©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Tumor Necrosis Factor--dependent Activation of a RelA Homodimer in Astrocytes
INCREASED PHOSPHORYLATION OF RelA AND MAD-3 PRECEDE ACTIVATION OF RelA (*)

(Received for publication, September 29, 1994; and in revised form, November 17, 1994)

J. Alan Diehl Wei Tong Grace Sun Mark Hannink (§)

From the Biochemistry Department, University of Missouri, Columbia, Missouri 65212

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Rel proteins are important intracellular mediators of cytokine-induced signal transduction. To understand how cytokines affect different cell populations in the brain, we have characterized Rel activation in astrocytes. A RelA homodimer is uniquely activated in cytokine-stimulated astrocytes. Cytokine-dependent phosphorylation of the RelA inhibitor MAD-3 occurred on discrete peptides prior to its dissociation from RelA. A transient hyperphosphorylation of RelA was also induced. Antioxidant treatment inhibited both RelA activation and phosphorylation of the RelAbulletMAD-3 complex. These results demonstrate that cytokine-dependent activation of the RelA homodimer involves phosphorylation of both RelA and its associated inhibitor. The sole activation of a RelA homodimer suggests that cytokines will activate a unique set of Rel-regulated genes in astrocytes.


INTRODUCTION

The Rel family is defined by a highly conserved 300-amino acid domain, referred to as the Rel homology domain. Within the Rel homology domain lie the amino acid residues necessary for sequence-specific DNA binding, dimerization, and association with members of the IkappaB family of proteins (inhibitor of kappaB; (1) ). The IkappaB proteins regulate the cellular location, sequence-specific DNA binding activity, and transcriptional activation properties of Rel proteins. Rel proteins modulate inducible expression of a variety of genes through cis-acting Rel binding sites. Cytokines, including tumor necrosis factor-alpha (TNFalpha) (^1)and interleukin-1, induce DNA binding and transcriptional activation by Rel proteins(2) . Induction of Rel activity by these stimuli is a post-translational event that targets an inactive cytosolic complex containing Rel family members and an IkappaB protein. These stimuli induce the phosphorylation and degradation of IkappaB followed by an increase in nuclear Rel DNA binding activity(3, 4) . Although in vitro phosphorylation of Rel proteins has been correlated with enhanced DNA binding activity(5, 6) , the role of Rel phosphorylation in the activation of the cytosolic RelbulletIkappaB complex in mammalian cells is not known. The demonstration that Toll-dependent phosphorylation of the dorsal morphogen from Drosophila correlates with increased nuclear import of Dorsal suggests that signal-dependent phosphorylation of Rel may also occur in mammalian cells(7) .

Rel proteins are important mediators of cytokine-dependent signal transduction in both lymphoid and non-lymphoid cells. As astrocytes are the primary cytokine-responsive cell in the brain(8) , we have examined cytokine-dependent activation of Rel proteins in astrocytes. We find that treatment of both primary rat astrocytes or the astrocyte cell line DITNC (9) with TNFalpha results in the rapid activation of DNA binding by a RelA homodimer. Induction of RelA DNA binding was preceded by the TNFalpha-dependent phosphorylation of both RelA and the RelA-specific IkappaB protein, MAD-3. The ability of an N-acetyl-L-cysteine (NAC) to inhibit signal-dependent phosphorylation of RelA and MAD-3 implicates a role for reactive oxygen intermediates upstream of this event. These results suggest that TNFalpha-dependent phosphorylation of RelA and MAD-3 is a critical step in the cytokine-dependent activation of the preexisting RelbulletIkappaB complex.


MATERIALS AND METHODS

Cell Culture, Transfection of DITNC Cells, and Luciferase Assays

Primary astrocytes were derived from 2-day-old rats(10) . Primary astrocytes and DITNC cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Transfections were performed using the calcium phosphate coprecipitation protocol(11) . Thirty hours post-transfection, media containing 10 ng/ml TNFalpha or media without TNFalpha was added to the cells. Seven hours after TNFalpha treatment, luciferase assays were performed on whole cell lysates as described previously(12) .

