relationship of protein kinase activities with normal embryogenesis. Casein kinase II (CKII) activity in developing mouse embryos shows a 3-4-fold activity increase at day 12 of gestation. Together with the CKII activity, increased phosphorylation of a 110-kDa protein is observed. Treatment of the embryo extracts with heparin, a highly specific inhibitor of CKII activity, results in a drastic reduction of the 110-kDa protein phosphorylation indicating that the protein might be a CKII-specific substrate. Rapidly proliferating mouse tumour cells also show an enhanced CKII activity. Here too, a 110-kDa phosphoprotein was the major phosphoryl acceptor. Partial proteolytic digestion shows that both proteins are identical. Other protein kinases tested (cAMP- and cGMP-dependent protein kinases) only show a basal level of enzyme activity with minor alterations throughout the different stages of embryogenesis investigated.     

A Casein Kinase-Like Kinase Phosphorylates β-Tubulin and May Be a Microtubule-Associated Protein

The hypothesis that casein kinase II (CKII) is a microtubule-associated protein kinase was investigated using a neuronal cell line and bovine brain. Heparin, an inhibitor of CKII, inhibited the phosphorylation of a PC12 cytosolic protein whose molecular weight was similar to that of beta-tubulin. Partially purified PC12 CKII was immunoreactive to an antibody directed against bovine CKII and was able to phosphorylate purified beta-tubulin in a heparin-inhibitable manner when the concentration of tubulin was less than 50 micrograms/ml. To better determine if CKII is a microtubule-associated protein kinase, bovine brain tubulin was chromatographed on FPLC Mono Q and phosphocellulose columns. Several tubulin casein kinase (TCK) activities were apparent. All TCK activities phosphorylated tubulin and casein, but none was able to phosphorylate the CKII-specific synthetic peptide RRREEETEEE. One of these TCK fractions was immunoreactive to the antibody directed against CKII, and this antibody labeled a 50-kDa molecular mass band that had a molecular mass distinctly different from those of the subunits of CKII. Thus, we suggest that a CKII-like protein, but not CKII, might be a microtubule-associated protein.     

Casein kinase II is elevated in solid human tumours and rapidly proliferating non-neoplastic tissue

Protein kinase CKII (i.e. casein kinase II, CKII, NII) is expressed at a higher level in rapidly proliferating tissues and in solid human tumours (e.g. colorectal carcinomas) when compared to the corresponding non-neoplastic colorectal mucosa. This could be shown by (a) Western blotting of cellular extracts from solid tumours followed by immunostaining with an anti-CKII polyclonal antibody, (b) immunohistochemical staining of cells from tissue sections and (c) by activity measurements using the CKII-specific synthetic peptide (RRRDDDSDDD). The maximum observed activity in the colorectal carcinomas investigated was up to eightfold higher in the tumour specimens than in the non-neoplastic tissue (i.e. colorectal mucosa). The activity range was between 33-350 U/mg protein and in the case of colorectal mucosa 13-106 U/mg protein. The amount of CKII determined in the individual tumours was in the range 0.4-1.6 nmol/g tissue.     

Developmental changes in the activity and cellular localization of hepatic casein kinase II in the rat

Abstract The activity and cellular localization of hepatic casein kinase II(CKII) was examined during late fetal development in the rat. Cultured fetal hepatocytes displayed constitutive CKII activity which was not further activated by growth factor exposure. Similarly, fetal liver CKII showed approximately fivefold greater activity than adult liver. The fetal hepatic activity was, to a large degree, localized to a nuclear fraction. Postnuclear cytosol preparations from fetal and adult liver showed similar CKII activity. In all cases, FPLC ion exchange chromatography followed by Western immunoblotting showed that immunoreactive CKII coincided with kinase activity. However, Parallel determinations of CKII activity and immunoreactive CKII levels showed a higher(five-to sixfold) CKII specific activity in nuclear extracts compared to cytosol. In summary, fetal hepatic CKII demonstrates coincident nuclear localization and activation. We hypothesize that the regulation of hepatic CKII is relevant to the mitogen-independent proliferation displayed by fetal rat hepatocytes. © Wiley-Liss, Inc.     

