蛋白激脢CK2訊息傳遞在大腦衍生營養因子抗細胞凋亡機制中所扮演的角色及機制探討---SRF及其下游基因Mcl-1表現的影響

(1)

行政院國家科學委員會專題研究計畫成果報告

蛋白激 CK2 訊息傳遞在大腦衍生營養因子抗細胞凋亡機

制中所扮演的角色及機制探討：對 SRF 及其下游基因 Mcl-1

表現的影響(第 2 年)

研究成果報告(完整版)

計畫類別：個別型計畫編號： NSC 96-2320-B-004-001-MY2 執行期間： 97 年 08 月 01 日至 98 年 07 月 31 日執行單位：國立政治大學神經科學研究所計畫主持人：趙知章計畫參與人員：此計畫無其他參與人員處理方式：本計畫涉及專利或其他智慧財產權，2 年後可公開查詢

中華民國 98 年 11 月 08 日

(2)

行政院國家科學委員會補助專題研究計畫

■ 成果報告

□期中進度報告

蛋白激酶CK2訊息傳遞在大腦衍生營養因子抗細胞凋亡機制中所扮演的

角色及機制探討：對SRF及其下游基因Mcl-1表現的影響

計畫類別：■ 個別型計畫 □ 整合型計畫

計畫編號：

NSC 96-2320-B-004 -001-MY2

執行期間： 96 年 08 月 01 日至 98 年 07 月 31 日

計畫主持人：趙知章

共同主持人：

計畫參與人員：趙知章、馬允立、李小媛

成果報告類型(依經費核定清單規定繳交)：□精簡報告 ■完整報告

本成果報告包括以下應繳交之附件：

□赴國外出差或研習心得報告一份

□赴大陸地區出差或研習心得報告一份

□出席國際學術會議心得報告及發表之論文各一份

□國際合作研究計畫國外研究報告書一份

處理方式：除產學合作研究計畫、提升產業技術及人才培育研究計畫、

列管計畫及下列情形者外，得立即公開查詢

■涉及專利或其他智慧財產權，□一年■二年後可公開查詢

執行單位：國立政治大學神經科學研究所

中華民國 98 年 10 月 30 日

(3)

Protein kinase CK2 mediates the signaling pathway of BDNF through phosphorylating NF-κB to increase the anti-apoptotic Bcl-xL gene expression

Chih C. Chao1, Yun L. Ma2 and Eminy H.Y. Lee2

1

Institute of Neuroscience, National Chengchi University, Taipei, Taiwan and

2

Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan

Address for correspondence: Dr. Eminy H.Y. Lee, Ph.D. Institute of Biomedical Sciences, Academia Sinica

Taipei 115, Taiwan Tel: +886-2-2789-9125

(4)

Abstract

Protein kinase CK2 (Casein kinase II) is a multifunctional serine/threonine protein kinase that is associated with the anti-apoptosis. The previous studies revealed that CK2 can be elevated and mediate the neurotrophic responses by brain-derived neurotrophic factor (BDNF) or glial cell line derived neurotrophic factor (GDNF). However, its action and the behind mechanisms are rarely focused on the nervous system. In the present studies, we found that BDNF increased the Bcl-xL gene expression correlative with CK2 activity and NF-κB phosphorylation in the hippocampal CA1 region. Transfection of the wildtype CK2α and the catalytically inactive CK2α156A mutant increased and decreased the Ser529 phosphorylation on NF-κB parallel with the anti-apoptotic Bcl-xL gene expression, respectively. Further, inhibition of CK2 would reverse the enhance effects of BDNF on Bcl-xL and NF-κB. Over-expression of mutant NFκBS529A would antagonize the increase effect on Bcl-xL mRNA either by BDNF treatment or by wildtype CK2α transfection. These results together suggest that CK2 might mediate an alternative signaling pathway for BDNF through directly phosphorylating NF-κB to increase the anti-apoptotic Bcl-xL gene expression.

Key words: brain derived neurotrophic factor, protein kinase CK2, NF-κB, Bcl-xL, anti-apoptosis

(5)

Introduction

Protein kinase CK2, which is highly conserved during evolution, is a multifunctional, constitutively active and ubiquitous serine/threonine protein kinase (Litchfield, 2003; Pinna and Meggio, 1997). The CK2 holoenzyme consists of subunits α, α’ (catalytic) and β (regulatory) which associate to form α2β2, α’2β2, and αα’β2 heterotetramers (Litchfield, 2003). It appears that CK2 has more than 300 substrates those are involved in organelle function, signal transduction, gene transcription in various cells and CK2 also plays a prominent role in ensuring cell survival by counteracting apoptosis (Meggio and Pinna, 2003). Evidence linking CK2 activity to inhibition of apoptosis showed that the inhibition of CK2 activity decreases cell viability and induces apoptosis in cancer cells (Ruzzene et al., 2002; Izeradjene et al., 2005). There are reports indicating that CK2 can directly phosphorylate the pro-apoptotic protein Bid and Bad to inhibit their cleavage by caspase (Desagher et al., 2001; Krippner-Heidenreich et al., 2001) or the ARC protein (apoptosis repressor with caspase recruitment domain) to increase its inhibitory effect on caspase activity (Li et al., 2002). Beside these direct effects, CK2 has also been found to involve in the signaling transduction pathways through phosphorylating Akt, NF-κB or β-catenin to enhance the cell survival (Di Maira et al., 2005; Manna et al., 2007b; Tapia et al., 2006). Many of the anti-apoptotic gene expressions (such as: Bcl-2, IAP, Mcl-1 and et

al) are found to be activated through the signaling pathways mediated by these

molecules (Toruner et al., 2006; Takada et al., 2005; Manna et al., 2007a).

