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Calorie restriction (CR) and the activation of autophagy extend healthspan by delaying the onset of age-associated diseases in most living organisms. Because protein kinase CK2 (CK2) downregulation induces cellular senescence and nematode aging, we investigated CK2's role in CR and autophagy. This study indicated that CR upregulated CK2's expression, thereby causing SIRT1 and AMP-activated protein kinase (AMPK) activation. CK2α overexpression, including antisense inhibitors of miR-186, miR-216b, miR-337-3p, and miR-760, stimulated autophagy initiation and nucleation markers (increase in ATG5, ATG7, LC3BII, beclin-1, and Ulk1, and decrease in SQSTM1/p62). The SIRT1 deacetylase, AKT, mammalian target of rapamycin (mTOR), AMPK, and forkhead homeobox type O (FoxO) 3a were involved in CK2-mediated autophagy. The treatment with the AKT inhibitor triciribine, the AMPK activator AICAR, or the SIRT1 activator resveratrol rescued a reduction in the expression of lgg-1 (the Caenorhabditis elegans ortholog of LC3B), bec-1 (the C. elegans ortholog of beclin-1), and unc-51 (the C. elegans ortholog of Ulk1), mediated by kin-10 (the C. elegans ortholog of CK2β) knockdown in nematodes. Thus, this study indicated that CK2 acted as a positive regulator in CR and autophagy, thereby suggesting that these four miRs' antisense inhibitors can be used as CR mimetics or autophagy inducers.

Cellular senescence is an irreversible form of cell cycle arrest. Senescent cells have a unique gene expression profile that is frequently accompanied by senescence-associated heterochromatic foci (SAHFs). Protein kinase CK2 (CK2) downregulation can induce trimethylation of histone H3 Lys 9 (H3K9me3) and SAHFs formation by activating SUV39h1. Here, we present evidence that the PI3K-AKTmTOR-reactive oxygen species-p53 pathway is necessary for CK2 downregulation-mediated H3K9me3 and SAHFs formation. CK2 downregulation promotes SUV39h1 stability by inhibiting its proteasomal degradation in a p53dependent manner. Moreover, the dephosphorylation status of Ser 392 on p53, a possible CK2 target site, enhances the nuclear import and subsequent stabilization of SUV39h1 by inhibiting the interactions between p53, MDM2, and SUV39h1. Furthermore, p21 Cip1/WAF1 is required for CK2 downregulation-mediated H3K9me3, and dephosphorylation of Ser 392 on p53 is important for efficient transcription of p21 Cip1/WAF1 . Taken together, these results suggest that CK2 downregulation induces dephosphorylation of Ser 392 on p53, which subsequently increases the stability of SUV39h1 and the expression of p21 Cip1/WAF1 , leading to H3K9me3 and SAHFs formation.

We investigated whether SIRT1 is associated with reactive oxygen species (ROS) accumulation during CK2 downregulationmediated senescence. SIRT1 overexpression suppressed ROS accumulation, reduced transcription of FoxO3a target genes, and nuclear export and acetylation of FoxO3a, which were induced by CK2 downregulation in HCT116 and MCF-7 cells. Conversely, overexpression of a dominant-negative mutant SIRT1 (H363Y) counteracted decreased ROS levels, increased transcriptional activity of FoxO3a, and increased nuclear import and decreased acetylation of FoxO3a, which were induced by CK2 upregulation. CK2 downregulation destabilized SIRT1 protein via an ubiquitin-proteasome pathway in human cells, whereas CK2 overexpression reduced ubiquitination of SIRT1. Finally, the SIRT1 activator resveratrol attenuated the accumulation of ROS and lipofuscin as well as lifespan shortening, and reduced expression of the DAF-16 target gene sod-3, which were induced by CK2 downregulation in nematodes. Altogether, this study demonstrates that inactivation of the SIRT1-FoxO3a axis, at least in part, is involved in ROS generation during CK2 downregulationmediated cellular senescence and nematode aging. [BMB Reports 2019; 52(4): 265-270].

