Regulation of Nuclear Factor kappa B Transactivation

IMPLICATION OF PHOSPHATIDYLINOSITOL 3-KINASE AND PROTEIN KINASE C zeta  IN c-Rel ACTIVATION BY TUMOR NECROSIS FACTOR alpha *

Angel G. Martin, Belén San-Antonio, and Manuel FresnoDagger

From the Centro de Biología Molecular "Severo Ochoa," Universidad Autónoma de Madrid, Cantoblanco, Madrid 28049, Spain

Received for publication, December 15, 2000


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Transactivation by c-Rel (nuclear factor kappa B) was dependent on phosphorylation of several serines in the transactivation domain, indicating that it is a phosphorylation-dependent Ser-rich domain. By Ser right-arrow Ala mutational and deletion analysis, we have identified two regions in this domain: 1) a C-terminal region (amino acids 540-588), which is required for basal activity; and 2) the 422-540 region, which responds to external stimuli as tumor necrosis factor (TNF) alpha  or phorbol myristate acetate plus ionomycin. Ser from 454 to 473 were shown to be required for TNFalpha -induced activation, whereas Ser between 492 and 519 were required for phorbol myristate acetate plus ionomycin activation. Phosphatidylinositol 3-kinase (PI3K) and protein kinase C (PKC) zeta  were identified as downstream signaling molecules of TNFalpha -activation of c-Rel transactivating activity. Interestingly, dominant negative forms of PI3K inhibited PKCzeta activation and dominant negative PKCzeta inhibited PI3K-mediated activation of c-Rel transactivating activity, indicating a cross-talk between both enzymes. We have identified the critical role of different Ser for PKCzeta - and PI3K-mediated responses. Interestingly, those c-Rel mutants not only did not respond to TNFalpha but also acted as dominant negative forms of nuclear factor kappa B activation.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Transcription factors belonging to the nuclear factor kappa B (NF-kappa B)1 family regulate several of the most important genes induced during T cell activation (for review, see Ref. 1). The NF-kappa B family of transcription factors is composed of homo- and heterodimers of a family of proteins, which include the Dorsal gene of Drosophila and the mammalian genes nfkappa b1, nfkappa b2, c-rel, relA (p65), and relB (for review, see Ref. 2). All members share a conserved 300-amino acid region in their N terminus that includes the dimerization, nuclear localization, and DNA binding regions. c-Rel, RelB, and RelA also have C-terminal transactivation domains, which strongly activate transcription from NF-kappa B sites. NF-kappa B is rapidly activated by the T cell receptor complex, but, at later phases of T cell activation, autocrine or paracrine secreted TNFalpha takes control of NF-kappa B activation (3). Tumor necrosis factor (TNF) alpha  is a pleiotropic cytokine with biological effects ranging from promoting growth and differentiation to induction of apoptosis. Those effects rely, at least in part, in the activation of the transcription factor NF-kappa B (for review, see Ref. 4). In T cells, the initial phase of NF-kappa B activation after T cell receptor triggering mainly relies on p65 translocation, whereas the later phase is controlled by c-Rel. We have previously shown that autocrine or paracrine TNFalpha secretion controls the c-Rel levels in T cells (3). Thus, c-Rel activation emerges as a key point for the later phase of T lymphocyte activation, a fact that is supported by the functional unresponsiveness of T lymphocytes from the c-Rel knock out mice (5, 6).

NF-kappa B activity is regulated, at least in part, by its subcellular localization. Thus, functional NF-kappa B complexes are held in the cytoplasm of resting T cells in an inactive state complexed with members of the Ikappa B family. In response to different activators, which include T cell receptor and TNFalpha , Ikappa B is phosphorylated by Ikappa B kinases (IKKs), and subsequently degraded, liberating the active NF-kappa B complex, which translocates to the nucleus and activates transcription (for review, see Ref. 7). Recently, a second level of regulation of NF-kappa B activity independent of Ikappa B, which relies in the activation of the transcriptional activity of p65, has been described (8-10). Thus, the catalytic subunit of protein kinase A was shown to be bound to inactive NF-kappa B complexes, and upon Ikappa B degradation this catalytic subunit phosphorylated p65, resulting in an enhanced transcription promoting activity (11). Moreover, TNFalpha treatment of cells results in phosphorylation of Ser529 in the transactivation domain of p65, resulting in the activation of the transcriptional activity of the protein (10). The small GTP-binding protein Ras enhanced p65/RelA transcriptional activity through a pathway that required the stress-activated protein kinase p38 or a related kinase (12), although it was not demonstrated whether this kinase was directly involved in activating NF-kappa B or instead a transcriptional co-activator. The activity of Ras as well as the atypical protein kinase C zeta  (PKCzeta ) has been also shown to be essential for the transcriptional activity of p65/RelA in endothelial cells (13). This activation relies in the phosphorylation of the N-terminal Rel homology domain and not on the C-terminal transactivation domain. PKCzeta was able to phosphorylate and activate IKK2 (14), thus demonstrating its direct implication in the NF-kappa B activation process by participating in Ikappa B degradation. A recently identified 62-kDa protein (named p62) might function as a bridge between PKCzeta and the TNF receptor-associated protein RIP (15). On the other hand, PI3K activity seems to be required for interleukin-1- and TNFalpha -induced NF-kappa B activity (16, 17). The PI3Ks are a family of lipid kinases that catalyze the addition of a phosphate group to the 3'-OH position of the inositol ring of phosphoinositides. The 3-phosphoinositides are second messengers that exert specific regulatory functions inside the cells (18). PI3K is composed of two different subunits, a regulatory subunit (p85) and a catalytic subunit, termed p110. Upon stimulation, p85 becomes associated to the cytosolic portion of tyrosine-phosphorylated receptors via its SH2 domains, which in turn promotes its association with the catalytic subunit p110 and its subsequent activation. The activation of PI3K triggers a signaling cascade that leads to the specific phosphorylation of p65/RelA subunit. This phosphorylation enhances p65-mediated transcription without affecting Ikappa B degradation, nuclear translocation of NF-kappa B, or the ability of NF-kappa B to bind to DNA (16).

Analysis of the transactivation domain of p65 by CD and NMR spectroscopy revealed no defined structure (19). Two differentiated acidic regions (termed TA1 and TA2) were identified as essential for its transcription promoting activity. Only TA2, however, was responsible for the activation by phorbol ester stimulation by a mechanism that involved phosphorylation of Ser residues (8). Moreover, the high Ser content of the transactivation domain of the avian c-Rel-related oncogene v-rel has been demonstrated to be essential for its transforming capabilities (20). Furthermore, the mutation of the Ser residue 471 in the human c-Rel transactivation domain abrogated TNFalpha -induced NF-kappa B activity in a Jurkat T cell clone (21). Taken together, all these works point to a key functional role of the regulation of the transactivation domain of c-Rel family proteins for NF-kappa B function.

In this work we have characterized the regulation of c-Rel transactivation domain. This domain seems to belong to the family of the phosphorylation-dependent Ser-rich acidic transactivation domains. We have revealed the critical role of several Ser residues for TNFalpha -dependent activation. Interestingly, mutations of those Ser residues not only abrogated c-Rel transactivating activity, but also acted as dominant negative forms of NF-kappa B activation, further stressing the importance of this regulation in the activity of NF-kappa B. Additionally, we have identified PI3K and PKCzeta as enzymes participating in the signaling route that leads to c-Rel activation by TNFalpha .

