Cell, Vol. 63, 603-614,

November

16, 1990, Copyright

0 1990 by Cell Press

The we/ Oncogene Encodes a KB Enhancer Binding Protein That Inhibits NFKB Function Dean W. Ballard: William H. Walker, Stefan Doerre: Prakash Sista: Jerry A. Molitor: Eric R Dixon,’ Nancy J. Petter: Mark Hannink,t* and Warner C. Greene’ * Howard Hughes Medical Institute and Departments of Medicine and Microbiology and Immunology Duke University Medical Center Durham, North Carolina 27710 t McArdle Laboratory for Cancer Research University of Wisconsin Madison, Wisconsin 53706

Summary Studies of NF-KB suggest that this enhancer binding activity corresponds to a family of at least four proteins (~50, ~55, ~75, and ~85) differentially induced with biphasic kinetics during T cell activation. While p55 and ~50 are closely related to the 50 kd DNA binding subunit of NFKB, p75 and p85 exhibit DNA binding propertles that distinguish them from this 50 kd poiypeptlde and its regulatory subunits IKB and ~55. All four members of this KB-specific protein family are structurally related to the v-Rel oncoprotein and one, ~85, appears identical to human c-Rel. v-Rel, but not nontransforming v-Rel mutants, binds to the KB enhancer and inhibits N&B-activated transcription from the IL-2 receptor a promoter and HIV-l LTR. These findings suggest a Rel-related family of KB enhancer binding proteins and raise the possibility that the transforming activity of v-Rel is linked to its inhibitory action on cellular genes under NFKB control. Introduction The KB enhancer represents a potent &-acting regulatory sequence present in many inducible cellular and viral transcription units, including those encoding the K light chain immunoglobulin (Sen and Baltimore, 1986a), the interleukin-2 receptor a (IL-2Ra) subunit (Bohnlein et al., 1988) and the type 1 human immunodeficiency virus (HIV-l) (Nabel and Baltimore, 1987). One transcription factor known to engage these related KB enhancers, a 50 kd polypeptide termed NF-KB (Sen and Baltimore, 1986a; Kawakami et al., 1988; Baeuerle and Baltimore, 1989) exists as an inactive cytoplasmic precursor in resting human T cells and as an active nuclear DNA binding protein in T cells stimulated with agents such as the tumor promoter phorbol 1Bmyristate 13-acetate (PMA) (Sen and Baltimore, 1986b; Bdhnlein et al., 1988); the cytokine tumor necrosis factor a (Lowenthal et al., 1989; Osborn et al., *Present address: Department of Biochemistry, souri, Columbia, Missouri 65201.

University

of Mis-

1989); or the bans-activator protein (Tax) of the type I human T cell leukemia virus (HTLV-I) (Ballard et al., 1988; Leung and Nabel, 1988; Ruben et al., 1988). Our recent DNA-protein cross-linking and proteolytic mapping studies have revealed that this NF-KB binding activity corresponds to a family of at least four inducible K6-specific proteins (designated ~50, ~55, ~75, and ~85). All of these proteins are sequestered in the cytoplasm of resting Tcelis but enter the nuclear compartment with distinctly biphasic kinetics following PMA stimulation (Molitor et al., 1990). Specifically, the ~55 and ~75 species are expressed in the nucleus within minutes following cellular activation, while the ~50 and ~85 species appear only after several hours of stimulation. Furthermore, partial proteolytic mapping of these NF-r&-like proteins has demonstrated distinct cleavage patterns for ~85, ~75, and p55 but a closely related pattern for ~50 and ~55. These findings suggest the possibility that the pleiotropic regulatory activities previously ascribed to a single 50 kd NF-KB factor may actually reflect the composite action of at least three gene products. However, the potential interplay of these proteins with each other, their mode of interaction with the KB enhancer (i.e., direct or indirect), and their relationship to the previously described NF-KB protein remained unknown. Recent biochemical studies of the latent cytoplasmic form of the 50 kd NF-KB protein have shown that the DNA binding activity of this factor is inhibited through its interaction with at least two proteins, a 35 kd inhibitory subunit termed IKB (Baeuerle and Baltimore, 1988a, 1988b; Ghosh and Baltimore, 1990; Zabel and Baeuerle, 1990) and a 65 kd “trans-modulator” subunit termed ~65 (Baeuerle and Baltimore, 1989). These studies have further suggested that neither of these two associated proteins appears to display DNA binding activity. in vitro reconstitution experiments have suggested that the ~65 protein is required for the cytoplasmic sequestration of NF-KB by IKB (Baeuerle and Baltimore, 1989). Cellular activation leads to phosphorylation of 1~6, which in turn promotes the dissociation and rapid translocation of NF-KB to its site of action in the nucleus (Ghosh and Baltimore, 1990). While the precise molecular mechanism for this posttranslational signal transduction event remains unresolved, it seems clear that NF-KB is capable of participating in protein-protein interactions with at least two different cellular polypeptides. During the course of these studies, Ghosh et al. (1990) and Kieran et al. (1990) reported the molecular cloning of cDNAs encoding the murine 50 kd NF-KB protein and a closely related 48 kd human protein termed KBFl. Remarkably, these proteins displayed homology with the v-rel oncogene product (v-Rel; p5gv-“‘) from the avian reticuloendotheliosis virus strain T (REV-T), as well as the chicken and human c-Rel proteins and the dorsal gene product, a ventral morphogen in Drosophila. In this regard, several similarities between NF-KB and v-Rel had been noted previously including their dual intracellular lo-