Cellular senescence is an irreversible form of cell cycle arrest and senescent cells have a unique gene expression profile that is frequently accompanied by senescence-associated heterochromatic foci (SAHF). Here, we present evidence that CK2 downregulation induces trimethylation of histone H3 Lys 9 (H3K9me3), selective binding of HP1γ to H3K9me3, formation of SAHF, and reduction of cyclin D1 expression in HCT116 and MCF-7 cells. CK2 downregulation-mediated H3K9me3 is associated with induction of H3K9 trimethylase SUV39h1 as well as reduction of H3K9 dimethylase G9a and GLP in cells. In addition, Pharmacological inhibition of SUV39h1 and G9a overexpression significantly attenuated induction of senescence-associated β-galactosidase (SA-β-gal) activity, H3K9me3 and SAHF formation in CK2-downregulated cells. Moreover, CK2 downregulation induced H3K9me3 in nematodes. Taken together, these results demonstrate that CK2 downregulation leads to H3K9me3 and SAHF formation by increasing SUV39h1 and decreasing G9a., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: The transcription factor nuclear factor (NF)-κB plays a pivotal role in the development of allergic airway inflammation. However, the mechanism of NF-κB activation in asthma remains to be elucidated., Methods: CK2α activation was assessed by CK2α phosphorylation and protein expression. Airway levels of histamine and cytokines were determined by ELISA. We used 2 (active and passive) forms of allergic pulmonary inflammation models. In the active form, the animals were immunized with ovalbumin (OVA) intraperitoneally, followed by an airway challenge with OVA. In the passive form, the animals were passively sensitized by intratracheal instillation with either anti-OVA IgE or anti-OVA IgG, followed by an airway challenge with OVA. The role of NADPH oxidase (NOX) in CK2α activation was assessed using NOX2-/- and NOX4-/- mice because NOX2 and NOX4 contribute to many inflammatory diseases., Results: The second airway challenge increased CK2α phosphorylation and protein expression in airway epithelial cells as well as nuclear translocation of the p50 and p65 subunits of NF-κB, all of which were inhibited by the CK2α inhibitor 4,5,6,7-tetrabromobenzotriazole and the antioxidant N-acetyl-L-cysteine. CK2α phosphorylation and protein expression were significantly impaired in NOX2-/-, but not in NOX4-/-, mice. Induction of passive sensitization using anti-OVA IgE activated neither CK2α nor NF-κB. In contrast, induction of passive sensitization using anti-OVA IgG activated both CK2α and NF-κB., Conclusions: These data suggest that Fcγ receptor/reactive oxygen species/CK2α is a key inducer of NF-κB activation in airway epithelial cells in a murine model of asthma., (© 2018 S. Karger AG, Basel.)

Studies show that a decrease in protein kinase CK2 (CK2) activity is associated with cellular senescence. However, the role of CK2 in organism aging is still poorly understood. Here, we investigated whether protein kinase CK2 (CK2) modulated longevity in Caenorhabditis elegans. CK2 activity decreased with advancing age in the worms. Knockdown of kin-10 (the ortholog of CK2β) led to a short lifespan phenotype and induced age-related biomarkers, including retardation of locomotion, decreased pharyngeal pumping rate, increased lipofuscin accumulation, and reduced resistance to heat and oxidative stress. The long lifespan of age-1 and akt-1 mutants was significantly suppressed by kin-10 RNAi, suggesting that CK2 acts downstream of AGE-1 and AKT-1. Kin-10 knockdown did not further shorten the short lifespan of daf-16 mutant worms but either decreased or increased the transcriptional activity of DAF-16 depending on the promoters of the target genes, indicating that CK2 is an upstream regulator of DAF-16 in C. elegans. Kin-10 knockdown increased production of reactive oxygen species (ROS) in the worms. Finally, the ROS scavenger N-acetyl-L-cysteine significantly counteracts the lifespan shortening and lipofuscin accumulation induced by kin-10 knockdown. Therefore, the present results suggest that age-dependent CK2 downregulation reduces longevity by associating with both ROS generation and the AGE-1-AKT-1-DAF-16 pathway in C. elegans.