The CK2 protein is much more abundant in the brain and testis than in any other tissues of adult rats and is suggested to play the important roles in specific neural functions (Nakajo et al., 1986; Blanquet 2002). The studies have shown that CK2 can phosphorylate a variety of molecules that are associated with neuronal growth and neuronal plasticity, such as growth-associated protein (GAP-43) and NR2B (Korshunova et al., 2007; Chung et al., 2004). Further, CK2 is shown to associate with long-term potentiation in the hippocampus and spatial memory of rats (Charriaut-Marlangue et al., 1991; Chao et al., 2007). Regarding the CK2 activity regulation, results of studies revealed that CK2 can be elevated and mediate the neurotrophic responses in hippocampal slices following brain-derived neurotrophic factor (BDNF) treatment or in the substaitia nigra by glial cell line derived neurotrophic factor (GDNF) infusion (Blanquet 2000; Chao et al., 2006). Both two neurotrophic factors are shown to increase the neuronal survival under injury. Up to now, there are many studies focusing on the BDNF signaling pathway underlying its neuroprotective mechanisms. Those results indicate that PI-3K/Akt or MAPK/ERK signaling pathway involves in its neuroprotective effects against either trophic

(6)

deprivation- or glutamate-induced apoptotic cell death and hypoxic-ischemic injury (Han et al., 2000; Chang et al., 2004; Rossler et al., 2004; Almeida et al., 2005). However, these may not be sufficient to explain BDNF’s pharmacological actions.

Although the survival effects of CK2 on cancer cells have been studied extensively, its action and mechanisms are rarely focused on the nervous system. According the clinical diagnosis from Alzheimer’s disease and schizophrenic patients, results have revealed the correlation of the change of CK2 activity and gene expression profile with the pathogenic processes (Aksenova et al., 1991; Dunckley et al., 2006; Paulson et al., 2003). Since BDNF is thought as one of the therapeutic candidates for neurodegenerative disorders (Schulte-Herbrüggen et al., 2007), it is worthy to investigate the possible mechanisms behind its neurotrophic effects. The aim of the present study was to examine whether CK2-mediated signaling pathway contributes to the neurotrophic action of BDNF on hippocampal neurons.

(7)

Materials and Methods

Animals. Adult male Sprague-Dawley rats (250-350 g) bred in the Institution of

Biomedical Sciences (IBMS), Academia Sinica, were used. They were maintained on a 12/12 hr light/dark cycle with food and water continuously available. For DNA transfection and drug administration, rats were subjected to stereotaxic surgery. Two 23-gauge, stainless-steel, thin-wall cannulae were implanted bilaterally to hippocampal CA1 area at the following coordinates: 3.5 mm posterior to the bregma; 2.5 mm lateral to the midline; and 3.4 mm ventral to the skull surface. Transfection and drug infusion were made at a rate of 0.4 μl/min and 0.8 μl was delivered to the each side. Animals were sacrificed 48 hr after DNA transfection or at different time points after drug infusion.

Plasmid DNA construction. Plasmid DNA was constructed as described previously

(Chao et al., 2006). The full-length RT-PCR products of rat brain CK2α and NF-κB gene were inserted in frame into the pcDNA3 with HA tag or pCMV vector with Flag tag, respectively, and their sequences were verified by DNA sequencing. Mutant plasmid CK2α156A and NFκBS529A were generated by site-directed mutagenesis (Promega). The S529 on NF-κB was identified as the phosphorylation site of CK2α (Wang et al., 2000). Branched polyethylenimine (25 kDa, Aldrich) was used as the transfection agent (Abdallah et al., 1996) and the equivalence of nitrogens per DNA phosphate was 10.

Drug infusion and Plasmid DNA transfection. To assess the dose-response effects of

recombinant human BDNF (Pepro Tech LTD.) on CK2 activity, NF-κB phosphorylation and Bcl-xL mRNA expression, 0.2 μg, 0.4 μg, and 1.2 μg of BDNF were bilaterally infused to the CA1. Animals were sacrificed 4 hr after infusion and the CA1 tissue was punched out for further assay.

To prove the role of CK2 in BDNF signaling, CK2α plasmid DNA transfections were first conducted to examine its effects on NF-κB phosphorylation and Bcl-xL mRNA expression. Animals were divided into three groups to receive 1.0 μg of pcDNA3-HA, wildtype CK2αWT and mutant CK2α156A DNA transfection. Animals were sacrificed 48 hr after DNA transfection.