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cells and Reagents-- Jurkat cells and COS-7 cells were grown in RPMI 1640 medium (Life Technologies, Inc.) supplemented with 5% heat-inactivated fetal bovine serum (FBS; Life Technologies, Inc.) and containing 100 µg/ml streptomycin, 100 units/ml penicillin, 2 mM L-glutamine, plus nonessential amino acids, at 37 °C in a 7% CO2-in-air atmosphere saturated with water vapor incubator.

Recombinant human tumor necrosis factor alpha  (TNFalpha ) was purchased from Genzyme (Cambridge, MA). Phorbol myristate acetate (PMA), calcium ionophore A23187 and HA1004, and L-1-chloro-3-[4-tosylamido]-4-phenyl-2-butanone (TPCK) were purchased from Sigma. Cyclosporin A, cypermethrin, and LY294002 were purchased from Biomol (Plymouth Meeting, PA). The inhibitors wortmannin and D609 were obtained from Calbiochem (San Diego, CA). The inhibitor SB203580 was a kind gift of Dr. J. M. Redondo (Centro de Biologia Molecular Severo Ochoa, Madrid, Spain). The lipid phosphatidylinositol 3,4,5-trisphosphate (PIP3) was obtained from Alexis Biochemicals (San Diego, CA).

Sera from rabbits hyperimmunized with peptides derived from human c-Rel (no. 265), kindly provided by Dr. Nancy Rice (NCI-FCRDC, Frederick, MD) were used to detect the corresponding protein on Western blots, used at a dilution of 1:10,000. Monoclonal anti-epitope HA antibody used for immunoprecipitation studies was purchased from Roche Molecular Biochemicals (Mannheim, Germany).

Plasmids-- The pNF3TK Luc reporter plasmid contains a trimer of the NF-kappa B-binding motif of the H-2k gene upstream of the TK minimal promoter and the luciferase reporter gene (22). The reporter Gal4 Luc contains five tandem repeats of the Gal4 element upstream from the luciferase reporter gene (kindly provided by Dr. J. M. Redondo). Expression plasmids encoding either wild-type or dominant-negative mutant of PKCzeta were kindly provided by Dr. J. Moscat (Centro de Biologia Molecular Severo Ochoa, Madrid, Spain). Delta p85 expresses deletion mutant of p85 subunit of human PI3K enzyme incapable of binding to catalytic subunit p110, rendering a dominant-negative form of the enzyme (kindly provided by Dr. J. Downward, Imperial Cancer Research Fund, London, United Kingdom).

Gal4 c-Rel-(309-588) wild type was made by cloning the corresponding c-Rel PCR fragment into the XhoI-BglII site of the Gal4 c-Jun-(1-166) plasmid, thus removing the c-Jun fragment. The template for PCR reactions was pRc-hc-Rel, which consists of pRcCMV with c-Rel cDNA inserted in the HindIII-XbaI site. Gal4 DNA binding domain (DBD) fusions with different c-Rel transactivation domain deletion mutants were made using the same approach as Gal4 c-Rel-(309-588). The fragments fused to Gal4 DBD were:Gal4-(309-318), Gal4-(309-372), Gal4-(309-421), Gal4-(309-455), Gal4-(309-497), Gal4-(309-540), Gal4-(422-588), Gal4-(456-588), Gal4-(498-588), Gal4-(541-588), Gal4-(498-540), Gal4-(456-497), and Gal4-(422-455).

Substitutions Serright-arrowAla were made with the QuickChange site-directed mutagenesis kit (Stratagene), using as template for mutation the Gal4-c-Rel deletion mutant Delta 7-(422-588). Mutation was confirmed by sequencing in each case. Substitutions were as follows: A1 = Ser426; A2 = Ser443, Ser444, Ser447; A3 = Ser454; A4 = Ser460; A5 = Ser463; A6 = Ser470, Ser471, Ser473; A7 = Ser484; A8 = Ser491, Ser494; A9 = Ser508, Ser509, Ser510, Ser511, Ser513; A10 = Ser518; A11 = Ser525, Ser527; A12 = Ser533, Ser536; A13 = Ser541; A14 = Ser546, Ser549, Ser551; A15 = Ser563, Ser566; A16 = Ser577, Ser579. Similar substitutions were made using pRc-hc-Rel as template.

Cell Transfection-- Jurkat T cells or COS-7 cells were washed once and resuspended at 106 cells/ml in Opti-MEM (Life Technologies, Inc.). Cells were transfected with the LipofectAMINE Plus reagent (Life Technologies, Inc.) preparing the LipofectAMINE Plus-plasmid mixtures in accordance with the manufacturer's instructions. The mixtures were incubated at 37 °C in a 7% CO2 incubator for 3 h before washing with fresh Dulbecco's modified Eagle's medium + 5% FBS, and incubated for another 18 h. The cells were then washed once with, and resuspended at the same concentration in, Dulbecco's modified Eagle's medium + 5% FBS. Culture medium with or without stimuli, as indicated in text (10 ng/ml TNFalpha or PMA (10 ng/ml) + calcium ionophore (1 µM)) was added to duplicate wells containing 0.5 ml of these cell suspensions, which were then incubated under the same conditions for 6 h. The cells were lysed with Passive Cell Culture Lysis Reagent (Promega, Madison, WI) and microcentrifuged at full speed for 5 min at 4 °C, and 20 µl of each supernatant was used to determine firefly luciferase activity in a Monolight 2010 luminometer (Analytical Luminescence Laboratory). The results were expressed as -fold increase in luminescence relative to the value obtained with the non-stimulated control after normalization with respect to protein concentration, determined by the bicinchoninic acid spectrophotometric method (Pierce). For normalization of transfection efficiency, cells were co-transfected with the reporter plasmid pTK Renilla (Promega) and luciferase activity recorded using the Dual Luciferase assay (Promega). Results are always expressed as values normalized to Renilla activity.

Western Blots and Immunoprecipitation-- Whole cell extracts (WCE) were made using TNT buffer as lysis buffer (20 mM Tris-HCl, pH 7.6, 200 mM NaCl, 1% Triton X-100) supplemented with protease inhibitors (2 µg/ml aprotinin, 2 µg/ml pepstatin, 2 µg/ml leupeptin, 0.1 mM benzamidine, and 0.5 mM phenylmethylsulfonyl fluoride) and phosphatase inhibitors (5 mM NaF, 1 mM Na3VO). For immunoprecipitation, WCE were incubated for 30 min at 4 °C with 1 µg of anti-HA antibody. Precipitates were collected on protein A-Sepharose (Amersham Pharmacia Biotech, Uppsala, Sweden), separated in a 10% SDS-PAGE, and subsequently transferred to a polyvinylidene difluoride membrane. Membranes were analyzed by Western blot. For Western blot, WCE were separated on a 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Immobilon, Amersham Pharmacia Biotech). Rabbit anti-human c-Rel was used as first antibody, and goat anti-rabbit IgG peroxidase as secondary antibody. The enhanced chemiluminescent (ECL) developing kit (Amersham Pharmacia Biotech) was used to identify the relevant band(s).