Cell

804

Figure 1. Purification of ~6 Enhancer Binding Proteins from HeLa Cell Cvtoolasmic Extracts . (A) Gel retardation profile of KB binding proteins. HeLa cytoplasmic extracts were treated with lauryldimethylamine oxide and initially applied to a poly(d[l-C])-Sepharose column. Flowthrough fractions were subjected to sequence-specific DNA affinity chromatography on Sepharose-coupled oligonucleotides composed of two kB-pd enhancer elements. Equal --p8S-c portions of protein fractions corresponding to -p75the column load (lane I), flowthrough (lane 2) and KCI step elutions (100 m M increments; lanes 3-10) from this column were mixed with * P66-A a 32P-radiolabeled kB-pd probe and analyzed -PM)\ for KB binding activity on native 5% polyacrylamide gels. The major KB binding activity eluting at 400-500 m M KCI is indicated by the arrow. (B) UV cross-linking analysis of affinity-purified KB binding proteins. Detergent-activated crude extracts (lane 4) or column fractions containing peak KB binding activity (lanes l-3) were mixed with the indicated photoreactive probes, irradiated with UV light, and analyzed on 75% SDS-poly acrylamide gels. The migration positions of the four major 32P-labeled DNA-protein adducts (~50, ~55, ~75, and ~85) and %-radiolabeled molecular weight markers are indicated. (C) SDS-PAGE analysis of electrophoretically purified proteins. Pooled eluted fractions from the KB column containing peak KB binding activity (lane 1) were precipitated with acetone and subjected to preparative SDS-PAGE. Protein species corresponding to p75 (lane 2) p66 (lane 3) and ~65 (lane 4) (as determined in subsequent DNA binding experiments; see Figures 2 and 3) were detected by shadowing with KCI, eluted from gel slices, and renatured essentially as previously described (Baeuerle and Baltimore, 1989). The analytical SDS gel showing the proteins recovered at each step was stained with silver. In the absence of bound oligonucleotide, the proteins corresponding to p75 and p55 migrate as 72 kd and 48 kd polypeptides, respectively.

calization and ability to interact with multiple other proteins (reviewed by Gilmore, 1990). Furthermore, the v-Rel oncoprotein had been shown to modulate the expression of several viral (Gelinas and Temin, 1988; Hannink and Temin, 1989) transcription units in vivo. We now demonstrate that the entire family of NF-KB proteins (~50, ~55, ~75, and ~85) is structurally related to v-Rel and that ~85 corresponds to human cellular Rel (hcRel; ~82~~-‘83. We have also found that the rapidly induced members of this protein family (~55 and ~75) each exhibit independent KB binding activity but also can associate with each other to form a unique heterodimeric complex that binds the KB enhancer. Finally, we show that the wild-type v-Rel oncoprotein also is a KB enhancer binding protein that functions as an inhibitor of NF-KBdirected gene expression. In contrast, we have found that nontransforming mutants of v-n?/ neither mediate these inhibitory effects nor display KB enhancer binding activity. These findings raise the provocative possibility that v-Relmediated transformation may be linked with the capacity of this oncoprotein to inhibit NF-KB action. Results Purification

of Cellular Proteins Binding

to the

KB Enhancer

Sequence analysis of the expanding number of functional KB enhancers present in various cellular and viral genes

has revealed a certain degree of nucleotide degeneracy within the KB core motif (consensus: GGGR[CAr]TYY[cAr]C; Lenardo and Baltimore, 1989; Greene et al., 1989). In our survey of various mutants of the wild-type KB en-