To study the interactive effects of CK2 inhibitor TBB (4,5,6,7-tetrabromobenzotriazole, Calbiochem) and BDNF on NF-κB phosphorylation and Bcl-xL mRNA expression, animals were divided into three groups to receive twice infusion of PBS; PBS followed by 0.4 μg of BDNF, or 10 ng of TBB followed

(8)

by 0.4 μg of BDNF. The interval between two infusions was 30 min. Animals were sacrificed 4 hr after the last infusion.

To examine the interaction between BDNF and NFκB on Bcl-xL mRNA expression, animals were divided into three groups to receive pCMV-Flag transfection (1.0 μg) followed by PBS infusion; pCMV-Flag transfection (1.0 μg) followed by BDNF (0.4 μg) infusion; or NFκBS529A transfection (1.0 μg) followed by BDNF (0.4 μg) infusion. The time schedule for these treatments was as follows: DNA transfection was performed on the first day and BDNF (or PBS) was infused 48 hr later. Animals were sacrificed 4 hr after BDNF infusion.

To examine the interaction between CK2α and NF-κB on Bcl-xL mRNA expression, animals were divided into three groups to receive pCMV-Flag and pcDNA3-HA transfection (1.0 μg each); pCMV-Flag+CK2αWT transfection (1.0 μg each); NFκBS529A+CK2αWT transfection (1.0 μg each). The interval between the two transfections was 1 hr. Animals were sacrificed 48 hr after DNA transfection.

CK2 activity assay. CK2 activity assay was described elsewhere (Chao et al., 2007).

Briefly, CK2 activity was measured in 5 μg protein aliquots from each sample by using the protein kinase CK2 assay kit (Upstate). The synthetic peptide and [γ-32

P]-ATP were used as the substrates and a kinase inhibitor cocktail was added to block the activity of other serine/threonine kinases. Total CK2 activity was calculated by subtracting the blank (without the peptide substrate) from the 32P radioactivity incorporated in the presence of the substrate.

Real-time PCR analysis. Real-time PCR analysis of Bcl-xL mRNA was performed by

using the Applied Biosystems 7500 Real-Time PCR System with standard TaqMan method. The house-keeping enzyme hypoxanthine phosphoribosyl transferase (HPRT) was used as an internal control. The cDNA was synthesized from 0.5 µg total RNA with 0.5 µg of oligo-dT by Superscript II reverse transcriptase (Invitrogen Inc.) and the commercial primers and fluorogenic probes for Bcl-xL and HPRT were purchased from Applied Biosystem Inc. The quantities of Bcl-xL and HPRT from the same sample were analyzed simultaneously but in separate tubes. Each assay was carried out in duplicate. The thermal conditions were: 2 min at 55 ℃ and 10 min at 95 ℃; followed by 40 cycles at 95 ℃ for 15 s and 60 ℃ for 1 min.

Western blot analysis. Fifteen μg of protein extract was subjected to SDS-PAGE and

(9)

TBS-T containing 2% BSA followed by anti-pS529NFκB (1:1000; Stressenge), anti-NFκB (1:1000; Santa Cruz Biotechnology), or anti-β-actin (1:10000, Chemicon) antibody and then with HRP-conjugated secondary antibody (1:8000, Amersham). The signals were detected by chemiluminescence and the density of each band was quantified by using the NIH Image J software.

Immunohistochemistry. The immunohistochemistry procedures were described in

detail elsewhere (Chao et al., 2007). The paraformaldehyde-fixed brains were cut into series of 20-μm coronal sections through the CA1 region. Sections were incubated with mouse anti-HA antibody (1:400, Upstate) and then with anti-mouse FITC-conjugated IgG (1:1000, Sigma). Image acquisition was obtained by using a confocal microscope (Bio-Rad).

Statistical analysis. Data for enzyme activity, western blot analysis and real-time PCR

determination were evaluated by Student’s t-test or one-way analysis of variance (ANOVA) followed by Dunnett’s t-test or the Newman-Keul’s method.

(10)

Results

Anti-apoptotic effects of BDNF on CA1 neurons

The effects of different doses of acute BDNF treatment on Bcl-xL mRNA expression in the CA1 are shown in Fig. 1A. There was an overall increase in Bcl-xL mRNA expression by BDNF treatments (F3,17 = 11.44, p<0.01). Further analyses

indicated that BDNF at 0.4 μg and 1.2 μg both markedly enhanced Bcl-xL mRNA expression (tD = 3.91 and 9.06, respectively, both p<0.01 by Dunnett’s t-test). Next we examine whether protein kinase CK2 and transcription factor NFκB were influenced by BDNF treatments. Statistical analyses showed that there was an overall significant effect of BDNF on CK2 activity (F3,17 = 23.3, p<0.01) (Fig. 1B). Further

analyses indicated that CK2 activity significantly enhanced by BDNF both at 0.4 μg and 1.2 μg (tD = 4.12 and 8.19, respectively, both p<0.01 by Dunnett’s t-test). The Ser529 phosphorylation on NF-κB was also found to increase significantly by BDNF treatment (F3,17 = 92.70, p<0.01) (Fig. 1C). Further analyses indicated that BDNF both

at 0.4 μg and 1.2 μg significantly enhanced Ser529 phosphorylation (tD = 6.37 and 15.90, respectively, both p<0.01 by Dunnett’s t-test). Fig. 1D is a representative illustration showing the needle placement and dye distribution in the CA1.