Solid Phase in Vitro Phosphorylation Assay-- c-Rel transactivation domain constructs from position 422-588 or 422-540 (using as template pRc-hc-Rel wild type) were cloned into the BamHI-EcoRI site of plasmid pGEX2T (Amersham Pharmacia Biotech) in order to express recombinant GST-c-Rel fusion protein. These recombinant proteins were purified from E. coli induced cultures according to the manufacturer's instructions. 25 µl of GSH-agarose-GST-c-Rel were used as substrate of an in vitro phosphorylation reaction in which whole cell extracts from non-stimulated or stimulated Jurkat cells were assayed. WCE were made from 106 Jurkat cells in 25 µl, as described above. The reaction mixture (kinase buffer) contained 20 mM Hepes, pH 7.6, 20 mM MgCl2, 20 mM beta -glycerophosphate, 20 µM ATP, and 1 µCi of [32P]ATP (specific activity, 3,000 Ci/mol). After 20 min at 30 °C, the reaction was terminated by washing with TNT buffer. Phosphorylated protein was boiled in 25 µl of Laemmli sample buffer and resolved in 10% SDS-PAGE, followed by autoradiography. For PKCzeta phosphorylation assay, anti-HA precipitates from WCE from transfected COS-7 cells were incubated in the kinase buffer described before, containing 1 µg of soluble recombinant GST c-Rel-(422-588).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mapping of the c-Rel Transactivation Domain

Mouse c-Rel transactivation domain has been previously localized in the C-terminal region of the protein, between positions 403 and 568 (23). In order to delineate the transcriptionally active region of human c-Rel we constructed several fusion plasmids between the Gal4 DBD and c-Rel, providing a system where transcriptional activity of this protein could be assayed without interference of Ikappa B association and/or degradation. The fragment of c-Rel from position 309 to 588 was fused to Gal4 DBD, and several deletion mutants of this region were generated (Fig. 1). These constructs were transfected into Jurkat T cells along with a 5xGal4 Luc reporter plasmid and luciferase activity was recorded. As a negative control, a construction which covered only the c-Rel fragment from position 309 to 318 fused to Gal4 DBD was used.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 1.   Deletion mapping of the C terminus of c-Rel. Left panel, structure of c-Rel transactivation domain deletion mutants. RHD, Rel homology domain; NLS, nuclear localization signal. Right panel, effect of deletion mutants on the transcriptional activity of c-Rel fusions to Gal4 DBD. Jurkat T cells were transfected with each of the mutants described and with a reporter plasmid containing five tandem repeats of Gal4 site upstream from the luciferase gene. Results are expressed as percentage of activity compared with the wild-type construct. Transfection efficiency was normalized using the Dual Luciferase assay (Promega). Additionally, similar amounts of the different construction were expressed in transfected cells as detected by EMSA assays (data not shown). The results shown are the mean ± S.D. of three independent experiments.

Constructions spanning the c-Rel region from 309 to 421 had no basal transcriptional activity, as the control construction Gal4-(309-318) (Fig. 1, right). A minimal transcriptional activation was observed when Gal4-(309-455) was transfected. By contrast, Gal4-(422-588) induced an activity of the Gal4 reporter that was 236% of the wild-type fusion, indicating that this region possessed all the transcriptionally active sequences of c-Rel and even behaved as a better autonomous transactivation domain that the whole c-Rel C-terminal region (from position 309 to 588). Progressive deletions toward the C terminus were introduced in this region (Gal4-(456-588), Gal4-(498-588), and Gal4-(541-588)), which increasingly reduced the transcriptional activity. However, the smallest construction Gal4-(541-588) still evidenced a significant transcription promoting activity. That opened the possibility of the co-existence of several subdomains within region 422-588. To test this, smaller fragments covering this region were fused to Gal4 DBD (Gal4-(498-540), Gal4-(456-497), and Gal4-(422-455)). As shown in Fig. 1, none of them were transcriptionally active, indicating that region 422-588 behaved as a single transcription activation domain.

Identification of the Regions in c-Rel Transactivation Domain Activated by TNFalpha and PMA + Ionophore

We used the Gal4 DBD fusion plasmids described in the preceding section to map the region responsible for the transcriptional activation of c-Rel by TNFalpha and compared it with the activation produced by PMA + ionophore. Table I lists the TNFalpha and PMA + ionophore inducibility of the different constructions. The region responsive to activation mapped between positions 422 and 540. Although region 540-588 showed a strong basal transcription activity (Fig. 1), it did not respond to stimulation, suggesting that this C-terminal region was necessary for the basal transcriptional activity of c-Rel but was not involved in TNFalpha or PMA + ionophore stimulation.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Stimulation of the transactivating activity of Gal4 DBD-c-Rel wild-type and deletion mutant fusions

Analysis of c-Rel Region 422-588 by Ser right-arrow Ala Substitutions

The above results indicate the region 422-588 includes the transcription promoting region of c-Rel, whereas the region 422-540 was responsible for integrating signals derived from activation by TNFalpha and PMA + ionophore. This region contains 33 Ser residues (20%), 20 acidic residues (12%), and 8 Pro residues (5%), suggesting that it could be an acidic transcription activation domain, despite not having any significant homology to conventional acidic and Pro-rich transactivation domains (24). The existence of 20% Ser residues could confer it with the properties of a transcription activation domain regulated by phosphorylation. Those Ser residues are strongly conserved between the human and murine proteins, suggesting the relevance of those residues for function. In order to study their functional relevance, we introduced Ser right-arrow Ala mutations into the Gal4-c-Rel-(422-588) (Fig. 2A) fusion. We subsequently assayed for the basal and the TNFalpha - or PMA/ionophore-induced transcriptional activity of the substitution mutants transfected into Jurkat T cells. However, we did not observe significant differences in the basal activity in any of the Ser right-arrow Ala substitutions (Fig. 2B), indicating that none of them is absolutely necessary for the basal transcriptional activity of c-Rel. Interestingly, when we assayed for activation by TNFalpha or PMA + ionophore, we observed that mutants A3, A4, A5, and A6 failed to respond to TNFalpha stimulation by increasing its transactivating activity, whereas mutants A8, A9, and A10 showed a reduced response to either PMA + ionophore or TNFalpha . Mutants A13-A16, included within the constitutively active region 541-588, showed a reduced response, as well. This results identified one region essential for the activation of c-Rel transcriptional capabilities by TNFalpha , which includes the Ser residues 454, 460, 463, 470, 471, and 473. Substitution of those residues abrogated the activation of c-Rel by TNFalpha . A second region including Ser residues 491, 494, 508, 509, 510, 511, 513, and 518 was identified to be involved in c-Rel activation, although it was not essential (Table II).


View larger version (43K):
[in this window]
[in a new window]
 
Fig. 2.   Activity of Ser right-arrow Ala substitutions of c-Rel-(422-588). A, structure of c-Rel transactivation domain with the indicated 16 Ser right-arrow Ala substitution mutants (A1-A16). B, effect of substitution mutants on the basal transcriptional activity of c-Rel fusions to Gal4 DBD. Jurkat T cells were transfected with each of the Ser right-arrow Ala mutants and co-transfected with a reporter plasmid, which contains five tandem repeats of the Gal4 site upstream the luciferase gene. Results are expressed as percentage of activity compared with the wild-type construct. Transfection efficiency was normalized using the Dual Luciferase assay (Promega). Additionally, similar amounts of the different constructions were expressed in transfected cells as detected by EMSA assays (data not shown). The results represents the mean ± S.D. of three independent experiments.