Cell Lysate Preparation, DNA Binding, and Immunoblot Assays

Whole cell lysates, nuclear extracts, and DNA binding experiments were prepared and performed as described previously (13, 14, 15) . The anti-p52 and anti-RelA sera were obtained from Santa Cruz Biotechnology, and the anti-p50 serum was obtained from W. Greene. For immunoprecipitations of the solution UV-cross-linked proteinbulletDNA adducts, the proteinbulletDNA adducts were diluted into radioimmune precipitation buffer containing the appropriate IgG and the immune complexes were analyzed as described below. Immunoblot analysis was carried with the ECL Western blotting kit (Amersham Corp.).

Metabolic Labeling, Immunoprecipitation Experiments, and V8 Protease Mapping

Cells were labeled with 200 µCi/ml [S]methionine/cysteine (EXPRES; DuPont) for 4 h in RPMI 1640 lacking methionine and cysteine and containing 10% dialyzed fetal calf serum. The cells were collected in radioimmune precipitation buffer (13) containing 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 0.4 mM sodium vanadate, 0.4 mM sodium fluoride, and 0.4 mM sodium pyrophosphate. Cellular debris was removed by centrifugation and 1.5 µg of the appropriate IgG was added to each supernatant. Immune complexes were collected with Staphylococcus aureus, and the immunoprecipitated proteins were analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. For metabolic labeling with P, astrocytes were starved in phosphate-free medium for 1.5 h prior to labeling with 500 µCi/ml [P]orthophosphate (DuPont) for 2 h. V8 protease digestions of the immunoprecipitated proteins were performed as described by Cleveland et al.(16) .


RESULTS

LPS Activation of a RelA Homodimer

Nuclear fractions from untreated and LPS-treated DITNC cells were examined for proteins that bound to an oligonucleotide containing a palindromic kappaB site by an electrophoretic mobility shift assay (EMSA; Fig. 1A). Examination of nuclear extracts by EMSA revealed an induction of DNA binding activity by LPS treatment (Fig. 1A, lanes1 and 6). Addition of antiserum raised against human RelA resulted in a proteinbulletDNA complex of decreased mobility (supershift) in the extracts from both treated and untreated cells (Fig. 1A, lanes5 and 10). Antisera raised against p50, p52, and c-Rel did not affect the proteinbulletDNA complex (Fig. 1A, lanes 3 and 4; data not shown). To confirm that RelA is the only activatable Rel protein in astrocytes, nuclear extracts from the LPS-treated cells were subjected to solution UV-cross-linking experiments followed by immunoprecipitation with Rel-specific antisera. Immunoprecipitation with anti-RelA serum resulted in the precipitation of a 75-kDa proteinbulletDNA adduct and several minor proteinbulletDNA adducts around 50 kDa (Fig. 1B; the smaller proteinbulletDNA adducts are proteolytic products of the larger 75-kDa proteinbulletDNA adduct). Normal rabbit serum and anti-p50 and anti-p52 sera failed to precipitate any of the proteinbulletDNA adducts (Fig. 1B). The DNA binding specificity of RelA was confirmed by competition experiments with oligonucleotides containing either wild type or mutant kappaB sites (data not shown). Stimulation of DITNC cells with either interleukin-1 (data not shown) or TNFalpha (see below) also resulted in the activation of DNA binding by a RelA homodimer. These results demonstrate that a RelA homodimer is the primary activatable Rel complex in astrocytes.