hancer present in the IL-2Ra promoter (GGGAATCTCC; designated KB-wt), we discovered a palindromic variant (GGGAATTCCC; designated KB-pd) that matches the consensus sequence, serves as a particularly potent binding site for the previously identified family of KB binding proteins (~50, ~55, ~75, and ~85) and acts as a fully functional enhancer element (Lowenthal et al., 1989; unpublished data). To characterize biochemically members of the KB family of factors further, sequence-specific DNA affinity chromatography (Kadonaga and Tjian, 1988) was performed using this KS-pd motif coupled to Sepharose and HeLa cell cytoplasmic extracts treated with lauryldimethylamine oxide. This zwitterionic detergent efficiently liberates sequestered forms of the p55 and ~75 KB enhancer binding proteins like that described for anionic detergent-mediated dissociation of NF-KB and IKB (Baeuerle and Baltimore, 1988a). As shown in the gel mobility shift profile in Figure lA, the peak of specific DNA binding activity was eluted from these KB-pd-Sepharose columns with 400500 mM KCI (lanes 8 and 7). To identify which of the KB binding proteins were present in these peak salt fractions, UV cross-linking analyses were performed using photoreactive forms of both the KBpd and KB-Wt probes as well as a functionally inactive KB mutant (ATCTATCTCC; KB-mu) (Leung and Nabel, 1988; Figure 16). These analyses revealed that cross-linked adducts corresponding to all four KB binding proteins identified in crude HeLa cytosolic extracts with the KB-wt probe (lane 4) were readily detected in affinity-purified preparations with the KB-pd probe (lane 3) but not with the nonfunctional KB-tTtU probe (lane 1). In keeping with the signif-

v-Rel Inhibits NF-KB Action 805

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icantly higher affinity binding site formed by the kB-pd sequence, the KB-wt probe detected primarily the more prevalent p75 and p55 proteins in these preparations (lane 2). When analyzed by silver staining following SDS-PAGE, the kB-Sepharose fractions containing the peak KB DNA binding activity appeared markedly enriched for three proteins migrating at 48 kd, 86 kd, and 72 kd (Figure lC, lane 1). These proteins were individually purified by preparative SDS-PAGE and reanalyzed (lanes 2-4). As discussed below, the 72 kd and 48 kd species correspond to the protein components present in the p75 and p55 crosslinked adducts and migrate with a slightly lower molecular weight due to the absence of covalently bound oligonucleotide. However, for consistency and to avoid confusion, we shall continue to refer to these proteins as p75 and p55 throughout this report. DNA Binding Properties of Purifled p55 and p75 Two characteristic properties of the recently purified 50 kd DNA binding subunit of NFKB include its cleavage by trypsin to a 40-42 kd proteolytic fragment that retains DNA binding activity and its enhanced binding activity in the presence of GTP (Baeuerle and Baltimore, 1989). Electrophoretically purified and renatured samples of p55 and p75 were studied for these properties in mobility shift assays. As shown in Figure 2A, the renatured p55 protein formed a nucleoprotein complex with the KB enhancer (designated complex 62) and was degraded by trypsin to yield a more rapidly migrating DNA-protein complex (lanes 1 and 3). In contrast, neither the full-length nor trypsin-truncated forms of p55 reacted with the &-mu probe (lanes 2 and

II

Binding

Properties

of ~55

(A and C) p55 and p75 each contain a trypsinresistant DNA binding fragment. Electrophoretically purified and renatured preparations of p55 (A) or ~75 (C)were incubated with radiolabeled probes corresponding to either functional KB (lanes 1 and 3) or nonfunctional mutant KB (lanes 2 and 4) enhancer sequences, followed by treatment with buffer (lanes 1 and 2) or trypsin (lanes 3 and 4). The resultant DNA-protein complexes were analyzed in mobility shift assays. (Band D) Effects of GTP on the binding of ~55 and ~75. Purified and renatured p55 (B) or p75 (D) proteins were incubated with a functional KB enhancer (xB-pd probe) in the absence (lane 1) or presence of 1-15 m M GTP (lanes 2-5). The resultant DNA-protein complexes were resolved on native 5% polyacrylamide gels and detected by autoradiography. The KBpd probe was used for p75 analyses since this sequence displays at least a 5-fold higher affinity than the KB-wt probe for this protein.