                              
View this table:
[in this window]
[in a new window]
 
Table II
Stimulation of the transactivating activity of Gal4 DBD-c-Rel wild-type and Ser right-arrow Ala mutants

Identification of the Signaling Route Involved in TNFalpha Activation of c-Rel Transactivating Activity

Implication of Phosphatidylinositol 3-Kinase (PI3K)-- We used several commercial inhibitors of putative signaling enzymes to define the route leading to c-Rel activation by TNFalpha . We first transfected Jurkat T cells with a NF-kappa B reporter plasmid and tested the effect of different inhibitors in TNFalpha -stimulated NF-kappa B activity. As a control, the proteasome inhibitor TPCK was used as a generic inhibitor of NF-kappa B activity since it interferes with the degradation of Ikappa B. Of the different inhibitors used, only the PI3K inhibitor wortmannin significantly inhibited TNFalpha stimulation of NF-kappa B activity (Fig. 3A). To corroborate the effect of wortmannin, another inhibitor of PI3K, LY294002, structurally unrelated to wortmannin, produced similar inhibition of NF-kappa B activity (Fig. 3B). Neither LY294002 was wortmannin affected cell viability (data not shown). However, NF-kappa B activity results from the combined effect of Ikappa B degradation and c-Rel and p65 activation. Thus, in order to study exclusively the implication of PI3K on c-Rel transactivating activity, we transfected Jurkat cells with the Gal4 c-Rel-(309-588) construct and tested the effect of the PI3K inhibitors. Both PI3K inhibitors prevented the transcription activity of c-Rel stimulated by TNFalpha (Fig. 3C).


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 3.   PI3K is involved in the TNFalpha -induced activation of the c-Rel transactivation domain. A and B, Jurkat (106) cells were transfected with 500 ng of the reporter NF-kappa B plasmid NF3TKLuc and cultures were treated with medium alone or with TNFalpha (10 ng/ml). The effect of the indicated inhibitors is shown. C, Jurkat cells were cotransfected with 0.5 µg of Gal4 c-Rel-(309-588) fusion construct and 10 ng of 5xGal4 luciferase reporter. The effect of the PI3K inhibitors wortmannin and LY294002 was assayed in B and C. Mean luciferase activity ± S.D. of three different experiments is shown.

PKCzeta Involvement in the Activation of the c-Rel Transactivation Domain-- PKCzeta has been recently found to be involved in the process of NF-kappa B activation by TNFalpha stimulation (25), so we studied the effect of this kinase on c-Rel activation. Jurkat T cells were co-transfected with the NF-kappa B reporter along with a plasmid that expressed either a wild-type or a dominant-negative mutant form of PKCzeta . Co-transfection of wild-type PKCzeta induced a strong increase in NF-kappa B-driven reporter activity, compared with cells co-transfected with an empty plasmid (Fig. 4). Furthermore, co-transfection of the PKCzeta dominant-negative mutant inhibited the activation of NF-kappa B transcriptional activity by TNFalpha (Fig. 4A). Parallel experiments were carried out assaying the activity of the Gal4 reporter driven by Gal4 c-Rel-(309-588) construct. Co-transfection of PKCzeta induced a strong activity of the Gal4 reporter, in a similar way as it did with the NF-kappa B reporter, and TNFalpha stimulation induced a still higher activation of the reporter (Fig. 4B). Interestingly, co-transfection of a PKCzeta dominant-negative mutant completely inhibited TNFalpha activation. Taken together, those results suggested the involvement of PKCzeta in the activation of the c-Rel transactivation domain by TNFalpha and subsequently in NF-kappa B activation.


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 4.   PKCzeta activates the transactivation domain of c-Rel. Jurkat cells (106) were transfected with 500 ng of the reporter NF-kappa B plasmid NF3TKLuc (A) or with 100 ng of 5xGal4 luciferase reporter along with 10 ng of Gal4 c-Rel-(309-588) fusion construct (B). Cultures were co-transfected with 500 ng of expression plasmids for PKCzeta wild-type, PKCzeta dominant negative mutant (mut-), PI3K dominant negative mutant (Delta p85), or empty vector (pcDNA3) and stimulated with TNFalpha (10 ng/ml) for 6 h. Luciferase activity is expressed normalized for transfection efficiency with the Dual Luciferase assay (Promega). Expression of wild-type and dominant negative PKCzeta was similar as detected by Western blot (data not shown). The results shown are the mean ± S.D. of three independent experiments.

Cross-talk between PI3K and PKCzeta -- The above results indicated that PI3K and PKCzeta were involved in the activation of the c-Rel transactivation domain by TNFalpha . On the other hand, the products of PI3K activity, PIP2 and PIP3, have been described to activate PKCzeta (26). Thus, these enzymes could be participating in the same signaling pathway leading to c-Rel activation by TNFalpha . To investigate this, Jurkat cells were transfected with a dominant-negative mutant of PI3K (termed Delta p85), resulting in the inhibition of both the NF-kappa B- and Gal4 c-Rel-driven activity induced by co-transfection of active PKCzeta (Fig. 4). Additionally, the PI3K inhibitors wortmannin and LY294002 inhibited the activation of NF-kappa B-dependent reporter activity (Fig. 5A), as well as activation of Gal4 c-Rel-driven activity (Fig. 5B) induced by transfection of wild-type PKCzeta . On the other hand, PIP3, the product of PI3K activity, was able to induce the activity of Gal4 reporter driven by Gal4 c-Rel when added exogenously. Addition of PIP3 to cultures of Jurkat cells co-transfected with wild-type PKCzeta , along with the Gal4 reporter and Gal4 c-Rel, resulted in a reporter activity similar to that observed after TNFalpha stimulation (Fig. 5C), suggesting that PIP3 is able to activate PKCzeta activity. However, PIP3 could not revert the inhibition produced by co-transfection of the dominant-negative mutant of PKCzeta (Fig. 5C). These data indicate that both PI3K and PKCzeta are necessary for the activation of c-Rel transactivation domain but do not allow establishment of the relative position of each other in the signaling pathway.


View larger version (32K):
[in this window]
[in a new window]
 
Fig. 5.   Cross-talk between PKCzeta and PI3K in the activation of the c-Rel transactivation domain. Jurkat cells (106) were transfected with 500 ng of the reporter NF-kappa B plasmid NF3TKLuc (A) or with 100 ng of 5xGal4 luciferase reporter along with 10 ng of Gal4 c-Rel-(309-588) fusion construct (B and C). Cultures were co-transfected with 500 ng of expression plasmids for PKCzeta wild-type, empty vector (pcDNA3) or PKCzeta dominant negative mutant (mut-, only in C). The effect of the PI3K inhibitors wortmannin and LY294002 in PKCzeta activation of the c-Rel transactivation domain was assayed (A and B). Effect of exogenously added PIP3 (20 ng/ml) was tested (C). Luciferase activity is expressed normalized for transfection efficiency with the Dual Luciferase assay (Promega). Expression of wild-type or mutant PKCzeta was similar in transfected cells as detected by Western blot (data not shown). The results shown are the mean ± S.D. of three independent experiments.