Figure 1: A, induction of RelA DNA binding activity. Nuclear fractions from untreated (lanes 1-5) and LPS-treated (lanes 6-10) DITNC cells were analyzed by EMSA with an oligonucleotide containing a palindromic kappaB site. To identify the Rel proteins present in the EMSA complex, antiserum raised against p50 (lanes3 and 8), p52 (lanes4 and 9), RelA (lanes6 and 10), or normal rabbit serum (lanes2 and 7) were added to the nuclear extract prior to the DNA binding reaction. The proteinbulletDNA complexes are denoted by the arrows to the left of the gel. B, immunoprecipitation of the RelAbulletDNA adduct. Nuclear extracts from LPS-treated DITNC cells were subjected to solution UV-cross-linking, followed by immunoprecipitation with antisera raised against RelA (lane 2), p50 (lane 3), p52 (lane 4), or normal rabbit serum (lane 1). The position of the full-length RelA proteinbulletDNA adduct is denoted by the arrow to the right of the gel. The position of molecular weight markers is indicated to the right of the gel. C, activation of kappaB-dependent gene expression. 5.0 µg of a plasmid containing only TATA and initiator sites (TATA-Inr) driving expression of the fire fly luciferase reporter gene or 5 µg of the same plasmid in which the kappaB binding sites from the HIV-1 long terminal repeat had been cloned in front of the TATA box were transfected into DITNC cells. Media containing 10 ng/ml TNFalpha or media without TNFalpha was added to the cells 30 h after transfection. Cell lysates were collected and assayed for luciferase activity 7 h post-transfection. Transfections were done in triplicate, and the results shown are the average of three independent experiments.



To determine if RelA DNA binding activity correlated with activation of kappaB-dependent transcription, two copies of the kappaB binding site from the HIV-1 long terminal repeat were cloned into a reporter plasmid that contains a canonical TATA and initiator sequences in front of the firefly luciferase reporter gene. TNFalpha treatment of the astrocytes transfected with the TATA-luciferase plasmid did not affect luciferase activity from this plasmid (Fig. 1C). However, treatment of astrocytes transfected with the kappaB-luciferase plasmid resulted in a 4-fold activation of luciferase activity over the basal luciferase activity in untreated cells expressing the kappaB-luciferase plasmid (Fig. 1C). Thus, activation of RelA DNA binding activity in astrocytes by cytokine stimulation correlates with kappaB-dependent transcriptional activation.

TNFalpha-dependent Phosphorylation and Degradation of MAD-3

We next examined the time course of RelA activation following treatment of DITNC cells with TNFalpha. DITNC were treated with TNFalpha for 0-60 min. Whole cell extracts were prepared from these cells and subjected to solution UV-cross-linking experiments. Maximal activation of RelA DNA binding was observed after 30 min of TNFalpha treatment (Fig. 2A). Immunoblot analysis of these cell lysates with antisera raised against RelA confirmed total levels of RelA were not altered by TNFalpha treatment (Fig. 2B). Immunoblot analysis with anti-MAD-3 serum demonstrated that the level of MAD-3 protein was transiently reduced after 10 min of stimulation with TNFalpha (Fig. 2C, lanes1 and 4). After 1 h of TNFalpha treatment, levels of MAD-3 were higher than detected prior to TNFalpha treatment (Fig. 2C, lane6), consistent with previous reports indicating that expression of MAD-3 is directly regulated by RelA(17, 18) . These results are consistent with the existence of a preexisting RelbulletMAD-3 complex, which, upon stimulation with TNFalpha, undergoes dissociation with subsequent degradation of free MAD-3 and activation of RelA DNA binding.


