Inhibition of the NF-jB transcriptional activity by protein kinase A Naoko Takahashi, Toshifumi Tetsuka, Hiroaki Uranishi and Takashi Okamoto Department of Molecular Genetics, Nagoya City University Medical School, Japan The cAMP-dependent protein kinase (PKA) signaling pathway plays a major role in a number of pathophysio- logical conditions. However, there have been conflicting evidences regarding the action of cAMP/PKA on nuclear factor- jB(NF-jB). In this study, we have explored the effect of cAMP/PKA on NF-jB activity and determined its molecular mechanism. PKA activating agents or expression of the catalytic subunit of PKA (PKAc) inhibited the NF-jB- dependent reporter gene expression induced by tumor nec- rosis factor a (TNFa). PKA activators affected neither IjBa phosphorylation, IjBa degradation, nor the NF-jB/DNA binding. Expression of PKAc inhibited the transactivation potential of Gal4-p65 (286–551) suggesting that the inhibi- tory action of PKA is through the C-terminal transactivation domain of p65 but not by phosphorylation of the consensus PKA recognition site containing serine at position 276. Overexpression of coactivators, CBP (CREB-binding pro- tein) and p300, failed to reverse the PKA-mediated inhibition of p65 transactivation. Thus, the inhibitory action of the cAMP/PKA pathway on the transcriptional activity of NF-jB appears to be exhibited by modifying the C-terminal transactivation domain of p65, either directly or indirectly. Keywords:NF-jB; PKA; cAMP; signal transduction. Nuclear factor jB (NF-jB) is an inducible cellular tran- scription factor that regulates a wide variety of cellular and viral genes including several cytokines, cell adhesion mole- cules and human immunodeficiency virus (HIV) [1–4]. Members of the NF-jB family in mammalian cells include the proto-oncogene c-Rel, Rel A (p65), Rel B, NF-kB1 (p50/105), and NF-kB2 (p52/p100) 1 . These proteins share a conserved 300 amino acids region known as the Rel homology domain, which is responsible for DNA binding, dimerization, and nuclear translocation [1–4]. In most cells, Rel family members form hetero- and homo-dimers with distinct specificities in various combinations. A common feature of the regulation of the NF-jB family is their sequestration in the cytoplasm as inactive complexes with a class of inhibitory molecules known as IjBs [1–4]. Treat- ment of cells with a variety of inducers such as phorbol ester, interleukin-1b (IL-1b) and tumor necrosis factor a (TNFa) results in phosphorylation, ubiquitination and degradation of the IjB proteins [3,5,6]. The degradation of IjB proteins exposes the nuclear localization sequence in the remaining NF-jB dimers, leading to nuclear translocation and subse- quent binding of NF-jB to the DNA cis-regulatory element of target genes [1–4]. In addition to the nuclear translocation and DNA-binding of NF-jB, its transcriptional activity is regulated by coactivators, CBP (CREB-binding protein) and p300, that associate with the C-terminal transactivation domain of p65. It has been demonstrated that these coactivators physically interact with p65 and promote its transcriptional activity [7,8]. On the other hand, elevation of intracellular cAMP induces the expression of numerous genes through the protein kinase A (PKA)-mediated phosphorylation of transcription factors, including the cAMP response ele- ment binding protein (CREB) [9]. Interestingly, cAMP inhibits the induction of a distinct set of NF-jB regulated genes. For example, cAMP inhibits IL-2 expression in activated T-lymphocytes. In monocytes, pharmacological agents that elevate intracellular cAMP, such as pentoxif- ylline, iloprost, dibutyryl cAMP (db-cAMP), forskolin (FSK), and isomethylxanthine, inhibit the TNFa produc- tion and tissue factor expression that are also under the control of NF-jB [10–14]. In endothelial cells, elevation of cAMP inhibits the induction of E-selectin and VCAM-1 mediated by IL-1b or TNFa [15,16]. Thus, elevation of intracellular cAMP and/or activation of PKA appear to modulate the transcriptional