Mapping of the Target Sites of PKCzeta and PI3K on the c-Rel Transactivation Domain

Results shown above indicate that TNFalpha -dependent activation of c-Rel transactivation domain relies on the Ser residues defined by the Ser right-arrow Ala substitution mutants A3, A4, A5, A6, A8, A9, and A10. In order to identify the exact residues that were affected by TNFalpha -induced PKCzeta activity, these mutants were co-transfected into Jurkat cells with the wild-type form of PKCzeta . As shown in Fig. 6A, co-transfection of PKCzeta along with wild-type Gal4 c-Rel-(422-588) induced an average of 2.5-fold induction of reporter activity. Co-transfection of Ser right-arrow Ala substitution mutants A3, A5, A6, A9, and A10 with PKCzeta produced a similar activation of the reporter. However, mutants A4 (Ser460) and A8 (Ser491, Ser494) were not activated by PKCzeta co-transfection. These results indicate that those sites were necessary for PKCzeta activation of c-Rel transactivation domain. Strikingly, both sites are palindromes of the sequence SNCS, not found in other part of the c-Rel transactivation domain.


View larger version (39K):
[in this window]
[in a new window]
 
Fig. 6.   Mapping of the target sites of PKCzeta and PI3K in the activation of the c-Rel transactivation domain. Jurkat cells (106) were transfected with 100 ng of 5xGal4 luciferase reporter along with 100 ng of the different TNFalpha -sensitive Ser right-arrow Ala substitution mutants together with 500 ng of expression plasmid for PKCzeta wild-type (A) or tested for the effect of exogenously added PIP3 (B). Luciferase activity was normalized for transfection efficiency with the Dual Luciferase assay (Promega). The results represent the increase above the control culture transfected with empty vector (A) or the non-stimulated control (B). A fragment of the sequence of the c-Rel transactivation domain is shown, and the relevant sites are included in boxes. Similar amounts of the different construction were expressed in transfected cells as detected by EMSA assays (data not shown). The results represent the mean ± S.D. of three independent experiments.

A similar approach was used to map the sites relevant in PI3K activation of c-Rel. Exogenous PIP3 was used to mimic PI3K activation. Wild-type Gal4 c-Rel-(422-588) was successfully activated by PIP3 treatment, as well as the Ser right-arrow Ala substitution mutants A4, A5, A9, and A10 (Fig. 6B). Substitution mutant A8 (Ser491, Ser494), which was not activated by PKCzeta , was not activated either by PIP3 treatment, while substitution mutants A3 (Ser454) and A6 (Ser470, Ser471, Ser473) displayed only a reduced activation. These results suggest that the positions defined by substitution mutant A8 may be the point of coincidence of PKCzeta and PI3K activation on c-Rel transactivation domain. Noteworthy, both substitution mutants A3 and A6 show a Ser residue close to an Asp residue, pointing out to a possible unique kinase dependent on PI3K activity.

c-Rel Transactivation Domain Mutants Act as Dominant Negative Forms in NF-kappa B Activation by TNFalpha

In order to test the functional significance of several of those mutations in c-Rel functioning as well as in NF-kappa B activation in T cells, we transfected expression plasmid of c-Rel mutants into Jurkat cells together with a NF-kappa B-luc reporter gene. Basal reporter activity was not altered by overexpression of any c-Rel protein (data not shown). As shown in Fig. 7, transfection of wild-type c-Rel slightly, although significantly, increased TNFalpha -induced stimulation. However, mutants A3, A4, A5, and A8 could not support TNFalpha -induced activation of NF-kappa B activity in contrast to wild-type c-Rel. More interestingly, those results indicate that overexpression of these mutant proteins acted as dominant negative forms for NF-kappa B activation (that includes both endogenous p65 and c-Rel), further pointing out to the importance of these pathways in NF-kappa B activity.


View larger version (10K):
[in this window]
[in a new window]
 
Fig. 7.   Functional activity of c-Rel mutants. Jurkat cells (106) were transfected with 500 ng of the indicated c-Rel cytomegalovirus-expressing plasmids together with 500 ng of the NF-kappa B reporter plasmid NF-3TK-luc and stimulated with TNFalpha (10 ng/ml) for 6 h. Luciferase activity was normalized with the Dual Luciferase assay (Promega) and represented as -fold induction over basal activity. The results shown are the mean ± S.D. of three independent experiments. Expression of all c-Rel proteins was similar as detected by Western blot data (data not shown).

Phosphorylation of c-Rel Region 422-588

The above results suggested that Ser residues in region 422-588 may be activated by TNFalpha through phosphorylation. Previous studies have shown that activity of c-Rel and other NF-kappa B proteins are indeed regulated by phosphorylation (27). In order to study the stimulation-dependent phosphorylation of this region, we made a recombinant GST fusion protein comprising region 422-588 of c-Rel transactivation domain. Solid phase phosphorylation assays using this recombinant protein revealed that extracts from non-stimulated cells already had strong c-Rel basal phosphorylation activity. Nonetheless, addition of extracts made from Jurkat cells stimulated either with TNFalpha or PMA + ionophore gave rise to a significant increase (about 2-fold) in the level of phosphorylation state of the recombinant protein (Fig. 8A). When a fusion construct lacking the region 541-588 was used, a great reduction in phosphorylation by unstimulated extracts was observed (Fig. 8B). Interestingly, this construct, comprising region 422-540, was more heavily phosphorylated by extracts from TNFalpha -stimulated Jurkat cells (about 5-fold increase). Those results suggest that region 541-588 retains most of the basal phosphorylation of c-Rel, but the phosphorylation dependent on TNFalpha activation resides in the region 422-540, thus corroborating the results obtained for transcriptional stimulation of the deletion mutants. In addition, PI3K inhibitors prevented the increase of the in vitro phosphorylation of c-Rel transactivation domain by cell extracts from TNFalpha -stimulated cells (Fig. 8C). Although both TNFalpha and PMA + ionophore stimulation induced the specific phosphorylation of the c-Rel transactivation domain, we could not detect any kinase activity in TNFalpha - or PMA/ionophore-treated cell extracts on this domain using "in-gel" kinase phosphorylation assays (data not shown).


View larger version (35K):
[in this window]
[in a new window]
 
Fig. 8.   TNFalpha -induced phosphorylation of c-Rel transactivation domain. A, recombinant GST c-Rel-(422-588) protein was used as substrate for in vitro solid-phase phosphorylation assays using WCE from cells treated with medium alone, TNFalpha (10 ng/ml), or PMA (10 ng/ml) + ionophore (1 µM) for 30 min, as indicated. B, recombinant GST c-Rel-(422-588) protein or GST c-Rel-(422-540) were used as substrates for in vitro solid-phase phosphorylation assays (left panel) using WCE from the same number of cells treated with medium alone or TNFalpha (10 ng/ml). The same amount of recombinant protein was run in a 10% SDS-PAGE and stained with Coomassie Blue to reveal the recombinant proteins (right panel). Intensity of the bands was quantified by densitometric scanning of the films and expressed as optical density (O.D.) in A or -fold increase above extracts from non-stimulated control in B. This set of results represents a representative experiment of the three performed. C, effect of PI3K inhibitors wortmannin and LY294002 on TNFalpha -induced phosphorylation of c-Rel. Recombinant GST-c-Rel-(422-588) was used as substrate for in vitro solid phase phosphorylation assays using WCE from non-stimulated cells or cells stimulated with TNFalpha or PMA + ionophore for 30 min, as indicated. The intensity of the bands was determined by densitometry of the film and the optical density (O.D.) units plotted. Results in panels A-C are the mean ± S.D. of three independent experiments. In C a representative experiment is shown.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The intermediate and late phase of T cell activation is controlled by the autocrine or paracrine effect of the cytokine TNFalpha , which in turn modulates the transcription factor NF-kappa B through the sustained activation of c-Rel (3). On the other hand, it is well established that IKK activation by TNFalpha stimulation, and subsequent Ikappa B degradation, is a pre-requisite for NF-kappa B activation (28-30). However, a second level of regulation of NF-kappa B activity that is independent of IKK degradation has been recently described. This second level involves the signal-dependent phosphorylation and activation of the transactivation domain of p65 (8, 10, 16, 31), although the exact mechanism has not yet been defined.