New ELISA-based method for the detection of O-GlcNAc transferase activity in vitro

O-GlcNAcylation is a dynamic, reversible, post-translational modification that regulates many cellular processes. O-GlcNAc transferase (OGT) is the sole enzyme transferring N-acetylglucosamine from uridine diphosphate (UDP)-GlcNAc to selected serine/threonine residues of cytoplasm and nucleus proteins. Aberrant of OGT activity is associated with several diseases, suggesting OGT as a novel therapeutic target. In this study, we created a new enzyme linked immunosorbent assays (ELISA)-based method for detection of OGT activity. First, casein kinase II (CKII), a well-known OGT substrate, was coated onto ELISA plate. Second, the GlcNAc transferred by OGT from UDP-GlcNAc to CKII was detected using an antibody to O-GlcNAc and then the horseradish peroxidase (HRP)-labeled secondary antibody. At last, 3,3′,5,5′-tetramethylbenzidine (TMB), the substrate of HRP, was used to detect the O-GlcNAcylation level of CKII which reflected the activity of OGT. Based on a series of optimization experiments, the RL2 antibody was selected for O-GlcNAc detection and the concentrations of CKII, OGT, and UDP-GlcNAc were determined in this study. ST045849, a commercial OGT inhibitor, was used to verify the functionality of the system. Altogether, this study showed a method that could be applied to detect OGT activity and screen OGT inhibitors.     

Casein Kinase II Activity in the Postischemic Rat Brain Increases in Brain Regions Resistant to Ischemia and Decreases in Vulnerable Areas

Abstract: Casein kinase II (CKII) is a protein kinase acting in the intracellular cascade of reactions activated by growth factor receptors, and that has a profound influence on cell proliferation and survival. In this investigation, we studied the changes in the activity and levels of CKII in the rat brain exposed to 10. 15 and 20 min of transient forebrain ischemia followed by variable periods of reperfusion. The cytosolic CKII activity decreased during reperfusion by ∼ 30 and ∼ 50% in the selectively vulnerable areas, striatum and the CA1 region of the hippocampus, respectively. In the resistant CA3 region of hippocampus and neocortex, the activity increased by ∼ 20 and ∼ 60%, respectively. The postischemic changes in CKII activity were dependent on the duration of the ischemic insult. The levels of CKII did not change after ischemia, suggesting that the enzyme is modulated by covalent modification or is interacting with an endogenous inhibitor/activator. Treatment of the cytosolic fraction from cortex of rats exposed to ischemia and 1 h of reperfusion with agarose-bound phosphatase decreased the activity of CKII to control levels, suggesting that CKII activation after ischemia involves a phosphorylation of the enzyme. The correlation between postischemic CKII activity and neuronal survival implies that preservation or activation of CKII activity may be important for neuronal survival after cerebral ischemia.     

Stimulation of enzymatic activity in filament preparations of casein kinase II by polylysine,melittin, and spermine

Summary Casein kinase II (CKII) has been purified from bovine heart tissue. Under conditions of low salt (0.05 M NaCl, 10 MM MgCl₂), CKII forms structured aggregates that appear as filaments similar to results obtained withDrosophila CKII [C.V.C. Glover (1986) J. Biol. Chem. 261:14349]. The aggregates have been analyzed by sucrose density gradients and electron microscopy. Filament preparations of the enzyme have reduced but measurable kinase activity. The addition of salt restores activity. Various modulators of CKII activity have been examined with the enzyme in the low salt, polymerized form. The polyamines spermine or spermidine stimulated CKII activity as much as six fold; putrescine had no effect. Polylysine of varying lengths activated CKII 4–6 fold. Melittin, the basic polypeptide from bee venom, was also an effective activator. Activation of filament preparations was also observed if the CKII specific peptide (RRREEETEEE) was used as the substrate in place of casein. These results with filament preparations provide an alternative in vitro system for the study of possible regulatory aspects of CKII.     

Growth of Theileria annulata and Theileria parva macroschizont-infected bovine cells in immunodeficient mice: effect of irradiation and tumour load on lymphocyte subsets.