Figure 2: A, induction of RelA DNA binding by TNFalpha. Whole cell lysates were prepared from DITNC cells treated with 10 ng/ml TNFalpha for 0-60 min. Equivalent volumes of each lysate were used for solution UV-cross-linking experiments. The proteinbulletDNA adducts were separated by SDS-polyacrylamide and visualized by autoradiography. The position of the RelAbulletDNA adduct is indicated by the arrow to the right of the gel, and the position of molecular weight markers is indicated to the left of the gel. B and C, anti-RelA immunoblot and anti-MAD-3 immunoblot of whole cell lysates prepared from the TNFalpha-treated DITNC cells. Equivalent volumes of the whole cell lysates prepared from untreated DITNC cells (lane1) or DITNC cells treated with TNFalpha for 2.5 min (lane2), 5 min (lane3), 10 min (lane4), 30 min (lane5), or 60 min (lane6) were electrophoresed through a 7.5% SDS-polyacrylamide gel. Protein samples were subsequently transferred to nitrocellulose and blotted with anti-RelA serum or anti-MAD-3 serum.



Although phosphorylation of MAD-3 prior to the activation of Rel DNA binding in vivo has been demonstrated previously(3, 4) , it is not clear if phosphorylation occurs before or after dissociation of the RelAbulletMAD-3 complex. To determine if RelA-associated MAD-3 underwent phosphorylation after TNFalpha treatment, DITNC cells were labeled with [P]orthophosphate or [S]methionine and stimulated with TNFalpha for 5 or 30 min. Whole cell lysates were subjected to immunoprecipitation with either anti-MAD-3 serum or anti-RelA serum. The anti-MAD-3 serum immunoprecipitated a 40-kDa protein from both P- and S-labeled astrocytes (Fig. 3A, lane1; top and middlepanels). A protein of identical mobility co-immunoprecipitated with RelA (Fig. 3A, lane 5; top and middlepanels). Five minutes after treatment with TNFalpha, P-labeled MAD-3 and RelA-associated MAD-3 proteins possessing an altered mobility were detected, while at 30 min following treatment, levels of P-labeled MAD-3 was dramatically reduced (Fig. 3A, lanes2 and 3 and lanes 6 and 7; middlepanels). The reduced level of P-labeled MAD-3 30 min following TNFalpha treatment is a reflection of MAD-3 proteolysis as demonstrated by the reduction in S-labeled MAD-3 following 30 min of TNFalpha treatment (Fig. 3A, lanes3 and 7; toppanel).


Figure 3: A, TNFalpha-dependent phosphorylation of MAD-3 and RelA-associated MAD-3. DITNC cells were metabolically labeled with S (top panel) or P (middlepanel) and left untreated (lanes1, 4, 5, and 8) or treated with 10 ng/ml TNFalpha for 5 min (lanes2 and 6), or 30 min (lanes3 and 7). Whole cell lysates were immunoprecipitated with either anti-MAD-3 serum (lanes 1-4) or anti-RelA serum (lanes 5-8). Lanes 4 and 8 are immunoprecipitations from S-labeled cells performed in the presence of excess peptide (lane 4, MAD-3 peptide; lane 8, RelA peptide). Gel slices containing MAD-3 and RelA-associated MAD-3 isolated from P-labeled cells were used for V8 protease mapping experiments (lower panel). MAD-3 and RelA-associated MAD-3 were digested in situ with 50 ng of V8 protease. The arrows to the left of the gel indicate the positions of TNFalpha-dependent phosphopeptides present in lanes2, 3, 6, and 7. B, TNFalpha-dependent phosphorylation of RelA. DITNC cells were metabolically labeled with S (toppanel) or P (middlepanel) and left untreated (lanes1 and 4) or treated with 10 ng/ml TNFalpha for 5 min (lane2) or 30 min (lane3). Whole cell lysates were subjected to immunoprecipitation with anti-RelA serum (lanes1-3) or anti-RelA serum plus excess peptide (lane4; peptide competition shown was performed on lysates from S-labeled cells). Gel slices containing P-labeled RelA were digested in situ with 50 ng of V8 protease (lowerpanel). Asterisks to the left of the gel indicate the positions of phosphopeptides that contained increased P incorporation in the presence of TNFalpha.