The transactivation domain of c-Rel has been previously defined as the region downstream the Rel homology domain to the C terminus of the protein (positions 309-588), as in this region reside all the transcription promoting capabilities of c-Rel (23, 32). The deletion analysis described in this work has indicated that positions 309-421 in the C terminus are dispensable for c-Rel-mediated transcription, further delimiting the location of this transactivation domain between positions 422 and 588 of the C terminus. However, a role of the region 309-421 in the regulation of c-Rel stability through degradation by the proteasome has been proposed (33). Furthermore, as the region 422-588 fused to the Gal4 DBD had a stronger transcriptional activity than the complete C-terminal region (309), this may indicate the existence of repressor sequences within the 309-421 region. Alternatively, region 422-588 could acquire a configuration in the Gal4 fusion that is more competent for transcriptional activation. Sequential deletions from position 422 to 588 (C terminus) were associated with parallel decrease in transcriptional activity. The region from position 541 to 588 still retained significant transcriptional activity, whereas smaller fragments (422-455, 456-497, and 498-540) showed no transcriptional activity, indicating that region 422-588 is a functional transactivation domain non divisible in smaller units. This is not the case of the Rel family member p65, where two different transcriptional activating regions were found in the transactivation domain, named TA1 and TA2 (8, 19).

Stimulation of the cells transfected with the Gal4 c-Rel fusion constructs revealed that only region 422-540 was activated by TNFalpha , as well as PMA + ionophore, stimulation. Region 541-588 was not further activated by these stimuli, even though it showed a strong basal transcriptional activity. Thus, those analyses indicate that this region is necessary for the function of c-Rel as a transcription factor but dispensable for inducible activation of this factor. In vitro solid phase phosphorylation assays demonstrated that this region (541) was strongly phosphorylated by cell extracts obtained from non-stimulated cells, thus supporting its role in basal transcriptional activity of c-Rel. In contrast, region 422-540 was weakly phosphorylated by cell extracts from unstimulated cells, whereas extracts from TNFalpha - or PMA/ionophore-stimulated Jurkat cells strongly phosphorylated it. This indicate that it is within this region where stimulus-induced activation occurs. These data suggested that the activation of this region requires extracellular signal-dependent phosphorylation.

This region (422) is negatively charged (15 acidic residues, 3 basic) and is rich in Pro and Ser. These characteristics resemble to acidic transactivation domains (24). Although this region is not conserved among different Rel family members, the acidic and Ser residues are well conserved between human and mouse c-Rel, suggesting a critical role for c-Rel functioning. This region does not show homology to other described acidic transactivation factors. However, its high Ser content is typical of other transcription factors like CREB, TCF/Elk-1, or STAT that are regulated by phosphorylation (34). Thus, phosphorylation may provide the additional negative charges necessary to constitute an acidic transactivation domain. The effect observed in Ser right-arrow Ala mutants of the transactivation domain of c-Rel corroborates this hypothesis. Although none of the mutants had any effect on the basal transcriptional activity of c-Rel, the substitution of the Ser residues at position 455 (mutant A3), 461 (mutant A4), 464 (mutant A5), or 470, 471, and 473 (mutant A6) completely prevented the activation of this domain by TNFalpha . Any of these positions is absolutely required and must be activated, as the substitution of any of them was enough to abrogate the activation of this domain by TNFalpha . Additionally, the substitution of the Ser residues at position 492 and 494 (mutant A8), 509-512 and 514 (mutant A9), or 519 (mutant A10) had a less pronounced inhibitory effect on both TNFalpha - and PMA/ionophore-induced activation of c-Rel transactivation domain, indicating that they may participate in c-Rel activation although they are probably not essential.

Transcription factors like NF-kappa B activate gene transcription through the interaction with basal transcriptional machinery, or with co-factors that modulate that machinery (35). In this regard, both c-Rel and v-Rel have been found to interact with the basal transcription factors TBP and TFIID directly through their transactivation domains (36). However, other reports indicate that only the first 50 amino acids were necessary for TBP interaction (37). p65 could interact with TBP and TFIID through its transactivation domain (8, 38). Furthermore, other proteins that are able to interact with NF-kappa B have been described: the HMG(I)Y nuclear protein (39), the SP1 factor (40), or a 40-kDa protein, which acts as a target for the quinone derivative E3330 in the inhibition of NF-kappa B activity (41). The mutations described in this work may be very useful for the study of the interactions of c-Rel with the transcriptional machinery and to define protein-protein interactions that regulates the process of transcription activation.

We were unable to identify a kinase activity, capable of phosphorylating c-Rel transactivation domain, by in-gel phosphorylation assays. However, only a fraction of cellular kinases can remain active after the renaturalization process required in those assays. Furthermore, no kinase that requires a co-factor or association with other proteins to form an active complex will remain active in those assays. On the other hand, the use of commercially available inhibitors revealed the dependence of NF-kappa B activation by TNFalpha on the activity of PI3K. PI3K activity was previously described as necessary for NF-kappa B activity in several cell types but not in lymphocytes (42). Furthermore, PI3K-dependent phosphorylation and activation of p65 has been described as a requisite for interleukin-1 activation of p65 transactivation domain, without affecting Ikappa B degradation or DNA binding activity of p65 complexes (16). In a similar manner, TNFalpha -dependent activation of NF-kappa B-induced transcription in HepG2 cells has been shown to depend on PI3K activity, which does not affect NF-kappa B binding to DNA or Ikappa B degradation induced by the cytokine (17). Our results indicate that a similar mechanism is taking place for c-Rel activation by TNFalpha . Thus, PI3K inhibitors wortmannin and LY294002 inhibited not only NF-kappa B dependent activity, but also c-Rel activation and phosphorylation of its transactivation domain, supporting a critical role of PI3K activity for TNFalpha activation of c-Rel. However, whether PI3K can associate with TNF receptors or any of its associated factors is not known yet. Furthermore, the PI3K metabolite, PIP3, was capable of activating Gal4 c-Rel when transfected into Jurkat cells but not NF-kappa B reporter activity (data not shown). Thus, TNFalpha might activate several signaling routes, leading to the direct modulation of transcriptional abilities of c-Rel, as well as to activation of IKKs for Ikappa B degradation and subsequent nuclear translocation of active NF-kappa B heterodimers. PI3K may therefore be implicated in the activation of c-Rel transactivation domain but not in the activation of IKK by TNFalpha . Nevertheless, one of the well known targets of PI3K activity, the protein kinase Akt, has been recently demonstrated to interact with IKK upon TNFalpha stimulation (43, 44). However, our results with PIP3 stimulation, as well as other recent reports looking directly at NF-kappa B binding activity (16, 17), suggest that PI3K may not be essential for IKK activation and subsequent nuclear translocation of NF-kappa B. Although those discrepancies cannot be explained yet, PI3K might differentially affect NF-kappa B activation depending on the cell type.