Bovine cells infected with macroschizonts of the protozoan parasites Theileria annulata and Theileria parva formed solid tumours when injected into irradiated Balb/c and irradiated Balb/c nude mice. T. annulata tumours grew more vigorously than T. parva tumours, when initiated with similar doses of infected cells in mice exposed to the same doses of gamma-irradiation. In irradiated Balb/c mice, tumours of both species of parasites began to regress 2-3 weeks after injection of cells but grew without regression in irradiated Balb/c nude mice. Haemorrhage and necrosis of tumours, induced by macrophages and neutrophils, were seen in both mouse strains but were insufficient to cause regression in Balb/c nude mice. Theileria-infected bovine cells failed to establish in C57 beige mice, which lack functional natural killer (NK) cells. Flow cytometry, using monoclonal antibodies to murine leukocyte/lymphocyte antigens, showed that the radiation dose required to allow establishment of T. annulata tumours in Balb/c mice caused a severe depletion of splenic lymphocytes. B cells, helper T and cytotoxic T cells showed differing levels of susceptibility to irradiation. The presence of a tumour promoted the recovery of lymphocyte populations: this recovery was accompanied by destruction of the tumour.     

Studies on beige-nude mice with low natural killer cell activity. 1. Introduction of the bg gene into nude mice and the characteristics of beige-nude mice

To improve the take-rate of human tumours in nude mice, a nude mouse strain with the bg gene was established. The introduction of the bg gene was confirmed by examination of giant granules of blood neutrophils. The genetic profile of beige-nude mice was the same as that of the C57BL/6 strain according to genetic screening. The natural killer cell activity in the beige-nude mice was much lower than that in ordinary nude mice but slightly higher than that in beige mice. The reproductivity of beige-nude mice was as high as that of ordinary nude mice.     

Inhibition of protein kinase CKII activity by resveratrol,a natural compound in red wine and grapes

Resveratrol is a phytoalexin found in grapes and other foods that has been shown to have anticancer and anti-inflammatory effects. Because protein kinase CKII is involved in cell proliferation and oncogenesis, we examined whether resveratrol could modulate CKII activity. Resveratrol was shown to inhibit the phosphotransferase activity of CKII with IC(50) of about 10 microM. Steady state studies revealed that resveratrol acted as a competitive inhibitor with respect to the substrate ATP. A value of 1.2 microM was obtained for the apparent K(i). Resveratrol also inhibited the catalytic reaction of CKII with GTP as substrate. Furthermore, resveratrol inhibits endogenous CKII activity on protein substrates in HeLa cell lysates. These results suggest that resveratrol is likely to function by inhibiting oncogenic disease, at least in part, through the inhibition of CKII activity.     

Xanthohumol inhibits inflammatory factor production and angiogenesis in breast cancer xenografts

Xanthohumol (XN), a natural polyphenol present in beer, is known to exert anti-cancer effects. However, its precise mechanisms are not yet clearly defined. The aim of this study was to investigate the effect of oral administration of XN in breast cancer xenografts in nude mice. Proliferation and apoptosis were first examined in MCF7 cell cultures after incubation with XN by trypan blue exclusion assay, [3H]-thymidine incorporation, KI67 immunostaining and TUNEL. Morphological and histological characteristics of tumours from XN-treated or control (vehicle-treated) mice were compared. Immunohistochemistry for proliferative, inflammatory and endothelial cell markers was performed and activation of nuclear factor kappa B (NFkappaB) pathway was assessed by ELISA. In vitro MCF7 cell proliferation decreased in a dose-dependent manner. Oral administration of XN to nude mice inoculated with MCF7 cells resulted in central necrosis within tumours, reduced inflammatory cell number, focal proliferation areas, increased percentage of apoptotic cells and decreased microvessel density. Anti-angiogenic effects of XN were further confirmed by immunoblotting for factor VIII expression in XN-treated tumours as compared to controls. Decreased immunostaining for NFkappaB, phosphorylated-inhibitor of kappa B and interleukin-1beta were also observed as well as a significant decrease in NFkappaB activity to 60% of control values. These novel findings indicate that XN is able to target both breast cancer and host cells, namely inflammatory and endothelial cells, suggesting its potential use as a double-edge anti-cancer agent.     