Objectives: Leukocyte NADPH oxidase, which is active in neutrophils, is a membrane-bound enzyme that catalyzes the reduction of oxygen to O2(-) by using NADPH as an electron donor. Previously, we reported that casein kinase 2 (CK2), a ubiquitous and highly conserved Ser/Thr kinase, is responsible for p47(phox) phosphorylation and that phosphorylation of p47(phox) by CK2 regulates the deactivation of NADPH oxidase., Methods: Here, we report that the residue Cys(196) of p47(phox) is a target of S-nitrosylation by S-nitrosothiol and peroxynitrite and that this modification enhanced phosphorylation of p47(phox) by CK2., Results: S-Nitrosylated p47(phox) enhanced CK2 b subunit binding, presumably due to alterations in protein conformation., Discussion: Taken together, we propose that S-nitrosylation of p47(phox) regulates the deactivation of NADPH oxidase via enhancement of p47(phox) phosphorylation by CK2.

An inhibitor of the protein kinase CKII (CKII) was purified from leaves of Glycine max (L.) Merrill and was identified as coumestrol by structural analysis. Coumestrol inhibited the phosphotransferase activity of CKII toward β-casein, with an IC50 of about 5 μM. It acted as a competitive inhibitor with respect to ATP as a substrate, with an apparent Ki value of 7.67 μM. Coumestrol at 50μM resulted in 50% and 30% growth inhibition of human breast cancer MCF-7 and colorectal cancer HCT116 cells, respectively. Coumestrol promoted senescence through the p53-p21(Cip1/WAF1) pathway by inducing reactive oxygen species (ROS) production in MCF-7 and HCT116 cells. The ROS scavenger N-acetyl-l-cysteine (NAC), NADPH oxidase inhibitor apocynin and p22(phox) siRNA almost completely abolished this event. Overexpression of CKIIα antagonised cellular senescence mediated by coumestrol, indicating that this compound induced senescence via a CKII-dependent pathway. Since senescence is an important tumour suppression process in vivo, these results suggest that coumestrol can function by inhibiting oncogenic disease, at least in part, through CKII inhibition-mediated cellular senescence., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Cellular senescence is a tumor suppression mechanism. We previously reported that CKII downregulation induces senescence in human lung fibroblast IMR-90 and colon cancer HCT116 cells. In this study, potential longevity drugs, including rapamycin, vitamin C, and vitamin E, blocked CKII downregulation-mediated senescence through reduction of reactive oxygen species (ROS) production in HCT116 cells. Since rapamycin is a mammalian target of rapamycin (mTOR) inhibitor, we examined the roles of mTOR and its upstream regulators phosphatidylinositol 3-kinase (PI3K) and AKT in CKII inhibition-mediated senescence. CKIIα knock-down or CKII inhibitor treatment strikingly increased phosphorylation of mTOR, p70S6K, an mTOR substrate, and AKT, whereas CKIIα overexpression reduced this phosphorylation event. This result indicated that CKII inhibition activated the PI3K-AKT-mTOR pathway. Further, pharmacological inhibition of PI3K and AKT attenuated ROS production and senescence in CKII-downregulated cells. Taken together, these results demonstrate, for the first time, that the PI3K-AKT-mTOR-ROS pathway is necessary for CKII inhibition-mediated cellular senescence., (Copyright © 2013 Elsevier Inc. All rights reserved.)