MAD-3 immunoprecipitated from P-labeled cells with anti-MAD-3 serum or co-immunoprecipitated with anti-RelA serum was subjected to one-dimensional V8 peptide mapping. Digestion of MAD-3 or RelA-associated MAD-3 from unstimulated cells with 50 ng of V8 protease resulted in five distinct phosphopeptides (Fig. 3A, lanes1 and 4; bottompanel). Digestion of MAD-3 or RelA-associated MAD-3 from cells treated with TNFalpha for either 5 or 30 min with 50 ng of V8 protease resulted in the appearance of two new phosphopeptides (Fig. 3A, lanes2 and 3 and lanes 5 and 6; bottompanel). These results demonstrate that MAD-3 undergoes TNFalpha-dependent phosphorylation prior to dissociation from RelA.

TNFalpha-dependent Phosphorylation of RelA

The involvement of Toll-dependent phosphorylation in the activation of Dorsal (7) prompted us to determine if phosphorylation of RelA is induced by TNFalpha. To this end, DITNC cells were labeled with [P]orthophosphate or [S]methionine and stimulated with TNFalpha for 5 or 30 min. Whole cell lysates were prepared and subjected to immunoprecipitation with anti-RelA serum. A protein of approximately 65 kDa was specifically immunoprecipitated with the anti-RelA serum from both [P]orthophosphate and [S]methionine-labeled cells (Fig. 3B, lane1; top and middlepanels). After 5 min of treatment with TNFalpha, increased levels of P-labeled RelA was detected (Fig. 3B, lane2; middlepanel). The [P]:[S] ratio of RelA increased 2.7-fold after treatment with TNFalpha for 5 min (an increase in the [P]:[S] ratio of RelA of approximately 3-fold was detected over multiple experiments). After 30 min of TNFalpha treatment, phosphorylation of RelA was reduced as compared to the 5-min time point, indicating that TNFalpha treatment results in a transient increase in RelA phosphorylation. Similar levels of S-labeled RelA were immunoprecipitated from DITNC cells with or without TNFalpha treatment (Fig. 3B, lanes1-3; toppanel). The immunoprecipitated P-labeled RelA was subjected to in situ digestion with V8 protease to determine if new phosphopeptides were apparent after treatment with TNFalpha. V8 protease digestion of RelA from TNFalpha-treated cells demonstrated an increased phosphorylation of two phosphopeptides that were weakly phosphorylated in untreated cells (Fig. 3B, lanes2 and 3; bottompanel). Thus, RelA undergoes transient hyperphosphorylation prior to activation of DNA binding.

TNFalpha-dependent Phosphorylation of RelA in Primary Rat Astrocytes

As cytokine-dependent phosphorylation of RelA prior to activation of DNA binding has not previously been observed, we reasoned that our detection of it in the DITNC cells might be due to immortalization of the astrocytes by large T antigen. To determine if TNFalpha-dependent phosphorylation of RelA prior to activation was a consequence of immortalization, we examined activation of RelA DNA binding (Fig. 4A) and inducible phosphorylation of RelA (Fig. 4B) by TNFalpha in primary astrocytes. Induction of RelA DNA binding was detected in whole cell lysates prepared from both primary astrocytes and in DITNC cells, with similar kinetics (Fig. 4A, compare lanes2 and 3 with lanes 5 and 6). Immunoblot analysis of the primary cell lysates with anti-RelA serum confirmed that TNFalpha did not affect levels of RelA (data not shown). To examine TNFalpha-dependent phosphorylation of RelA, primary astrocytes were labeled with [P]orthophosphate and treated with TNFalpha for 0, 5, or 10 min followed by immunoprecipitation with anti-RelA serum (Fig. 4B). Increased phosphorylation of RelA was detected after 5 min of TNFalpha treatment followed by a reduction after 10 min of TNFalpha treatment (Fig. 4B). Loss of RelA-associated MAD-3 is apparent after 10 min of TNFalpha treatment (data not shown). These results demonstrate that TNFalpha-dependent phosphorylation of RelA and activation of DNA binding by a RelA homodimer occurs in primary astrocytes as well as in the DITNC cells with similar kinetics.