Casein kinase II stimulates rat liver mitochondrial glycerophosphate acyltransferase activity

Rat liver mitochondrial glycerophosphate acyltransferase (mtGAT) possesses 14 consensus sites for casein kinase II (CKII) phosphorylation. To study the functional relevance of phosphorylation to the activity of mtGAT, we treated isolated rat liver mitochondria with CKII and found that CKII stimulated mtGAT activity approximately 2-fold. Protein phosphatase-lambda treatment reversed the stimulation of mtGAT by CKII. Labeling of both solubilized and non-solubilized mitochondria with CKII and [gamma-32P]ATP resulted in a 32P-labeled protein of 85kDa, the molecular weight of mtGAT. Our findings suggest that CKII stimulates mtGAT activity by phosphorylation of the acyltransferase. The significance of this observation with respect to hormonal control of the enzyme is discussed.     

Retention of endocrine function by an insulin-secreting pancreatic islet cell tumour from Syrain hamster through serial transplantation in nude mice.

An insulin-secreting islet cell tumour of the Syrian hamster has been transplanted serially in the congenitally immune-deficient nude mouse, in order to test the potential usefulness of this mouse mutant as a graft carrier of heterologous tumours with stable differentiated phenotypes. The incidence of tumour growth was very high, and the hamster tumour retained its functional and histologic characteristics during consecutive passages in nude mice. These results show that nude mice may be useful carriers of differentiated tumours from non-inbred species including man, and for the isolation of cell lines from such tumours.     

The human cytomegalovirus virion possesses an activated casein kinase II that allows for the rapid phosphorylation of the inhibitor of NF-kappaB, IkappaBalpha

We documented that the NF-kappaB signaling pathway was rapidly induced following human cytomegalovirus (HCMV) infection of human fibroblasts and that this induced NF-kappaB activity promoted efficient transactivation of the major immediate-early promoter (MIEP). Previously, we showed that the major HCMV envelope glycoproteins, gB and gH, initiated this NF-kappaB signaling event. However, we also hypothesized that there were additional mechanisms utilized by the virus to rapidly upregulate NF-kappaB. In this light, we specifically hypothesized that the HCMV virion contained IkappaBalpha kinase activity, allowing for direct phosphorylation of IkappaBalpha following virion entry into infected cells. In vitro kinase assays performed on purified HCMV virion extract identified bona fide IkappaBalpha kinase activity in the virion. The enzyme responsible for this kinase activity was identified as casein kinase II (CKII), a cellular serine-threonine protein kinase. CKII activity was necessary for efficient transactivation of the MIEP and IE gene expression. CKII is generally considered to be a constitutively active kinase. We suggest that this molecular characteristic of CKII represents the biologic rationale for the viral capture and utilization of this kinase early after infection. The packaging of CKII into the HCMV virion identifies that diverse molecular mechanisms are utilized by HCMV for rapid NF-kappaB activation. We propose that HCMV possesses multiple pathways to increase NF-kappaB activity to ensure that the correct temporal regulation of NF-kappaB occurs following infection and that sufficient threshold levels of NF-kappaB are reached in the diverse array of cells, including monocytes and endothelial cells, infected in vivo.     

Effect of chemotherapy on human tumor heterografts and lymphocytes in athymic mice

Sensitivity of human serially transplanted tumours (melanoma, hepatoma, lung, stomach and bladder cancer) to monochemotherapy was studied in nude mice. The following groups of nude mice were examined: intact and those with human xenografts (control and mice treated with alkylating agents). Changes in peripheral blood morphology have been revealed and functional activity of the energetic enzymes (SDH and alpha-GPDH) has been cytochemically discovered.     

Toxicity of anticancer agents, growth and chemosensitivity of human tumour xenografts in a segregating stock of AF nude mice.