We previously demonstrated that downregulation of protein kinase CKII induces cellular senescence in human colon cancer HCT116 cells. To investigate the role of microRNAs (miRNAs) in CKII downregulation during senescence, we employed computational algorithms. Four miRNAs (miR-186, miR-216b, miR-337-3p, and miR-760) were predicted to be miRNAs against CKIIα mRNA. Mimics of all four miRNAs jointly downregulated CKIIα expression in HCT116 cells. Reporter analysis and RT-PCR have suggested that these four miRNAs may stimulate degradation of CKIIα mRNA by targeting its 3' untranslated regions (UTRs). The four miRNA mimics increased senescent-associated β-galactosidase (SA-β-gal) staining, p53 and p21(Cip1/WAF1) expression, and reactive oxygen species (ROS) production. In contrast, concomitant knockdown of the four miRNAs by antisense inhibitors increased the CKIIα protein level and suppressed CKII inhibition-mediated senescence. Finally, CKIIα overexpression antagonized senescence induced by the four miRNA mimics. Therefore, the present results show that miR-186, miR-216b, miR-337-3p, and miR-760 cooperatively promote cellular senescence through the p53-p21(Cip1/WAF1) pathway by CKII downregulation-mediated ROS production in HCT116 cells., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Cellular senescence is thought to be an important tumor suppression process in vivo. We have previously shown that p53 activation is necessary for CKII inhibition-mediated cellular senescence. Here, CKII inhibition induced acetylation of p53 at K382 in HCT116 and HEK293 cells. This acetylation event was suppressed by SIRT1 activation. CKIIα and CKIIβ were co-immunoprecipitated with SIRT1 in a p53-independent manner. Maltose binding protein pull-down and yeast two-hybrid indicated that SIRT1 bound to CKIIβ, but not to CKIIα. CKII inhibition reduced SIRT1 activity in cells. CKII phosphorylated and activated human SIRT1 in vitro. Finally, SIRT1 overexpression antagonized CKII inhibition-mediated cellular senescence. These results reveal that CKII downregulation induces p53 stabilization by negatively regulating SIRT1 deacetylase activity during senescence., (Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Elevated phospholipase D (PLD) expression prevents cell cycle arrest and apoptosis. However, the roles of PLD isoforms in cell proliferation and apoptosis are incompletely understood. Here, we investigated the physiological significance of the interaction between PLD2 and protein kinase CKII (CKII) in HCT116 human colorectal carcinoma cells. PLD2 interacted with the CKIIβ subunit in HCT116 cells. The C-terminal domain (residues 578-933) of PLD2 and the N-terminal domain of CKIIβ were necessary for interaction between the two proteins. PLD2 relocalized CKIIβ to the plasma membrane area. Overexpression of PLD2 reduced CKIIβ protein level, whereas knockdown of PLD2 led to an increase in CKIIβ expression. PLD2-induced CKIIβ reduction was mediated by ubiquitin-dependent degradation. The C-terminal domain of PLD2 was sufficient for CKIIβ degradation as the catalytic activity of PLD2 was not required. Taken together, the results indicate that the C-terminal domain of PLD2 can regulate CKII by accelerating CKIIβ degradation in HCT116 cells.

We have shown that protein kinase CKII (CKII) inhibition induces senescence through the p53-dependent pathway in HCT116 cells. Here we examined the molecular mechanism through which CKII inhibition activates p53 in HCT116 cells. CKII inhibition by treatment with CKII inhibitor or CKIIalpha small-interfering RNA (siRNA) increased intracellular hydrogen peroxide and superoxide anion levels. These effects were significantly blocked by pretreatment of cells with the antioxidant N-acetylcysteine. Additionally, NADPH oxidase (NOX) inhibitor apocynin and p22(phox) siRNA significantly reduced p53 expression and suppressed the appearance of senescence markers. CKII inhibition did not affect mitochondrial superoxide generation. These data demonstrate that CKII inhibition induces superoxide anion generation via NOX activation, and subsequent superoxide-dependent activation of p53 acts as a mediator of senescence in HCT116 cells after down-regulation of CKII., (Copyright 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Protein kinase CKII (CKII), a heterotetramer composed of two catalytic (alpha or alpha') subunits and two regulatory (beta) subunits, plays a critical role in cell proliferation and anti-apoptosis. Recently, capsaicin was shown to trigger apoptosis. Therefore, we examined the effect of capsaicin on CKII activity. Although capsaicin induced apoptotic death in HeLa cells, CKII activity was increased in the cytosolic fraction of HeLa cells after treatment. Capsaicin did not change the expression of the CKIIalpha and CKIIbeta proteins. Capsaicin stimulated the catalytic activity of recombinant CKII tetramer, but not the CKIIalpha subunit. Moreover, capsaicin enhanced the autophosphorylation of CKIIalpha and CKIIbeta. Taken together, our data suggest that capsaicin stimulates the phosphotransferase activity of CKII holoenzyme by interacting with the CKIIbeta subunit.