activity of NF-jBina negative way. In this report, we examined whether cAMP and PKA modulates NF-jB activity and found that the cAMP/PKA signaling pathway inhibits the NF-jB activity by acting on the transactivation domain of p65. Possible mechanisms for this inhibitory action are discussed. EXPERIMENTAL PROCEDURES Reagents Forskolin (FSK), 8-bromo-cAMP (Br-cAMP), dibutyryl cAMP (db-cAMP) were purchased from Wako (Osaka, Japan). Recombinant human TNFa and recombinant human IL-1b were purchased from Roche (Tokyo, Japan). Correspondence to T. Okamoto, Department of Molecular Genetics, Nagoya City University Medical School, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan. Fax: + 81 52 859 1235, Tel.: + 81 52 853 8205, E-mail: tokamoto@med.nagoya-cu.ac.jp Abbreviations:NF-jB,nuclearfactorjB; PKA, cAMP-dependent protein kinase; PKAc, catalytic subunit of PKA; FSK, forskolin; db-cAMP, dibutyryl cAMP; Br-cAMP, 8-Bromo-cAMP; CREB, cAMP response element binding protein; CBP, CREB binding pro- tein; TNFa, tumor necrosis factor a;IL-1b, interleukin-1b;C/EPBb, CCAAT/enhancer-binding protein b. (Received 20 May 2002, revised 9 July 2002, accepted 29 July 2002) Eur. J. Biochem. 269, 4559–4565 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03157.x Cell cultures Jurkat T-lymphocytes were maintained in RPMI1640 medium supplemented with 10% fetal bovine serum. HeLa and human embryonic kidney 293 cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. Primary culture of rat mesangial cells were prepared as described previously [17], and maintained in RPMI1640 medium supplemented with 15% fetal bovine serum and 0.15 IUÆmL )1 insulin. Plasmids Mammalian expression vectors, pCMV-p300, pRSV-CBP, and pRSV-CREB were generous gifts from Drs David Livingston (Dana-Farber Cancer Institute), Shunsuke Ishii (Riken) and Masatoshi Hagiwara (Tokyo Medical and Dental University), respectively. The constructions of luciferase reporter 4 jB-luc, pCMV-p65, pGal4-p65 (1–551), and pGal4-p65 (286–551) expression plasmids were described previously [18]. The pGal4-CREB (1–280), pCRE-luc containing four tandem repeats of the CREB- binding sites, pGal4-luc containing five tandem repeats of Gal4-binding sites, and the mammalian expression vector pCMV-PKAc encoding the PKA catalytic subunit were obtained from Stratagene (La Jolla, CA). Transfection conditions and luciferase assay Transient transfection of Jurkat T-lymphocytes was accomplished by using 5 lL of SuperFect transfection reagent (Qiagen) per ml of culture medium and a total of 1 lg of plasmid DNA. Blank control plasmids pcDNA3.1(–) and pUC19 were used to equalize the amount of DNA for each transfection. SuperFect-DNA complexes were allowed to form for 10 min at room temperature in serum-free medium before being added to the cells. Cells were incubated with the complex for 24 h, stimulated with 10 ngÆmL )1 of human recombinant TNFa for an additional 24 h and harvested. Gene expression was measured by luciferase activity as previously described [18]. Luciferase assays were performed and relative light units were determined with an AutoLumat LB9507 luminometer (EG & G Berthold, Japan). Transfection efficiency was monitored by Renilla luciferase activity with pRL-TK plasmid containing TK promoter as an internal control. All luciferase activities shown in transient transfection assays are corrected by the internal control activity of Renilla luciferase by pRL-TK. Electrophoretic mobility shift assay (EMSA) Jurkat T-lymphocytes were pretreated with FSK (50 l M )or vehicle (DMSO) for 30 min, and then stimulated with TNFa (10 ngÆmL )1 ) for 30 min. Nuclear extracts were prepared as previously described [19–21]. The double stranded oligonucleotide probe for NF-jB was synthesized and endlabeled by [c- 32 P]-ATP. The jB sequence was taken from the human immunodeficiency virus long-terminal repeat (HIV-LTR). The jB sequence used was forward (5¢-TTTCTAGGGACTTTCCGCCTGGGGACTTTCCAG-3¢) and complement (5¢-TTTCTGGAAAGTCCCCAGGCG GAAAGTC CCTAG-3¢). The EMSA was performed as previously