Figure 4: A, solution UV-cross-linking of primary astrocytes and DITNC cells treated with TNFalpha. Primary rat astrocytes or DITNC cells were left untreated (lanes1 and 4) or treated with 10 ng/ml TNFalpha for 10 min (lanes2 and 5) or 30 min (lanes 3 and 6). Whole cell lysates were prepared and subjected to solution UV-cross-linking experiments. The position of the RelAbulletDNA adduct is indicated by the arrow to the right of the gel. The position of the molecular weight markers is indicated to the left of the gel. B, TNFalpha-dependent phosphorylation of RelA in primary astrocytes. Primary astrocytes were metabolically labeled with [P]orthophosphate and left untreated (lane1) or treated with 10 ng/ml TNFalpha for 5 (lane2) or 10 min (lane3). Whole cell lysates were prepared and subjected to immunoprecipitation with anti-RelA serum. The position of RelA is indicated by the arrow to the right of the gel slice.



Inducible Phosphorylation of RelA and MAD-3 Is Inhibited by Antioxidants

Antioxidants such as NAC are potent inhibitors of Rel activation and IkappaB degradation(4, 17) . The precise mechanism(s) by which antioxidants inhibit Rel activation and IkappaB degradation are not known. To determine if NAC inhibited activation of RelA in astrocytes, DITNC cells were either treated with TNFalpha for 1 h, treated with TNFalpha for 1 h after pretreatment with 30 mM NAC for 1 h, or left untreated. Whole cell lysates were prepared and assayed for RelA DNA binding by solution UV-cross-linking. Treatment with TNFalpha for 1 h resulted in a substantial induction of RelA DNA binding (Fig. 5A). Pretreatment of cells with NAC resulted in a significant reduction in the activation of RelA DNA binding (Fig. 5A, lane3), implicating the involvement of reactive oxygen species as intermediates for TNFalpha-induced activation of the RelA homodimer in these cells.


Figure 5: A, NAC inhibits TNFalpha-dependent activation of DNA binding by the RelA homodimer. DITNC cells were left untreated (lane1), treated with 10 ng/ml TNFalpha for 60 min (lane2), or pretreated with 30 mM NAC for 1 h prior to addition of 10 ng/ml TNFalpha for 60 min (lane3). Whole cell lysates from these three treatments were subjected to solution UV-cross-linking experiments. The position of the RelAbulletDNA adduct is indicated by the arrow to the right of the gel, and the position of molecular weight markers is indicated to the left of the gel. B, NAC inhibits TNFalpha-dependent phosphorylation of RelA and dissociation of RelA-associated MAD-3. DITNC cells were left untreated (lane1), treated with 10 ng/ml TNFalpha for 5 min (lane 2), or pretreated with 30 mM NAC for 1 h prior to addition of 10 ng/ml TNFalpha for 5 min (lane3). Whole cell lysates were collected and subjected to immunoprecipitation with anti-RelA serum. The positions of RelA and MAD-3 are indicated to the right of the gel, and the positions of molecular weight markers are indicated to the left of the gel. C, NAC inhibits TNFalpha-dependent phosphorylation of MAD-3. DITNC cells were treated as in B. Whole cell lysates prepared from these cells were subjected to immunoprecipitation with anti-MAD-3 serum. MAD-3 was isolated from a one-dimensional preparative SDS-polyacrylamide gel and subjected to in situ digestion in a second SDS-polyacrylamide gel with 50 ng of V8 protease. The arrows to the right of the gel indicate the positions of the TNFalpha-dependent phosphopeptides that are present in lane2.