We have previously shown that the down-regulation of protein kinase CKII activity is tightly associated with cellular senescence of human fibroblast IMR-90 cells. Here, we examined the roles of p53 and p21(Cip1/WAF1) in senescence development induced by CKII inhibition using wild-type, isogenic p53-/- and isogenic p21-/- HCT116 human colon cancer cell lines. A senescent marker appeared after staining for senescence-associated beta-galactosidase activity in wild-type HCT116 cells treated with CKII inhibitor or CKIIalpha siRNA, but this response was almost abolished in p53- or p21(Cip1/WAF1)-null cells. Increased cellular levels of p53 and p21(Cip1/WAF1) protein occurred with the inhibition of CKII. CKII inhibition upregulated p53 and p21(Cip1/WAF1) expression at post-transcriptional level and transcription level, respectively. RB phosphorylation significantly decreased in cells treated with CKII inhibitor. Taken together, this study shows that the activation of the p53-p21(Cip1/WAF1) pathway acts as a major mediator of cellular senescence induced by CKII inhibition.

Over-expression of phospholipase D (PLD) 1 or PLD2 down-regulated CKII activity in NIH3T3 cells. The same results were found with catalytically inactive mutants of PLD isozymes, indicating that the catalytic activity of PLD is not required for PLD-mediated CKII inhibition. Consistent with this, 1-butanol did not alter CKII activity. The reduction in CKII activity in PLD-over-expressing NIH3T3 cells was due to reduced protein level, but not mRNA level, of the CKIIbeta subunit. This PLD-induced CKIIbeta degradation was mediated by ubiquitin-proteasome machinery, but MAP kinase and mTOR were not involved in CKIIbeta degradation. PLD isozymes interacted with the CKIIbeta subunit. Immunocyto-chemical staining revealed that PLD and CKIIbeta colocalize in the cytoplasm of NIH3T3 cells, especially in the perinuclear region. PLD binding to CKIIbeta inhibited CKIIbeta autophosphory-lation, which is known to be important for CKIIbeta stability. In summary, the current data indicate that PLD isozymes can down-regulate CKII activity through the acceleration of CKIIbeta degradation by ubiquitin-proteasome machinery.

Previously we reported that down-regulation of CKII activity is tightly associated with cellular senescence and that the mRNA and protein levels of CKII alpha decrease during senescence. The present study demonstrates that the mRNA and protein levels of CKII alpha' also decrease during senescence. Knockdown of CKII alpha' in IMR-90 cells by RNA interference induced the senescent phenotype. Treatment of senescent IMR-90 cells with a demethylating agent 5-aza-2'-deoxycytidine induced CKII alpha and CKII alpha' expression, suggesting that DNA hypermethylation might be involved in the silencing of CKII alpha and CKII alpha' genes in senescent cells. However, bisulfite sequencing analysis revealed that the methylation status of the CpG islands within the reported CKII alpha and CKII alpha' promoters was not associated with senescence. Instead, senescence-dependent hypermethylation was observed in the region ranging from position +1112 to +1128 of the CKII alpha gene and at positions -527 and +829 of the CKII alpha' gene. In addition, this study indicates that DNA methylation-dependent down-regulation of transcription factors Sp1, Ets1 and NF-kappaB might be involved in silencing of the CKII alpha and CKII alpha' genes during cellular senescence.