Effects of manipulation CK2α in CA1 neurons

The above results showed the correlations between CK2 activity, NF-κB phosphorylation and Bcl-xL mRNA expression by BDNF treatment. We further examined whether manipulations of CK2α indeed influences the NF-κB phosphorylation and Bcl-xL mRNA expression in CA1. The results from Fig. 2A revealed that Ser529 phosphorylation on NF-κB was significantly increased by wildtype CK2αWT DNA transfection (F2,22 = 69.91, p<0.01; tD = 8.12, p<0.01 by

Dunnett’s t-test) while decreased by mutant CK2α156A DNA transfection (tD = 4.71,

p<0.01). The expression of Bcl-xL mRNA was also significantly increased by

wildtype CK2αWT DNA transfection (F2,22 = 127.6, p<0.01; tD = 10.12, p<0.01 by

Dunnett’s t-test) while decreased by mutant CK2α156A DNA transfection (tD = 7.10,

p<0.01) (Fig. 2B). Immunohistochemistry staining against HA confirmed the

transfection and expression of CK2αWT in CA1 neurons (Fig.2C).

Effects of CK2 inhibitor TBB and BDNF on CA1 neurons

The results above do not reveal whether the CK2 signaling pathway contributes to the anti-apoptotic effect of BDNF on CA1 neurons. We addressed this issue in the present experiment. The concentration of CK2 inhibitor TBB used in the present study only slight altered CK2 basal activity in the CA1 (t1,7 = 1.78, p>0.05) (Fig. 3A).

(11)

phosphorylation on NFκB (F2,17 = 7.47, p<0.01; q = 5.46, p<0.01 by Newman-Keul’s

method), but this effect is significantly blocked by prior TBB treatment (q = 3.10,

p<0.05). BDNF also consistently increased Bcl-xL mRNA expression (F2,17 = 20.84,

p<0.01; q = 9.02, p<0.01 by Newman-Keul’s method), while prior administration of

TBB markedly antagonized this BDNF’s effect (q = 5.75, p<0.0) (Fig. 3C).

Interactive effects of mutant NFκBS529A with BDNF or CK2αWT on Bcl-xL expression in CA1 neurons

The above results showed that CK2 mediates the anti-apoptotic signaling of BDNF. We therefore examine whether this signaling pathway through the NF-κB molecule. In the study of mutant NFκBS529A transfection with BDNF, results revealed that BDNF markedly increased the Bcl-xL mRNA expression (F2,11 = 26.58,

p<0.01; q = 10.25, p<0.01 by Newman-Keul’s method), while the NFκBS529A

transfection significantly antagonized this effect of BDNF (q = 6.11, p<0.01) (Fig. 4A). In analyzing the effects of co-transfection of CK2αWT and NFκBS529A, the expression of Bcl-xL mRNA was shown to increase significantly by CK2αWT transfection (F2,12 = 7.79, p<0.01; q = 5.12, p<0.05 by Newman-Keul’s method),

whereas NFκBS529A transfection blocked the effect of CK2αWT (q = 4.58, p<0.05) (Fig. 4B).

(12)

Discussion

In the present study, we have shown that BDNF increased the Bcl-xL gene expression correlative with CK2 activity and NF-κB phosphorylation in the hippocampal CA1 in a dose-dependent manner. The MAPK/ERK or PI-3K/Akt signaling pathways have been indicated to mediate the neuroprotective effects of BDNF (Han et al., 2000; Chang et al., 2004; Rossler et al., 2004; Almeida et al., 2005). CK2 was revealed to be regulated by B-Raf/MAPK/ERK pathway possibly through PP1 or PP2A in hippocampal slices by BDNF combined with pharmacological drugs treatment (Blanquet, 2000). However, a previous study had shown that CK2 directly phosphorylates PP2A and up-regulates its activity (Heriche et al., 1997). Further, a recent study also identified that CK2 contributes in kinase suppressor of Ras (KSR1) complex by functioning as Raf N-region serine kinase (Ritt et al., 2007). Disruption of the KSR1/CK2 interaction or inhibition of CK2 activity significantly reduces the growth-factor-induced phosphorylation of B-Raf and correlates with impaired Raf, MEK, and ERK activation. In addition, Ck2 was found to partially involve in PI-3K/Akt signaling pathways by directly phosphorylating Akt on ser473 (Di Maira et al., 2005). It seems that CK2 might play a cross-talk role between different signaling pathways and mediate an alternative signaling pathway for BDNF (see the below discussion about NF-κB).