described [19–21]. Nuclear extracts were incuba- ted in 10 lL EMSA buffer containing the radiolabeled jB oligonucleotide probe. The samples were analyzed by 6% nondenaturing PAGE. Western blot analysis Jurkat T-lymphocytes were pretreated with or without FSK (50 l M ) for 30 min and then stimulated with TNFa (10 ngÆmL )1 )orIL-1b (10 ngÆmL )1 ). After stimulation, cells were washed twice with ice-cold NaCl/Pi and lysed in TOTEX buffer (20 m M Hepes/KOH, pH 7.9, 350 m M NaCl, 20% glycerol, 1% NP-40, 1 m M MgCl 2 ,0.5m M EDTA, 0.1 m M EGTA, 1 m M dithiothreitol, 20 m M 2-gly- cerophosphate, 0.1 m M Na 3 VO 4 ,1m M phenylmethane- sulfonyl fluoride, and 2.5 lgÆmL )1 each of aprotinin, leupeptin and pepstatin). Equal amounts of samples (20 lg of total protein) were subjected to 10% SDS/PAGE and transferred onto a nitrocellulose membrane (Hybond- C; Amersham Pharmacia Biotech). The membranes were probed with anti-(IjBa) serum, anti-(IjBa-P) serum (New England Bio Laboratories, Beverly, MA) and proteins were visualized by enhanced chemiluminescence (Amersham Life Science Inc., Cleveland, OH). RESULTS cAMP/PKA inhibits TNFa-induced NF-jB activation To examine the effect of cAMP/PKA on TNFa-induced NF-jB activity, Jurkat T-lymphocytes were treated with PKA activating agents, FSK, db-cAMP and Br-cAMP. Stimulation with these agents decreased TNFa-induced NF-jB reporter gene expression in a dose dependent manner (Fig. 1A–C). To test whether these inhibitory effects of PKA activating agents are not limited for 4 kB- luc, we examined the effects of FSK and db-cAMP on HIV- LTR-luc which contains two NF-jB binding sites. As shown in Fig. 1D, FSK and db-cAMP inhibited the TNFa- induced HIV-LTR activation. To further test whether this inhibitory effect of cAMP was mediated by PKA, we transfected the catalytic subunit of PKA (PKAc) into Jurkat and HeLa cells along with the jB-dependent luciferase reporter. Transfection of PKAc led to a dramatic decrease in the TNFa-induced expression from the jB-reporter gene in both cells (Fig. 1E and F). These data indicate that elevation of intracellular cAMP and subsequent activation of PKA lead to the inhibition of NF-jB activity. To further examine the effect of PKA on NF-jB activity, we cotransfected Jurkat T-lymphocytes and HeLa cells with plasmids expressing PKAc and p65 subunit of NF-jB,and measured the level of NF-jB-dependent luciferase gene expression. Transfection of p65 alone resulted in high levels of luciferase expression, whereas cotransfection of p65 with PKAc led to a dramatic decrease in the expression of the reporter gene in both cells (Fig. 2A and B) supporting an idea that PKA generally inhibits NF-jB-mediated tran- scription. One of the major nuclear PKA substrates is the transcription factor CREB (cAMP response element-bind- ing protein) which binds to the cAMP response element (CRE) in cAMP-inducible genes [9]. The phosphorylation of CREB by PKA results in increased transactivation. To examine whether the inhibitory effect of PKAc on 4560 N. Takahashi et al.(Eur. J. Biochem. 269) Ó FEBS 2002 transcription factor is specific for NF-jB, we cotransfected the reporter plasmid pCRE-luc with PKAc and measured CRE-dependent luciferase activity in Jurkat T-lymphocytes. As expected, overexpression of PKAc led to an increase in the CRE-dependent gene expression (Fig. 2C). PKA activation does not affect IjB degradation nor DNA-binding activity of NF-jB In the unstimulated cells, NF-jB is sequestrated in the cytoplasm as inactive complexes with IjBs, which are phosphorylated and degraded upon stimulation of cells with inducers of NF-jB,suchasTNFa and IL-1b [1–4]. As Neumann et al. indicated that elevation of cAMP reduces NF-jB activity, possibly by stabilizing IjBa [22], we investigated whether the activation of PKA could inhibit IjBa degradation. Jurkat T-lymphocytes were stimulated with TNFa (20 ngÆmL )1 ) in the presence or absence of FSK (50 l M ), and IjBa were detected by immunoblotting. As demonstrated in Fig. 3A, there was no difference in IjBa degradation in the