On the other hand, PKCzeta has been implicated in NF-kappa B activation, through mechanisms that involve IKK activation (14, 45, 46) or directly through the phosphorylation and activation of p65 (13). Thus, PKCzeta may play a dual role in the activation of NF-kappa B, activating IKK and also participating in the activation the transactivation domain of members of the Rel family. Our results clearly support this hypothesis, indicating that PKCzeta , besides participating in IKK activation, is also involved in the activation of c-Rel transactivation domain. Thus, co-transfection experiments into Jurkat cells of PKCzeta wild-type with Gal4 c-Rel showed a strong potentiation of c-Rel transcriptional promoting capabilities. Furthermore, a dominant-negative mutant of PKCzeta abrogated TNFalpha -induced c-Rel activation. Surprisingly, PKCzeta activation of c-Rel transactivation domain was inhibited in the presence of wortmannin and LY294002, both inhibitors of PI3K activity, as well as the co-transfection of a dominant-negative mutant of PI3K, suggesting that PI3K might act downstream of PKCzeta . In addition, the activation of c-Rel transactivation domain by PIP3, a product of PI3K activity, was inhibited by a dominant-negative mutant of PKCzeta . However, PKCzeta has been shown to be activated by the protein kinase PDK-1, an effector of PI3K activity (47), suggesting that PKCzeta would be downstream of PI3K in the route of c-Rel activation. A possible explanation for this apparent different positioning in the signaling pathway of PI3K and PKCzeta is that both pathways are parallel and required for activity.

The fact that PIP3 and active PKCzeta seem to act on different Ser residues of the c-Rel transactivation domain would be in agreement with the above hypothesis. The Ser right-arrow Ala substitution analysis showed that positions defined by the mutants A3, A4, A5, and A6 are essential and in lesser extent by the mutants A8, A9, and A10 for c-Rel activation by TNFalpha . Thus, the failure to activate one of them resulted in the inability of c-Rel to be activated by TNFalpha . Mapping of the sites activated by PKCzeta and PIP3 revealed that the only Ser residues substituted in mutant A8 are the point of convergence for PKCzeta and PI3K activation. However, PKCzeta also required the Ser residue defined by mutant A4, whereas PIP3 required Ser residues defined by mutants A3 and A6. Thus, the inhibition of either PKCzeta or PI3K would result in an inhibition of c-Rel transactivation domain. The sites defined by mutants A4 and A8 showed a striking palindromic similarity (SNCS for A4 and SCNS for A8). Mutant A5, however, which substituted the second Ser residue downstream mutant A4, was not essential for PKCzeta activation. Thus the Ser residue close to Asn (Ser460 and Ser494) may be the actual target of PKCzeta activation. Furthermore, A8, which defined the point of convergence between PI3K and PKCzeta , mutants A3 (Ser454) and A6 (Ser471, Ser473, and Ser474), failed to be activated by PIP3. These mutants involve Ser residues that are in close proximity of an Asp residue, suggesting that the same kinase might be activating both sites. Furthermore, a natural mutant of Ser471 to Asn produced a form of c-Rel that could not be activated by TNFalpha stimulation (21), suggesting that PI3K may be an essential part of the signaling mechanisms activated by TNFalpha , resulting in the activation of c-Rel transactivation domain. Furthermore, the different responses of several Ser mutants to the different stimuli clearly discard nonspecific effects of the pathways or activators used.

More interestingly, those mutants not only are defective in transactivating activity, but they prevent NF-kappa B-dependent reporter activity. Those results indicate that they act as dominant negative forms of NF-kappa B activation, either by recruiting the kinases required for phosphorylation of transactivation domains of c-Rel and/or p65 or by binding to NF-kappa B sites on DNA and then prevent active NF-kappa B complexes (either p65 or endogenous c-Rel) for binding. The fact that the spontaneous Ser471 mutant failed to activate at all NF-kappa B (21) tends to support the first hypothesis.

In summary, our results have revealed an important level of TNFalpha -induced NF-kappa B activation mediated through PI3K and PKCzeta , which are absolutely required for c-Rel transactivating activity (Fig. 9).


View larger version (31K):
[in this window]
[in a new window]
 
Fig. 9.   Model of activation of c-Rel transactivation domain by TNFalpha .


    ACKNOWLEDGEMENTS

We thank María Chorro and Lucía Horrillo for excellent technical assistance.

    FOOTNOTES

* This work was supported by grants from Dirección General de Investigación Científica y Técnica, Fondo de Investigaciones Sanitarias, Comunidad Autónoma de Madrid, and Fundación Ramón Areces.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 34-913978413; Fax: 34-913974799; E-mail: mfresno@cbm.uam.es.