The transcription factor NF-κB is indicated to participate in the anti-apoptotic mechanisms in many cell types (Burstein and Duckett, 2003; Janssens and Tschopp, 2006). After activation, the phosphorylated NF-κB will translocate into the nucleus and bind to the cognate DNA sequences present in gene promoters. The decrease in NF-κB/RelA phosphorylation by PI-3K or MAPK/ERK inhibitor was revealed to increase the cell death in HTLV-1-transformed cells or pancreatic cancer cell line (Jeong et al., 2005; Rengifo-Cam et al., 2007). The inhibition of CK2 phosphorylation on NF-κB/RelA by chemical compound P3-25 was also shown to increase the

sensitivity of apoptosis in constitutive NF-κB-expressing cells (Manna et al., 2007b). Further, the NF-κB/RelA is indicated to be phosphorylated on Ser529 by CK2 and can be responsible for increased NOSII gene transcription (Wang et al., 2000; Chantome et al., 2004). Our present studies also found that transfection of wildtype CK2α increased, whereas mutant CK2α156A decreased, the Ser529 phosphorylation on NF-κB. It is though that NF-κB plays a key role in the protection of cells against apoptosis possibly through the activation of the anti-apoptotic genes. In addition, two functional NF-κB DNA binding sites are found to cluster in the promoter region of murine bcl-x (Glasgow et al., 2001). In the previous studies, the inhibition of NF-κB was found to reverse the enhance effects of Inhibitor of apoptosis protein hRFI or

(13)

platelet activating factor on the Bcl-2 and Bcl-xL mRNA levels (Konishi et al., 2006; Heon Seo et al., 2006). These above studies can support our present finding that the over-expression of mutant NFκBS529A would antagonize the increase effect on Bcl-xL mRNA either by BDNF treatment or by CK2αWT transfection. The present findings also suggest that NF-κB might exist in an alternative signaling pathway for the neuroprotective mechanism of BDNF. The classical pathway for NF-κB activation requires the presence of IKKβ and NEMO and leads to phosphorylation of IκBα, liberating an active NF-κB heterodimer (Janssens and Tschopp, 2006). The present finding from us and the results of Chantome et al. (2004) provide the evidence that NF-κB can be directly phosphorylated by CK2 to trigger the gene expression.

The neuroprotective effects of BDNF are well characterized in many studies (Schulte-Herbrüggen et al., 2007). The up-regulation of anti-apoptotic Bcl-2 family members is one of the actions for BDNF to promote the cell survival. Among those studies, BDNF was found to increase the Bcl-xL protein level under excitotoxicity- or ischemia-induced injury, which could counter the translocation of apoptotic protein Bax and Bad (Almeida et al., 2005; Perez-Navarro et al., 2005; Miyawaki et al., 2008). However, the increase degree of Bcl-xL protein in those studies is not enormous as that of mRNA level in our present study. This diversity might involve in the post-transcriptional regulation for this protein. Recently, the study about the repeated unpredictable stress has shown that Bcl-xL mRNA, not Bcl-2 mRNA, was significantly decreased in hippocampal subfields (Kosten et al., 2008). Our study also found that the increase degree in Bcl-2 mRNA (~1.4 folds) was not as enormous as that of Bcl-xL by BDNF treatment (unpublished data). How to increase the Bcl-xL protein level by BDNF might be one of the considerations to improve its potential therapeutic effects.

In summary, our results suggest that CK2 might mediate an alternative signaling pathway for BDNF through directly phosphorylating NF-κB to increase the anti-apoptotic Bcl-xL gene expression. This sheds light on new therapeutic strategies for BDNF application.

Acknowledgement: This work was supported by a Grant (NSC 96-2320-B-004 -001 -MY2) from the National Science Council of Taiwan and by Research Fund from IBMS, Academia Sinica, Taiwan.

(14)

References

Abdallah B, Hassan A, Benoist C, Goula D, Behr JP, Demeneix BA (1996) A powerful onviral vector for in vivo gene transfer into the adult mammalian brain: polyethylenimine. Hum Gene Ther 7: 1947-1954.

Aksenova MV, Burbaeva GS, Kandror KV, Kapkov DV, Stepanov AS (1991) The decreased level of casein kinase-2 in brain cortex of schizophrenic and Alzheimer’s disease patients. FEBS Lett 279: 55-57.

Almeida RD, Manadas BJ, Melo CV, Gomes JR, Mendes CS, Grãos MM, Carvalho RF, Carvalho AP, Duarte CB (2005) Neuroprotection by BDNF against glutamate-induced apoptotic cell death is mediated by ERK and PI3-kinase pathways. Cell Death Differ 12: 1329-1343.

Blanquet PR. (2000) Identification of two persistently activated neurotrophin- regulated pathways in rat hippocampus. Neuroscience 95: 705-719.

Blanquet PR (2002) Casein kinase 2 as a potentially important enzyme in the nervous system. Prog Neurobiol 60: 211-246.

Burstein E, Duckett CS (2003) Dying for NF-kappaB? Control of cell death by transcriptional regulation of the apoptotic machinery. Curr Opin Cell Biol. 15: 732-737.

Chang SH, Poser S, Xia Z (2004) A novel role for serum response factor in neuronal survival. J Neurosci 24: 2277-2285.

Chantome A, Pance A, Gauthier N, Vandroux D, Chenu J, Solary E, Jeannin JF, Reveneau S (2004) Casein kinase II mediated phosphorylation of NF-kappaB p65 subunit enhances inducible nitric-oxide synthase gene transcription in vivo. J Biol Chem 279: 23953–23960.

Chao CC, Chiang CH, Ma YL, Lee EH (2006) Molecular mechanism of the neurotrophic effect of GDNF on DA neurons: role of protein kinase CK2. Neurobiol Aging 27: 105-118.