presence or absence of FSK. Similar observation was obtained with HeLa cells (data not shown). In addition, in primary culture of rat glomerular mesangial cells, FSK did not affect the IL-1b-induced IjBa phospho- rylation and subsequent degradation (Fig. 3B and C). These data indicate that PKA activation by FSK does not affect TNFa-orIL-1b-induced IjBa degradation in most cells. To confirm that PKA activation does not modify DNA- binding activity of NF-jB, we performed EMSA using nuclear extract prepared from Jurkat cells. FSK did not inhibit TNFa-induced DNA-binding of NF-jB (Fig. 3D). Thus, it is likely that the inhibitory effect of PKA on NF-jB is not due to the inhibition of DNA-binding activity of NF-jB. PKA impairs transactivation potential of p65 p65 protein has a putative consensus PKA recognition site (RRPS) located approximately 25 amino acids N-terminal from the nuclear localizing signal (NLS) in the Rel homology domain [1–4]. (Fig. 4A). Zhong et al. reported that the phosphorylation of this serine residue (at 276 amino acid position) could modulate the transcriptional activity of p65 [23]. Thus we have examined whether the phosphory- lation of p65 at Ser276 is involved in the mechanism by which PKA inhibits NF-jB activation. In Fig. 4B, we examined the transcriptional activity of pGal4-p65 (1–551), containing the full-length p65, and pGal4-p65 (286–551), containing the transactivation domain of p65 but lacking the Rel homology domain and the RRPS site (Ser276) of p65, in response to PKAc overexpression. When we cotransfected Jurkat T-lymphocytes with pCMV-PKAc and either pGal4-p65 (1–551) or pGal4-p65 (286–551) and measured the Gal4-dependent luciferase reporter gene expression, PKAc inhibited the Gal4-dependent luciferase activity induced by either pGal4-p65 (1–551) or pGal4-p65 Fig. 1. The effect of PKA activating agents and PKAc on NF-jB-dependent transcription inducedbyTNFa. (A–C) Suppression of NF-jB-dependent gene expression (4 jB-luc) in Jurkat cells by cAMP elevating agents including FSK (A), db-cAMP (B), and Br-cAMP (C). (D) Inhibition of TNFa- induced HIV-LTR-luc activation in Jurkat cells by FSK and db-cAMP. (E and F) Inhi- bition of TNFa-induced NF-jB-dependent gene expression (4 jB-luc) in Jurkat (E) and HeLa cells (F). Cells were transfected with 100 ng of 4 jB-luc or HIV-LTR-luc reporter plasmid and/or 150 ng of pCMV-PKAc expression plasmid. After 24 h of transfection, cells were stimulated with 10 ngÆmL )1 of TNFa for 24 h in the presence or absence of the PKA activating agent. Cells were then harvested and subjected to luciferase assay. The data are presented as the fold-increase in luciferase activities relative to control trans- fection (no TNF stimulation). Values are the mean of two independent transfections (A–C) or the mean ± SE of three independent transfections (D–F). Experiments were repea- ted at least three times with the same results. Ó FEBS 2002 PKA inhibits NF-jB transactivation (Eur. J. Biochem. 269) 4561 (286–551) almost to a similar extent (Fig. 4B). These results excluded the possibility that the activation of PKA inhibits NF-jB by direct phosphorylation of the consensus PKA recognition site at Ser276 of p65 but suggested that PKA inhibits NF-jB by acting at the C-terminal transactivation domain of p65 subunit. These results also excluded the possibility that PKAc directly affected the NF-jB DNA binding. In contrast, PKAc augmented the transcriptional activity of Gal4-CREB (1–280) containing the CREB transactivation domain and the possible PKA-mediated phosphorylation site (Fig. 4C). Thus, the inhibitory effect of PKAc on transcription appeared to be specific for NF-jB subunit p65. CREB and coactivator CBP/p300 are not involved in the PKA-mediated inhibition of NF-jB To examine if CREB, a well-known nuclear substrate of PKA, is involved in the PKA-mediated inhibition of p65 activity, Jurkat cells were transfected with pRSV-CREB in addition to pCMV-p65 and 4 jB-luc (Fig. 5A). However, CREB did not affect the transcriptional activity of p65. As both CREB and