Published, JBC Papers in Press, February 15, 2001, DOI 10.1074/jbc.M011313200

    ABBREVIATIONS

The abbreviations used are: NF-kappa B, nuclear factor kappa B; DBD, DNA binding domain, FBS, fetal bovine serum; GST, glutathione S-transferase; IKK, Ikappa B kinase; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; PI3K, phosphatidylinositol 3-kinase; PMA, phorbol myristate acetate; TNF, tumor necrosis factor; TPCK, L-1-chloro-3-[4-tosylamido]-4-phenyl-2-butanone; WCE, whole cell extract; PKC, protein kinase C; HA, hemagglutinin; PIP3, phosphatidylinositol 3,4,5-trisphosphate.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Baldwin, A. S., Jr. (1996) Annu. Rev. Immunol. 14, 649-683[CrossRef][Medline] [Order article via Infotrieve]
2. Ghosh, S., May, M. J., and Kopp, E. B. (1998) Annu. Rev. Immunol. 16, 225-260[CrossRef][Medline] [Order article via Infotrieve]
3. Pimentel-Muiños, F. X., Mazana, J., and Fresno, M. (1995) Eur. J. Immunol. 25, 179-186[Medline] [Order article via Infotrieve]
4. Wallach, D., Varfolomeev, E. E., Malinin, N. L., Goltsev, Y. V., Kovalenko, A. V., and Boldin, M. P. (1999) Annu. Rev. Immunol. 17, 331-367[CrossRef][Medline] [Order article via Infotrieve]
5. Gerondakis, S., Strasser, A., Metcalf, D., Grigoriadis, G., Scheerlinck, J. Y., and Grumont, R. J. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 3405-3409[Abstract/Free Full Text]
6. Liou, H. C., Jin, Z., Tumang, J., Andjelic, S., Smith, K. A., and Liuo, M. L. (1999) Int. Immunol. 11, 361-371[Abstract/Free Full Text]
7. May, M. J., and Ghosh, S. (1998) Immunol. Today 19, 80-88[CrossRef][Medline] [Order article via Infotrieve]
8. Schmitz, M. L., dos Santos Silva, M. A., and Baeuerle, P. A. (1995) J. Biol. Chem. 270, 15576-15584[Abstract/Free Full Text]
9. Schmitz, M. L., and Baeuerle, P. A. (1995) Immunobiology 193, 116-127[Medline] [Order article via Infotrieve]
10. Wang, D., and Baldwin, A. S., Jr. (1998) J. Biol. Chem. 273, 29411-29416[Abstract/Free Full Text]
11. Zhong, H., Voll, R. E., and Ghosh, S. (1998) Mol. Cell 1, 661-671[Medline] [Order article via Infotrieve]
12. Norris, J. L., and Baldwin, A. S., Jr. (1999) J. Biol. Chem. 274, 13841-16846[Abstract/Free Full Text]
13. Anrather, J., Csizmadia, V., Soares, M. P., and Winkler, H. (1999) J. Biol. Chem. 274, 13594-13603[Abstract/Free Full Text]
14. Lallena, M. J., Díaz-Meco, M. T., Bren, G., Pay, C. V., and Moscat, J. (1999) Mol. Cell. Biol. 19, 2180-2188[Abstract/Free Full Text]
15. Sanz, L., Sánchez, P., Lallena, M. J., Díaz-Meco, M. T., and Moscat, J. (1999) EMBO J. 18, 3044-3053[Abstract/Free Full Text]
16. Sizemore, N., Leung, S., and Stark, G. R. (1999) Mol. Cell. Biol. 19, 4798-4805[Abstract/Free Full Text]
17. Reddy, S. A., Huang, J. H., and Liao, W. S. (2000) J. Immunol. 164, 1355-1363[Abstract/Free Full Text]
18. Toker, A., and Cantley, L. C. (1997) Nature 387, 673-676[CrossRef][Medline] [Order article via Infotrieve]
19. Schmitz, M. L., dos Santos Silva, M. A., Altmann, H., Czisch, M., Holak, T. A., and Baeuerle, P. A. (1994) J. Biol. Chem. 269, 25613-25620[Abstract/Free Full Text]
20. Chen, C., Agnës, F., and Gèlinas, C. (1999) Mol. Cell. Biol. 19, 307-316[Abstract/Free Full Text]
21. Martín, A. G., and Fresno, M. (2000) J. Biol. Chem. 275, 24383-24391[Abstract/Free Full Text]
22. Yano, O., Kanellopoulos, J., Kieran, M., Le Bail, O., Israel, A., and Kourilsky, P. (1987) EMBO J. 6, 3317-3324[Abstract]
23. Bull, P., Morley, K. L., Hoekstra, M. F., Hunter, T., and Verma, I. M. (1990) Mol. Cell. Biol. 10, 5473-5485[Medline] [Order article via Infotrieve]
24. Hahn, S. (1993) Cell 72, 481-483[Medline] [Order article via Infotrieve]
25. Domínguez, I., Sanz, L., Arenzana-Seisdedos, F., Díaz-Meco, M. T., Virelizier, J. L., and Moscat, J. (1993) Mol. Cell. Biol. 13, 1290-1295[Abstract]
26. Standaert, M. L., Galloway, L., Karnam, P., Bandyopadhyay, G., Moscat, J., and Farese, R. V. (1997) J. Biol. Chem. 272, 30075-30082[Abstract/Free Full Text]
27. Naumann, M., and Scheidereit, C. (1994) EMBO J. 13, 4597-4607[Abstract]
28. Mercurio, F., Zhu, H., Murray, B. W., Shevchenko, A., Bennett, B. L., Li, J., Young, D. B., Barbosa, M., Mann, M., Manning, A., and Rao, A. (1997) Science 278, 860-866[Abstract/Free Full Text]
29. Mercurio, F., and Manning, A. M. (1999) Curr. Opin. Cell Biol. 11, 226-232[CrossRef][Medline] [Order article via Infotrieve]
30. Zandi, E., Rothwarf, D. M., Delhase, M., Hayakawa, M., and Karin, M. (1997) Cell 91, 243-252[Medline] [Order article via Infotrieve]
31. Zhong, H., SuYang, H., Erdjument-Bromage, H., Tempst, P., and Ghosh, S. (1997) Cell 89, 413-424[Medline] [Order article via Infotrieve]
32. Ishikawa, H., Asano, M., Kanda, T., Kumar, S., Gélinas, C., and Ito, Y. (1993) Oncogene 8, 2889-2896[Medline] [Order article via Infotrieve]
33. Chen, E., Hrdlickova, R., Nehyba, J., Longo, D. L., Bose, H. R., Jr., and Li, C. C. (1998) J. Biol. Chem. 273, 35201-35207[Abstract/Free Full Text]
34. Karin, M. (1994) Curr. Opin. Cell Biol. 6, 415-424[Medline] [Order article via Infotrieve]
35. Greenblatt, J. (1992) Nature 360, 16-17[CrossRef][Medline] [Order article via Infotrieve]
36. Xu, X., Prorock, C., Ishikawa, H., Maldonado, E., Ito, Y., and Gelinas, C. (1993) Mol. Cell. Biol. 13, 6733-6741[Abstract]
37. Kerr, L. D., Ransone, L. J., Wamsley, P., Schmitt, M. J., Boyer, T. G., Zhou, Q., Berk, A. J., and Verma, I. M. (1993) Nature 365, 412-419[CrossRef][Medline] [Order article via Infotrieve]
38. Schmitz, M. L., Stelzer, G., Altmann, H., Meisterernst, M., and Baeuerle, P. A. (1995) J. Biol. Chem. 270, 7219-7226[Abstract/Free Full Text]
39. Thanos, D., and Maniatis, T. (1992) Cell 71, 777-789[Medline] [Order article via Infotrieve]
40. Hirano, F., Tanaka, H., Hirano, Y., Hiramoto, M., Handa, H., Makino, I., and Scheidereit, C. (1998) Mol. Cell. Biol. 18, 1266-1274[Abstract/Free Full Text]
41. Hiramoto, M., Shimizu, N., Sugimoto, K., Tang, J., Kawakami, Y., Ito, M., Aizawa, S., Tanaka, H., Makino, I., and Handa, H. (1998) J. Immunol. 160, 810-819[Abstract/Free Full Text]
42. Kaliman, P., Canicio, J., Testar, X., Palacín, M., and Zorzano, A. (1999) J. Biol. Chem. 274, 17437-17444[Abstract/Free Full Text]
43. Romashkova, J. A., and Makarov, S. S. (1999) Nature 401, 86-90[CrossRef][Medline] [Order article via Infotrieve]
44. Ozes, O. N., Mayo, L. D., Gustin, J. A., Pfeffer, S. R., Pfeffer, L. M., and Donner, D. B. (1999) Nature 401, 82-85[CrossRef][Medline] [Order article via Infotrieve]
45. Folgueira, L., McElhinny, J. A., Bren, G. D., MacMorran, W. S., Díaz-Meco, M. T., Moscat, J., and Paya, C. V. (1996) J. Virol. 70, 223-231[Abstract]
46. Muller, G., Ayoub, M., Storz, P., Rennecke, J., Fabbro, D., and Pfizenmaier, K. (1995) EMBO J. 14, 1961-1969[Abstract]
47. Chou, M. M., Hou, W., Johnson, J., Graham, L. K., Lee, M. H., Chen, C. S., Newton, A. C., Schaffhausen, B. S., and Toker, A. (1998) Curr. Biol. 8, 1069-1077[Medline] [Order article via Infotrieve]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.