Chao CC, Ma YL, Lee EH (2007) Protein kinase CK2 impairs spatial memory formation through differential cross talk with PI-3 kinase signaling: activation of Akt and inactivation of SGK1. J Neurosci 27: 6243-6248.

Charriaut-Marlangue C, Otani S, Creuzet C, Ben-Ari Y, Loeb J (1991) Rapid activation of hippocampal casein kinase 2 during long-term potentiation. Proc Natl Acad Sci USA 88: 10232-10236.

Chung HJ, Huang YH, Lau LF, Huganir RL (2004) Regulation of the NMDA receptor complex and trafficking by activity-dependent phosphorylation of the NR2B subunit PDZ ligand. J Neurosci 24: 10248-10259.

(15)

Desagher S, Osen-Sand A, Montessuit S, Magnenat E, Vilbois F, Hochmann A, Journot L, Antonsson B, Martinou JC (2001) Phosphorylation of bid by casein kinases I and II regulates its cleavage by caspase 8. Mol Cell 8: 601-611.

Di Maira G, Salvi M, Arrigoni G, Marin O, Sarno S, Brustolon F, Pinna LA, Ruzzene M (2005) Protein kinase CK2 phosphorylates and upregulates Akt/PKB. Cell Death Differ 12: 668-677.

Dunckley T, Beach TG, Ramsey KE, Grover A, Mastroeni D, Walker DG, LaFleur BJ, Coon KD, Brown KM, Caselli R, Kukull W, Higdon R, McKeel D, Morris JC, Hulette C, Schmechel D, Reiman EM, Rogers J, Stephan DA (2006) Gene expression correlates of neurofibrillary tangles in Alzheimer's disease. Neurobiol Aging 27: 1359-1371.

Glasgow JN, Qiu J, Rassin D, Grafe M, Wood T, Perez-Pol JR (2001) Transcriptional regulation of the BCL-X gene by NF-kappaB is an element of hypoxic responses in the rat brain. Neurochem Res 26: 647-659.

Han BH, Holtzman DM (2000) BDNF protects the neonatal brain from hypoxic-ischemic injury in vivo via the ERK pathway. J Neurosci 20, 5775-5781. Heon Seo K, Ko HM, Kim HA, Choi JH, Jun Park S, Kim KJ, Lee HK, Im SY (2006)

Platelet-activating factor induces up-regulation of antiapoptotic factors in a melanoma cell line through nuclear factor-kappaB activation. Cancer Res 66: 4681-4686.

Heriche JK, Lebrin F, Rabilloud T, Leroy D, Chambaz EM, Goldberg Y (1997) Regulation of protein phosphatase 2A by direct interaction with casein kinase 2alpha. Science 276: 952-955.

Izeradjene K, Douglas L, Delaney A, Houghton JA (2005) Casein kinase II (CK2) enhances death-inducing signaling complex (DISC) activity in TRAIL-induced apoptosis in human colon carcinoma cell lines. Oncogene 24: 2050-2558.

Janssens S, Tschopp J (2006) Signals from within: the DNA-damage-induced NF-kappaB response. Cell Death Differ 13: 773-784.

Jeong SJ, Pise-Masison CA, Radonovich MF, Park HU, Brady JN (2005) Activated AKT regulates NF-kappaB activation, p53 inhibition and cell survival in HTLV-1-transformed cells. Oncogene 24: 6719-6728.

Konishi T, Sasaki S, Watanabe T, Kitayama J, Nagawa H (2006) Overexpression of hRFI inhibits 5-fluorouracil-induced apoptosis in colorectal cancer cells via activation of NF-kappaB and upregulation of BCL-2 and BCL-XL. Oncogene 25: 3160-3169.

Korshunova I, Novitskaya V, Kiryushko D, Pedersen N, Kolkova K, Kropotova E, Mosevitsky M, Rayko M, Morrow JS, Ginzburg I, Berezin V, Bock E (2007)

(16)

GAP-43 regulates NCAM-180-mediated neurite outgrowth. J Neurochem 100: 1599-1612.

Kosten TA, Galloway MP, Duman RS, Russell DS, D'Sa C (2008) Repeated unpredictable stress and antidepressants differentially regulate expression of the bcl-2 family of apoptotic genes in rat cortical, hippocampal, and limbic brain structures. Neuropsychopharmacology 33: 1545-1558.

Krippner-Heidenreich A, Talanian RV, Sekul R, Kraft R, Thole H, Ottleben H, Luscher B (2001) Targeting of the transcription factor Max during apoptosis: phosphorylation-regulated cleavage by caspase-5 at an unusual glutamic acid residue in position P1. Biochem J 358: 705-715.

Li PF, Li J, Muller EC, Otto A, Dietz R, von Harsdorf R (2002) Phosphorylation by protein kinase CK2: a signaling switch for the caspase-inhibiting protein ARC. Mol Cell 10: 247-258.

Litchfield DW (2003) Protein kinase CK2: structure, regulation and role in cellular decisions of life and death. Biochem J 369: 1-15.