p65 use coactivators CBP/p300 [7,8], the limiting amount of CBP/p300 might account for the PKA- mediated inhibition of p65 transactivation. To test this hypothesis, pRSV-CBP expressing CBP was cotransfected with pCMV-PKAc and pCMV-p65. However, overexpres- sion of CBP did not reverse the PKA-mediated inhibition of p65 transactivation (Fig. 5B). Similar results were obtained with p300 overexpression (data not shown). These obser- vations indicate that PKA-mediated inhibition of p65 transactivation is not likely through the sequestration of CBP/p300. DISCUSSION In this study we have observed reproducibly that the cAMP/ PKA signaling pathway inhibits transcriptional activity of NF-jB. Although a previous report by others [22] suggested that PKA inhibited NF-jB activation by stabilizing IjB, PKA activating agents such as FSK did not significantly affect the TNFa-induced IjBa degradation or DNA- binding activity of NF-jB (Fig. 3). These findings clearly indicated that PKA inhibits NF-jB activation at a step downstream of IjB degradation. Sequestration of CBP/ p300 coactivators by CREB is not likely because CBP/p300 overexpression did not abolish the inhibitory effect of PKA on NF-jB(Fig.5B). 2 In this study, we have demonstrated the evidence that the C-terminal transactivation domain of p65 is responsible for the inhibition of NF-jBmediatedby the cAMP/PKA signaling (Fig. 4). Fig. 2. Inhibition of NF-jB (p65)-dependent gene expression by PKAc. Jurkat (A) or HeLa (B) cells were transfected with 100 ng of 4 jB-luc, 50 ng of pCMV-p65 and/or 150 ng of pCMV-PKAc. (C) Activation of CREB-dependent gene expression by PKAc. Jurkat cells were trans- fected with 100 ng of pCRE-luc and various amounts of pCMV- PKAc. The data are presented as the fold-increase in luciferase activ- ities relative to control transfection. Values are the mean ± SE of three independent transfections. Fig. 3. Forskolin does not affect IjBa degradation or DNA-binding activity of NF-jB. (A) Jurkat cells were pretreated with 50 l M of FSK for 30 min, and then stimulated with 10 ngÆmL )1 of TNFa for indi- catedtimeperiods.CellswereharvestedandsubjectedtoWesternblot analysis. (B) The time course of IL-1b-induced IkBa degradation and phosphorylation in rat glomerular mesangial cells. Mesangial cells were stimulated with IL-1b (10 ngÆmL )1 ) for indicated time periods. Cells were harvested and subjected to Western blot analysis with anti- IjBa antibody or the antibody against phosphorylated IjBa at Ser32 (IjBa-P). (C) Effect of FSK on the IL-1b-induced IjBa degradation and phosphorylation in rat mesangial cells. Cells were pretreated with 50 l M ofFSKfor30min,andthenstimulatedwithIL-1b (10 ngÆmL )1 ) for 5 and 15 min. Cells were harvested and total cell lysates were subjected to Western blot analysis. (D) Effect of FSK on the NF-jB DNA-binding activity. Jurkat cells were pretreated with or without 50 l M FSK for 30 min, and then simulated with TNFa (10 ngÆmL )1 ) for 30 min. Cells were harvested and nuclear extracts were subjected for EMSA analysis with the jB probe. 4562 N. Takahashi et al.(Eur. J. Biochem. 269) Ó FEBS 2002 There have been conflicting evidences regarding the action of PKA on the NF-jB activity. Although the majority of reports [10–14] including this study, demonstra- ted the inhibitory actions of PKA on the NF-jB dependent gene expression, some reports such as Zhong et al. [23,24] showed that PKAc, associated with the cytosolic NF-jB/ IjB complex, enhanced NF-jB-dependent gene expres- sion by transient luciferase reporter assay. This marked difference may be explained by use of distinct reporter plasmids. It is noteworthy that several commonly used reporter plasmids contain CRE sites that mediate induction of reporter gene expression in cells stimulated with cAMP- increasing agents [25]. It is also possible that NF-jB- dependent gene expression may be enhanced or inhibited depending on the level of PKA enzyme activity. Induction of C/EBPb 3 (CCAAT/enhancer-binding pro- tein b) by cAMP/PKA might be involved in cAMP/PKA- mediated