Nopp140, a highly phosphorylated nucleolar protein, negatively regulates CK2, a kinase essential for cell proliferation. We quantitatively analyzed the interaction between two subunits of CK2 and Nopp140 and characterized the mechanism by which InsP(6) inhibits the interaction. Nopp140 specifically binds to the catalytic subunit of CK2 (CK2alpha) with a dissociation constant of (K(d)) of 4nM, which interferes with the catalytic activity of CK2. The C-terminal region of Nopp140 is determined as CK2alpha-binding region by a yeast two-hybrid method as well as a direct measurement of the interaction between CK2alpha and deletion mutants of Nopp140. InsP(6) specifically binds to CK2alpha and disrupts the interaction between CK2alpha and Nopp140 with an IC(50) value of 25 microM, thereby attenuating the Nopp140-mediated repression of CK2 activity.

Telomere maintenance is essential for continued cell proliferation and chromosome stability. Telomeres are maintained by telomerase and a collection of associated proteins. The telomeric protein telomeric repeat binding factor 1 (TRF1) negatively regulates telomere length by inhibiting access of telomerase at telomere termini. Here we report that TRF1 interacts with the beta subunit of casein kinase 2 (CK2) and serves as a substrate for CK2. CK2-mediated phosphorylation is required for the efficient telomere binding of TRF1 in vitro and in vivo. Inhibition of CK2 by the CK2 inhibitor 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole decreased the ability of TRF1 to bind telomeric DNA. The resulting telomere-unbound form of TRF1 was then ubiquitinated and degraded by the proteasome. Partial knockdown of CK2 by small interfering RNA resulted in removal of TRF1 from telomeres and subsequent degradation of TRF1. Mapping of the CK2 target site identified threonine 122 as a substrate in TRF1. A threonine to alanine change at this position led to a diminished DNA binding due to reduced dimerization of TRF1. In addition, phosphorylation of threonine 122 seemed critical for TRF1-mediated telomere length control. Our findings suggest that CK2-mediated phosphorylation of TRF1 plays an important role in modulating telomere length homeostasis by determining the levels of TRF1 at telomeres.

The CKII inhibitory compound was purified from the fruit of Xanthium strumarium by organic solvent extraction and silica gel chromatography. The inhibitory compound was identified as 3,4-dihydroxybenzaldehyde by analysis with FT-IR, FAB-Mass, EI-Mass, (1)H-NMR and (13)C-NMR. 3,4-dihydroxybenzaldehyde inhibited the phosphotransferase activity of CKII with IC(50) of about 783 microM. Steady-state studies revealed that the inhibitor acts as a competitive inhibitor with respect to the substrate ATP. A value of 138.6 microM was obtained for the apparent K(i). Concentration of 300 microM 3,4-dihydroxybenzaldehyde caused 50% growth inhibition of human cancer cell U937. 3,4-dihydroxybenzaldehyde-induced cell death was characterised with the cleavage of poly(ADP-ribose) polymerase and procaspase-3. Furthermore, the inhibitor induced the fragmentation of DNA into multiples of 180 bp, indicating that it triggered apoptosis. This induction of apoptosis by 3,4-dihydroxybenzaldehyde was also confirmed by using flow cytometry analysis. Since CKII is involved in cell proliferation and oncogenesis, these results suggest that 3,4-dihydroxybenzaldehyde may function by inhibiting oncogenic disease, at least in part, through the inhibition of CKII activity.

CKII plays a significant role in cell proliferation and cell cycle control. In this report, yeast two-hybrid assay and pull-down assay demonstrate that CKBBP2/CRIF1 associates with the beta subunit of CKII in vitro and in vivo. Recombinant CKBBP2/CRIF1 is phosphorylated in vitro by purified CKII and by CKII inhibitor apigenin-sensitive protein kinase in HEK293 cell extract. Phosphoamino acid analysis and mutational analysis indicate that CKII phosphorylates serine at residue 221 within CKBBP2/CRIF1. Furthermore, serine to alanine mutation at residue 221 abrogates the phosphorylation of CKBBP2/CRIF1 observed in HEK293 cell extract, indicating that CKII is a major kinase that is responsible for phosphorylation of CKBBP2/CRIF1. As compared with the wild-type CKBBP2/CRIF1 or nonphosphorylatable mutant CKBBP2(S221A) (in which the serine-221 is replaced by alanine), overexpression of CKBBP2(S221E) in COS7 cells promotes cell proliferation. Taken together, the present results suggest that CKII may be involved in cell proliferation, at least in part, through the phosphorylation of serine-221 within CKBBP2/CRIF1.