Manna SK, Aggarwal RS, Sethi G, Aggarwal BB, Ramesh GT. (2007a) Morin (3,5,7,2',4'-Pentahydroxyflavone) abolishes nuclear factor-kappaB activation induced by various carcinogens and inflammatory stimuli, leading to suppression of nuclear factor-kappaB-regulated gene expression and up-regulation of apoptosis. Clin Cancer Res 13: 2290-2297.

Manna SK, Manna P, Sarkar A (2007b) Inhibition of RelA phosphorylation sensitizes apoptosis in constitutive NF-kappaB-expressing and chemoresistant cells. Cell Death Differ 14: 158-1570.

Meggio F, Pinna LA (2003) One-thousand-and one-substrates of protein kinase CK2. FASEB J 17: 349-368.

Miyawaki T, Mashiko T, Ofengeim D, Flannery RJ, Noh KM, Fujisawa S, Bonanni L, Bennett MV, Zukin RS, Jonas EA (2008) Ischemic preconditioning blocks BAD translocation, Bcl-xL cleavage, and large channel activity in mitochondria of postischemic hippocampal neurons. Proc Natl Acad Sci USA 105: 4892-4897.

Nakajo S, Hagiwara T, Nakaya K, Nakamura Y (1986) Tissue distribution of casein kinases. Biochem Int 13: 701-707.

Paulson L, Martin P, Persson A, Nilsson CL, Ljung E, Westman-Brinkmalm A, Eriksson PS, Blennow K, Davidsson (2003) Comparative genome- and proteome analysis of cerebral cortex from MK-801-treated rats. J Neurosci Res 71: 526-533. Perez-Navarro E, Gavaldà N, Gratacòs E, Alberch J (2007) Brain-derived

neurotrophic factor prevents changes in Bcl-2 family members and caspase-3 activation induced by excitotoxicity in the striatum. J Neurochem 92: 678-691.

(17)

Pinna LA, Meggio F (1997) Protein kinase CK2 ("casein kinase-2") and its implication in cell division and proliferation. Prog Cell Cycle Res 3: 77-97.

Rengifo-Cam W, Umar S, Sarkar S, Singh P (2007) Antiapoptotic effects of progastrin on pancreatic cancer cells are mediated by sustained activation of nuclear factor-{kappa}B. Cancer Res 67: 7266-7274.

Ritt DA, Zhou M, Conrads TP, Veenstra TD, Copeland TD, Morrison DK (2007) CK2 is a component of the KSR1 scaffold complex that contributes to Raf kinase activation. Curr Biol 17: 179-184.

Rossler OG, Giehl KM, Thiel G (2004) Neuroprotection of immortalized hippocampal neurones by brain-derived neurotrophic factor and Raf-1 protein kinase: role of extracellular signal-regulated protein kinase and phosphatidylinositol 3-kinase. J Neurochem 88: 1240-1252.

Ruzzene M, Penzo D, Pinna LA (2002) Protein kinase CK2 inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB) induces apoptosis and caspase-dependent degradation of haematopoietic lineage cell-specific protein 1 (HS1) in Jurkat cells. Biochem J 364: 41-47.

Schulte-Herbruggen O, Braun A, Rochlitzer S, Jockers-Scherübl MC, Hellweg R (2007) Neurotrophic factors -- a tool for therapeutic strategies in neurological, neuropsychiatric and neuroimmunological diseases? Curr Med Chem 14: 2318-2329.

Takada Y, Murakami A, Aggarwal BB. (2005) Zerumbone abolishes NF-kappaB and IkappaBalpha kinase activation leading to suppression of antiapoptotic and metastatic gene expression, upregulation of apoptosis, and downregulation of invasion. Oncogene 24: 6957-6969.

Tapia JC, Torres VA, Rodriguez DA, Leyton L, Quest AF (2006) Casein kinase 2 (CK2) increases survivin expression via enhanced beta-catenin-T cell factor/lymphoid enhancer binding factor-dependent transcription. Proc Natl Acad Sci USA 103: 15079-15084.

Toruner M, Fernandez-Zapico M, Sha JJ, Pham L, Urrutia R, Egan LJ (2006) Antianoikis effect of nuclear factor-kappaB through up-regulated expression of osteoprotegerin, BCL-2, and IAP-1. J Biol Chem 281: 8686-8696.

Wang D, Westerheide SD, Hanson JL, Baldwin AS Jr (2000) Tumor necrosis factor alpha-induced phosphorylation of RelA/p65 on Ser529 is controlled by casein kinase II. J Biol Chem 275: 32592-32597.

(18)

Figure Legends

Fig. 1 BDNF treatments increased the Bcl-xL expression, CK2 enzyme activity and NFκB phosphorylation. BDNF dose-dependently increased (A) Bcl-xL mRNA expression, (B) CK2 enzyme activity and (C) phosphorylation of Ser529 on NFκB in the CA1. Animals received bilateral infusions of 0.2, 0.4, or 1.2 μg BDNF in the CA1 and were sacrificed 4 hr later. Data are expressed as mean±SEM. n=4-6 each group. Statistical significance was evaluated by one-way analysis of variance (ANOVA) followed by Dunnett’s t-test. * p<0.05, ** p<0.01. (D) A representative illustration showed the needle placement and dye distribution (0.3% methylene blue) in the CA1.