As TNFalpha treatment causes increased phosphorylation of both RelA and MAD-3 prior to activation of RelA DNA binding, we reasoned that the TNFalpha-dependent phosphorylation of the RelAbulletMAD-3 complex might also be inhibited by NAC. To determine if NAC inhibited TNFalpha-dependent phosphorylation of RelA or MAD-3, DITNC cells were labeled with [P]orthophosphate and pretreated with 30 mM NAC prior to treatment with TNFalpha. Whole cell lysates prepared from these cells were subjected to immunoprecipitation with anti-RelA serum. Pretreatment of astrocytes with NAC significantly decreased the TNFalpha-dependent phosphorylation of RelA and inhibited dissociation of MAD-3 (Fig. 5B, lane3; data not shown). To determine if NAC inhibited TNFalpha-dependent phosphorylation of MAD-3, MAD-3 was immunoprecipitated from cells labeled with [P]orthophosphate and subjected to one-dimensional V8 peptide mapping. One-dimensional V8 peptide mapping of MAD-3 from cells treated with TNFalpha resulted in the appearance of two TNFalpha-dependent phosphopeptides (Fig. 5C, lane2). In cells pretreated with NAC, the TNFalpha-dependent phosphopeptides were no longer apparent in one-dimensional V8 peptide maps of MAD-3 (Fig. 5C, lane3). Thus, NAC inhibits TNFalpha-dependent phosphorylation of both MAD-3 and RelA.


DISCUSSION

Induction of Rel activity by cytokines is a post-translational event that targets an inactive, preexisting cytosolic complex containing Rel family members and an IkappaB protein(3, 4) . The ability of several protein kinases to phosphorylate IkappaBalpha and thereby relieve IkappaBalpha-mediated inhibition of Rel DNA binding in vitro suggests that phosphorylation of IkappaBalpha is a mechanism by which activation of the preexisting RelbulletIkappaB complex occurs in vivo(18, 19, 20) . The demonstration of signal-dependent phosphorylation of IkappaB prior to activation of the RelbulletIkappaB complex in vivo has provided additional support for this model(3, 4) . The function of signal-dependent phosphorylation of IkappaBalpha is not known, but it is postulated to trigger dissociation of the RelbulletIkappaBalpha complex or to act as a signal for the proteolytic degradation of IkappaBalpha.

In this study, we find that treatment of rat astrocytes with TNFalpha results in the signal-dependent phosphorylation of RelA-associated MAD-3. TNFalpha treatment resulted in the appearance of two new MAD-3-derived phosphopeptides, as determined by V8 protease mapping experiments. This result indicates that sites of TNFalpha-dependent phosphorylation are distinct from the sites of constitutive phosphorylation in MAD-3. Constitutive phosphorylation of the avian IkappaBalpha homolog, p40, is localized to a serine-rich C-terminal region between amino acids 282 and 301. (^2)This serine-rich region is highly conserved between p40 and MAD-3 and V8 protease digestion of P-labeled MAD-3 and p40 isolated from untreated cells resulted in similar one-dimensional peptide maps (data not shown). The detection of new MAD-3-derived phosphopeptides upon TNFalpha treatment indicates that signal-dependent phosphorylation of MAD-3 occurs N-terminal to serine 283 of MAD-3, possibly within the ankyrin domain. Signal-dependent phosphorylation of MAD-3 within the ankyrin domain would be consistent with a role for phosphorylation in the dissociation of the RelbulletIkappaB complex.