An investigation of the usefulness of a segregating stock of nude mice [AF nude mice (AF-nu)] for screening anticancer agents was undertaken. The toxicity of anticancer agents, takes and growth rates of human tumour xenografts and chemosensitivities of xenografts in AF-nu were studied and compared with those in BALB/cA nude mice (BALB/cA-nu). The results showed differences in the pattern of mortalities of AF-nu and BALB/cA-nu administered a range of anticancer agents. Body weight changes in the two nude mouse strains differed in the case of 5-fluorouracil, but not for nimustine, adriamycin and vincristine. All tumours transplanted in AF-nu grew as in BALB/cA-nu. Growth rates of 2 xenografts (gastric cancer and glioblastoma) were not significantly different between the 2 nude mouse strains, but those of 2 lung tumour xenografts were significantly greater in AF-nu than those in BALB/cA-nu. There were no significant differences in chemosensitivities of human tumours in AF-nu and BALB/cA-nu (consistency rate as evaluated by our criteria was 88%). From these results, it is suggested that AF-nu are more suitable for anticancer agent screening and experimental chemotherapy of human tumour xenografts than BALB/cA-nu because of lower costs and high reproductive rate. Although they are genetically heterogeneous, sets of experimental animals sharing the same gene pool can be produced routinely.     

Isolation and characterization of recombinant human casein kinase II subunits alpha and beta from bacteria

cDNA encoding the casein kinase II (CKII) subunits alpha and beta of human origin were expressed in Escherichia coli using expression vector pT7-7. Significant expression was obtained with E. coli BL21(DE3). The CKII subunits accounted for approximately 30% of the bacterial protein; however, most of the expressed proteins were produced in an insoluble form. The recombinant CKII alpha subunit was purified by DEAE-cellulose chromatography, followed by phosphocellulose and heparin-agarose chromatography. The recombinant CKII beta subunit was extracted from the insoluble pellet and purified in a single step on phosphocellulose. From 10 g bacterial cells, the yield of soluble protein was 12 mg alpha subunit and 5 mg beta subunit. SDS/PAGE analysis of the purified recombinant proteins indicated molecular masses of 42 kDa and 26 kDa for the alpha and beta subunits, respectively, in agreement with the molecular masses determined for the subunits of the native enzyme. The recombinant alpha subunit exhibited protein kinase activity which was greatest in the absence of monovalent ions. With increasing amounts of salt, alpha subunit kinase activity declined rapidly. Addition of the beta subunit led to maximum stimulation at a 1:1 ratio of both subunits. Using a synthetic peptide (RRRDDDSDDD) as a substrate, the maximum protein kinase stimulation observed was fourfold under the conditions used. The Km of the reconstituted enzyme for the synthetic peptide (80 microM) was comparable to the mammalian enzyme (40-60 microM), whereas the alpha subunit alone had a Km of 240 microM. After sucrose density gradient analysis, the reconstituted holoenzyme sedimented at the same position as the mammalian CKII holoenzyme.     

Enhanced casein kinase II activity in human tumour cell cultures

Casein kinase II (CKII) activity is enhanced as much as 2–3 fold in established and 4–5-fold in transformed human cell lines when compared to that of fibroblasts and primary human tumour cell cultures where CKII activity never exceeded a basic level. The high activity of CKII in transformed cells and in established cell lines was reduced to about the same basic level after treatment with heparin, a highly specific inhibitor of CKII activity. The activity of the cAMP-dependent protein kinase was virtually the same in fibroblasts and various human tumour cell lines investigated.     

Activation of mammalian DNA ligase I through phosphorylation by casein kinase II.

Mammalian DNA ligase I has been shown to be a phosphoprotein. Dephosphorylation of purified DNA ligase I causes inactivation, an effect dependent on the presence of the N-terminal region of the protein. Expression of full-length human DNA ligase I in Escherichia coli yielded soluble but catalytically inactive enzyme whereas an N-terminally truncated form expressed activity. Incubation of the full-length preparation from E. coli with purified casein kinase II (CKII) resulted in phosphorylation of the N-terminal region and was accompanied by activation of the DNA ligase. Of a variety of purified protein kinases tested, only CKII stimulated the activity of calf thymus DNA ligase I. Tryptic phosphopeptide analysis of DNA ligase I revealed that CKII specifically phosphorylated a major peptide also apparently phosphorylated in cells, implying that CKII is a protein kinase acting on DNA ligase I in the cell nucleus. These data suggest that DNA ligase I is negatively regulated by its N-terminal region and that this inhibition can be relieved by post-translational modification.