Fig. 2 Effects of CK2α DNA transfection on NFκB phosphorylation and Bcl-xL expression. CK2αWT DNA transfection increased whereas CK2α156A mutant DNA transfection decreased (A) phosphorylation of Ser529 on NFκB and (B) Bcl-xL mRNA expression in the CA1. Animals separately received different plasmid DNA transfection in the CA1 and were sacrificed 2 days later. Data are expressed as mean±SEM. n=8-10 each group. Statistical significance was evaluated by one-way analysis of variance (ANOVA) followed by Dunnett’s t-test, ** p<0.01. (C) Immunohistochemical staining showing CK2αWT DNA transfection to the CA1. Anti-HA tag antibody and FITC-conjugated IgG secondary antibody were used. Arrow indicates the area of transfection, scale bar equals 300 μm.

Fig. 3 CK2 inhibitor TBB blocked the effects of BDNF on NFκB phosphorylation and Bcl-xL expression. (A) Basal CK2 activity was not significantly altered by CK2 inhibitor TBB treatment alone. BDNF treatments increased (B) phosphorylation of Ser529 on NFκB and (C) Bcl-xL mRNA expression but these effects were antagonized by TBB. Animals received bilateral infusions of 10 ng TBB (or PBS) followed by 0.4 μg BDNF in the CA1 and were sacrificed 4 hr after the last infusion. The interval between two infusion was 30 min. Data are expressed as mean±SEM. n=6-7 each group. Statistical significance was evaluated by one-way analysis of variance (ANOVA) followed by Newman-Keul’s method. * p<0.05, ** p<0.01; a: compared with the PBS+PBS group, b: compared with the PBS+BDNF group.

Fig. 4 Transfection of mutant NFκBS529A antagonized the effects of BDNF or CK2αWT on Bcl-xL expression. (A) The Bcl-xL mRNA expression in the CA1 was increased by BDNF infusion, but this effect was blocked by mutant NFκBS529A Transfection. Animals received bilateral infusion of 0.4 μg BDNF following 48 hr after 1 μg of NFκBS529A DNA transfection in the CA1 and were sacrificed 4 hr after

(19)

BDNF infusion. (B) The Bcl-xL mRNA expression in the CA1 was increased by CK2αWT DNA transfection, but this effect was also antagonized by mutant NFκBS529A Transfection. Animals bilaterally received NFκBS529A DNA transfection followed by CK2αWT DNA transfection (1 μg each) in the CA1 and were sacrificed 48 hr later. The interval between two transfections was 1 hr. Data are expressed as mean±SEM. n=4-5 each group. Statistical significance was evaluated by one-way analysis of variance (ANOVA) followed by Newman-Keul’s method. *

p<0.05, ** p<0.01; a: compared with the corresponding control group, b: compared

with the (A) BDNF or (B) CK2αWT group.

(20)

0 100 200 400 600 800 1000 1200 PBS 0.2 μg 0.4 μg 1.2 μg Ratio of Bcl-xL/HPRT (% of c o ntrol )

∗∗

(A) 0 50 100 150 200 250 CK2 enzyme activity (% of c o ntro l)

∗∗

PBS 0.2 μg 0.4 μg 1.2 μg (B) (C) (D)

Fig 1

0 50 100 150 200 250 300 Ratio of pNF κB/ NF κB (% of c o ntrol ) PBS 0.2 μg 0.4 μg 1.2 μg

∗∗

0.2 μg PB S pS529-NFκB β-actin NFκB 0.4 μg 1.2 μg

(21)

0 20 40 60 80 100 120 140 160 180 Ratio of pNF κB/ NF κB (% of c o ntro l) pcDNA3 CK2αWT CK2α156A

∗∗

Ratio of Bcl-xL/HPRT (% of c o ntrol ) 0 25 50 75 100 125 150 175 200

∗∗

pcDNA3 CK2αWT CK2α156A (A) (B) (C)

Fig 2

CK 2α-W T PcD NA3 pS529-NFκB β-actin NFκB CK 2α-1 56A

(22)

0 20 40 60 80 100 120 PBS TBB CK2 enzyme activity (% of c o ntrol ) 0 25 50 75 100 125 150 175 200 PBS + PBS PBS + BDNF TBB + BDNF Ratio of pNF κB/ NF κB (% of c o ntrol )

∗∗

a

∗

b

Fig 3

(A) (B) (C) 50 100 150 200 250 300 350 400 Ratio of Bcl-xL/HPRT (% of c o ntrol )

∗∗

a 0

∗∗

b PBS + PBS PBS + BDNF TBB + BDNF PBS +P BS pS529-NFκB β-actin NFκB PBS +B DN F TBB +B DNF

(23)

0 50 100 150 200 250 300 350 400 pCMV + PBS pCMV + BDNF NFκBS529A + BDNF Ratio of Bcl-xL/HPRT (% of c o ntrol )

∗∗

a

∗∗

b 0 25 50 75 100 125 150 175 200 pCMV + pcDNA3 pCMV + CK2αWT NFκBS529A + CK2αWT Ratio of Bcl-xL/HPRT (% of c o ntrol )