We have found that RelA is also subject to TNFalpha-dependent phosphorylation prior to activation of RelA DNA binding. A 2-3-fold increase in RelA phosphorylation was detected after 5 min of TNFalpha treatment. This is the first demonstration that signal-dependent phosphorylation of RelA occurs prior to activation of RelA DNA binding. The fact that TNFalpha-dependent phosphorylation of RelA precedes the induction of RelA DNA binding suggests that TNFalpha-dependent phosphorylation is important for the activation of RelA DNA binding. This conclusion is supported by the concurrent inhibition of both TNFalpha-dependent phosphorylation of RelA and induction of RelA DNA binding by NAC. At least two roles can be envisioned for the TNFalpha-dependent phosphorylation of RelA. First, phosphorylation may occur while RelA is still associated with MAD-3 and thus be required for dissociation of the RelAbulletMAD-3 complex. In favor of this hypothesis, TNFalpha-dependent phosphorylation of RelA occurs in parallel with TNFalpha-dependent phosphorylation of MAD-3. Alternatively, TNFalpha-dependent phosphorylation of RelA may occur immediately after dissociation of the RelAbulletMAD-3 complex and thus play a functional role in the nuclear import of RelA. In favor of this latter possibility, Toll-dependent phosphorylation of Dorsal correlates with its increased nuclear import during Drosophila embryogenesis (7) . Furthermore, a serine to glutamic acid mutation within the conserved phosphorylation site for cyclic AMP-dependent protein kinase of avian c-Rel allowed increased nuclear import of avian c-Rel(21) . A direct correlation between phosphorylation of this cyclic AMP-dependent protein kinase site, IkappaB association, and nuclear transport remains to be established.

Antioxidants such as NAC inhibit both the activation of Rel DNA binding and the proteolytic degradation of IkappaB(4, 17) , although the precise mechanisms by which they function is not known. Our results demonstrate that NAC inhibits the TNFalpha-dependent phosphorylation of both RelA and MAD-3. Several mechanisms by which NAC inhibits TNFalpha-dependent phosphorylation of RelA and MAD-3 can be proposed. First, antioxidants may function early in the signal transduction pathway and thereby prevent activation of the protein kinase(s) that targets RelA and MAD-3. Second, RelA and MAD-3 might be substrates for one or more cytokine-inducible protein kinases that are directly inhibited by NAC. The recent identification of a Rel-associated kinase that demonstrates substrate specificity for Rel proteins suggests that multiple protein kinases may target the RelAbulletMAD-3 complex(5) . The role of this putative Rel kinase in the cytokine-dependent activation of Rel DNA binding remains to be established. Identification of the protein kinase(s) that phosphorylate RelA and MAD-3 in vivo will be critical to fully elucidate the regulatory steps that ultimately lead to RelA activation.

The Rel protein family currently comprises seven distinct polypeptides that are capable of forming a variety of homo- and heterodimeric complexes with distinct functional properties (reviewed in (1) ). Most cell types that have been examined contain multiple Rel DNA binding complexes(4) . For example, neurons contain both constitutive and inducible forms of the canonical NF-kappaB heterodimer(22) . In contrast, we have found that astrocytes contain a single, cytokine-inducible Rel DNA binding complex that comprises a RelA homodimer. RelA homodimers have previously been detected in lymphoid cells, but as only one of multiple Rel DNA binding complexes(4) . As RelA homodimers have distinct DNA binding specificities (23) and transcriptional activation properties (24) relative to other homodimeric and heterodimeric combinations of Rel proteins, activation of a RelA homodimer will likely result in the expression of a distinct set of cytokine-inducible genes in astrocytes.


FOOTNOTES

*
This work was supported by Public Health Service Grants CA55027 (to M. H.) and NS30178 (to G. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 314-882-7971; Fax: 314-884-4597.

(^1)
The abbreviations used are: TNFalpha, tumor necrosis factor-alpha; NAC, N-acetyl-L-cysteine; LPS, lipopolysaccharide; EMSA, electrophoretic mobility shift assay; HIV-1, human immunodeficiency virus, type 1.

(^2)
R. Sachden and M. Hannink, manuscript in preparation.


ACKNOWLEDGEMENTS

We are grateful to R. Sachdev for assistance with the peptide mapping experiments using avian p40. We are also grateful to D. W. Ballard and W. C. Greene for the anti-p50 serum and to B. Fahl for the luciferase reporter plasmid.


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