Elevated expression of protein casein kinase II (CKII) stimulated basal phospholipase D (PLD) activity as well as PMA-induced PLD activation in human U87 astroglioma cells. Moreover, CKII-selective inhibitor, emodin and apigenin suppressed PMA-induced PLD activation in a dose-dependent manner as well as basal PLD activity, suggesting the involvement of CKII in the activation of both PLD1 and PLD2. CKII was associated with PLD1 and PLD2 in co-transfection experiments. Furthermore, CKII induced serine/threonine phosphorylation of PLD2 in vivo, and the multiple regions of PLD2 were phosphorylated by CKII in vitro kinase assay using glutathione S-transferase-PLD2 fusion protein fragments. Elevated expression of CKII or PLD increased cell proliferation but pretreatment of cells with 1-butanol suppressed CKII-induced cell proliferation. These results suggest that CKII is involved in proliferation of U87 cells at least in part, through stimulation of PLD activity.

Protein kinase CKII (CKII) plays a critical role in cell growth and proliferation. In this study, we examine how CKII activity is regulated during cellular senescence. Our results demonstrate that CKII activity apparently decreases during both replicative and H2O2-induced senescence in human diploid fibroblast IMR-90 cells. The mRNA and protein levels of CKIIalpha decreases significantly during replicative and H2O2-induced senescence, while only slight reduction in those of CKIIbeta is observed during replicative senescence. Treatment of IMR-90 cells with CKII inhibitors 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole and apigenin led cells to acquire a senescent phenotype as judged by the senescence-associated beta-galactosidase marker and overexpression of p53 and p21(Waf-1). Knockdown of CKIIalpha in IMR-90 cells by RNA interference also dramatically induced the senescent phenotype. In parallel, CKII activity was transcriptional downregulated in rat liver and testis with advancing age. Taken together, these results suggest that downregulation of CKII activity is tightly associated not only with cellular senescence but also with organism aging.

Protein kinase CKII is composed of two catalytic (alpha or alpha') subunits and two regulatory (beta) subunits. The beta subunit mediates tetramer formation through beta-beta homodimerization and alpha-beta heterodimerization. In a previous study R26 and R75, point mutants of CKIIb defective in beta-beta dimerization, were isolated. In the present work we characterized these CKIIbeta mutants in vitro. Purified R26 and R75 bound to CKIIalpha but were defective in binding to CKIIbeta. R75 stimulated the catalytic activity of CKII whereas R26 gave little stimulation, and poly-L-lysine increased the stimulation of catalytic activity by R26 or R75. Circular dichroism and intrinsic fluorescence data pointed to different conformational changes in R26 and R75. Molecular modeling of these mutants provides an explanation of the difference in their ability to interact with CKIIbeta and to activate CKIIalpha.

The CKII (EC 2.7.1.37) inhibitory compound was purified from the root barks of Cudrania tricuspidata and identified as (2S)-2-[2,4-dihydroxy-5-(3-methyl-but-2-enyl)-phenyl]-5,7-dihyroxy-6-(3-methyl-but-2-enyl)chroman-4-one (euchrestaflavanone B). Euchrestaflavanone B was shown to inhibit the phosphotransferase activity of CKII with IC50 of about 78 microM. Steady-state studies revealed that euchrestaflavanone B acted as a competitive inhibitor with respect to the substrate ATP. A value of 16.4 microM was obtained for the apparent Ki. Concentration of 0.8 microM euchrestaflavanone B caused 50% growth inhibition of human cancer cells U937 and HeLa. Euchrestaflavanone B-induced cell death was characterized with the cleavage of poly(ADP-ribose) polymerase and procaspase-3, indicating that the inhibitor triggered apoptosis. Because protein kinase CKII is involved in cell proliferation and oncogenesis, these results suggest that euchrestaflavanone B may function by inhibiting oncogenic disease, at least in part, through the inhibition of CKII activity.