inhibition of NF-jB activity. In our preliminary experiments, cAMP elevating agents induced C/EBPb and formed a complex with NF-jB, and overexpression of C/EBPb inhibited the transactivation potential of p65, Gal4-p65 (1–551) and Gal4-p65 (286–551) (data not shown). A number of reports suggest a physiological role of C/EBPb in mediating the PKA signaling. For instance, PKA stimulates C/EBPb transcription in hepatocytes, neuronal cells and in fibroblasts [26]. In addition, C/EBPb is induced through PKA signaling pathway upon stimula- tion with human chorionic gonadotropin in rat granulosa Fig. 5. Effects of CREB and CBP on transcriptional activity of p65. (A) Effect of CREB on the p65-mediated transcription. Jurkat cells were transfected with 4 jB-luc reporter plasmid (100 ng), pCMV-p65 and/ or pRSV-CREBs. After 24 h of transfection, cell lysates were harves- ted and the luciferase assay was performed. (B) Effect of CBP over- expression on the PKAc-mediated inhibition of p65 activity. Jurkat cells were transfected with 4 jB-luc, pCMV-p65 (50 ng), pCMV- PKAc (150 ng), and various amounts of pRSV-CBP (50, 150, 500 ng). The relative luciferase activity as compared with control (4 jB-luc reporter plasmid alone) is shown. Values are the mean ± SE of three independent transfections. Fig. 4. Inhibition of NF-jB activity by PKAc through the C-terminal transactivation domain of p65. (A) Functional configuration of p65 protein. Location of rel-homology domain (RHD), putative PKA phosphorylation site (Ser276), and two transactivation domains (TA1 and TA2) are indicated. (B) Inhibition of p65 transcriptional activity by PKAc. In order to evaluate the effect of PKAc in Jurkat cells, either pGal4-p65 (1–551), encoding a fusion protein of Gal4 DNA-binding domain and the full-length p65 (amino acids 1–551), or pGal4-p65 (286–551), that lacking the RHD and the putative PKA phosphory- lation site was cotransfected with pCMV-PKAc and the gene expres- sion from the pGal4-luc reporter plasmid (100 ng) was assessed. (C) Activation of CREB activity by PKAc. Jurkat cells were transfected with Gal4-CREB (1–280), pCMV-PKAc, and pGal4-luc reporter plasmid. Data are presented as percentage inhibition (B) or percentage activation (C) relative to control transfection without pCMV-PKAc. Values are the means of two independent transfections. Similar results were obtained repeatedly. Ó FEBS 2002 PKA inhibits NF-jB transactivation (Eur. J. Biochem. 269) 4563 cells and in rat Leydig cells [27,28]. C/EBPb can physically interact with NF-jB [29], and form a repressor protein complex with NF-jB and estrogen receptor in the context of interleukin-6 promoter [30]. These findings collectively suggest that C/EBPb may mediate the inhibitory effects of cAMP/PKA on NF-jB activity by interacting with p65 at least in part. However, the effect of C/EBPb on NF-jB- dependent transcription may be context-dependent because in some promoters such as IL-6 and IL-8, in which NF-jB binding site and C/EBP binding site are very closely located, C/EBPb synergistically activates the NF-jB-dependent transcription [31]. Thus, further studies are needed to clarify theroleofC/EBPb in controlling the transcriptional activity of NF-jB. The C-terminal transactivation domain of p65 has been shown to be phosphorylated at Ser529 and Ser536 by various upstream stimuli [32–35]. 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Jang, M.K., Goo, Y.H., Sohn, Y.C., Kim, Y.S., Lee, S.K., Kang, H.,Cheong,J.&Lee,J.W.(2001)Ca 2+ /calmodulin-dependent protein kinase IV stimulates nuclear factor-kappa B transactiva- tion via phosphorylation of the p65 subunit. J. Biol. Chem. 276, 20005–20010. Ó FEBS 2002 PKA inhibits NF-jB transactivation (Eur. J. Biochem. 269) 4565 . Inhibition of the NF-jB transcriptional activity by protein kinase A Naoko Takahashi, Toshifumi Tetsuka, Hiroaki Uranishi and Takashi Okamoto Department. stimulates the transactivation potential of the RelA/ p65 subunit of NF-kappa B through utilization of the Ikappa B kinase and activation of the mitogen-activated