4) indicating sequence-specific binding. UV cross-linking analysis of the trypsin-resistant DNA binding fragment of p55 revealed a nucleoprotein complex of approximately 44 kd (data not shown). This result is consistent with the size of the previously described trypsin-resistant core of NF-KB (Baeuerle and Baltimore, 1989) when the presence of the photoreactive cross-linking probe is considered. Furthermore, like NF-KB, the DNA binding activity of purified p55 was markedly enhanced in the presence of 5-10 mM GTP (Figure 28, lanes l-5). Based on these findings and its rapid kinetics of posttranslational induction in activated T cells (Molitor et al., 1990), we conclude that the polypeptide giving rise to the p55 cross-linked adduct is probably identical to the previously described 50 kd NF-KB protein (Sen and Baltimore, 1986b; Kawakami et al., 1988; Baeuerle and Baltimore, 1989). Similar studies were performed with the electrophoretitally purified and renatured p75 protein. As shown in Figure 2C, this protein bound to the KB-pd probe (lane 1) and with lower affinity to the KB-wt probe (data not shown), but failed to bind to the functionally inactive kB-mu probe. Cross-competition studies using unlabeled wild-type and mutated KB enhancer sequences fully confirmed the KBspecific nature of p75 binding (data not shown). Notably, the electrophoretic mobility of the nucleoprotein complex formed by renatured p75 (designated as complex Bl’) was significantly less than the B2 complex formed by ~55. Like ~55, the p75 polypeptide also yielded a trypsin-resistant DNA binding core (lanes 3 and 4); however, unlike ~55, the cross-linked adduct containing this p78derived proteolytic fragment migrated at 38 kd rather than 44 kd. In further contrast to ~55, the binding of p75 to the KB enhancer

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Figure 3. The ~55 and p75 Proteins Interact to Form a Novel &-Specific Protein Complex

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was not enhanced by the addition of GTP (Figure 2D, lanes l-5). Together with the structural differences for p75 and p55 previously discerned by partial proteolytic mapping of the corresponding UV cross-linked adducts (Molitor et al., 1990) these data strongly suggest that p75 is distinct from p55 and thus represents a novel KB enhancer binding protein. The ~55 and p75 Proteins Associate to Form a Distinct KB-Specific Nucleoprotein Complex To explore the possibility that p55 and ~75 may interact with each other as well as with the KB enhancer, a series of additional renaturation experiments was performed. As shown in Figure 3A, purified and renatured preparations of p75 alone consistently yielded the slowly migrating 61’ complex (lane 1). As expected, UV cross-linking analysis of the protein component(s) present in the Bl’complex revealed only the presence of p75 (Figure 38, lane 1). Purified and renatured preparations of p55 alone gave rise to a more rapidly migrating 82 complex (Figure 3A, lane 3) which only contained the p55 protein (Figure 3B, lane 3). In contrast to the Bl’ complex, this 82 complex comigrated with one of two nucleoprotein complexes formed with crude nuclear extracts from Jurkat T cells stimulated with PMA for 16 hr (Figure 3A, lane 7). When the ~75 and p55 preparations were renatured together and examined directly in mobility shift (Figure 3A, lane 4) and crosslinking (Figure 3B, lane 4) assays, a new DNA-protein complex of intermediate electrophoretic mobility (designated as complex Bl) was readily detected. Of note, the migration of this new Bl complex precisely coincided with nucleoprotein complexes formed using nuclear extracts isolated from Jurkat cells following 10 min or 16 hr of activation with PMA (Figure 3A, lanes 6 and 7) or the complex generated by detergent treatment of cytosolic extracts (Molitor et al., 1990).

-D75

(A and B) Reconstitution of the 81 complex. DNA binding reactions were performed using the xB-pd probe and renatured ~75 (lane I), p66 (lane 2) ~55 (lane 3) combinations of these proteins renatured together (lanes 4 and 5) or crude nuclear extracts from Jurkat T cells stimulated with PMA for 10 min (lane 6) or 16 hr (lane 7). The resultant nucleoprotein complexes were then directly analyzed in both mobility shift (A) and UV cross-linking (B) assays. (C) Protein composition of the Bl complex. Cross-linked adducts corresponding to the reconstituted Bl complex (lane 1) or the Bl complexes formed with crude nuclear extracts from Jurkat T cells stimulated with PMA for 10 min (lane 2) or 16 hr (lane 3) were selectively excised from a native polyacrylamide gel following in situ UV cross-linking and subjected to SDS-PAGE (lane 4). The positions of the ~55. ~75, and ~85 cross-linked adducts are indicated with arrows.

To determine what proteins were present in this reconstituted Bl complex, in situ UV cross-linking analysis was performed on the 81 complex alone (see Experimental Procedures). As shown in Figure 3C, both p75 and ~55 were readily detected in the reconstituted 81 complex (lane 1) as well as in the Bl complex formed with crude nuclear extracts from PMA-stimulated Jurkat T cells (lanes 2 and 3). This result, coupled with the observation that Bl complex formation required corenaturation of p75 and ~55, strongly suggests that the ~55 and p75 proteins associate to form noncovalent heterodimers or even higher order structures. Based on the slower mobility of the Bl’ complex compared with the Bl and 82 complexes, it seems likely that purified p75 may also engage the KB enhancer as a homodimer (or larger multimer) similar to that described for the 50 kd DNA binding subunit of NF-KB (Baeuerle and Baltimore, 1969). Renaturation studies were also performed with the 66 kd protein recovered from the r&-pd DNA affinity columns (Figure lC, lane 3). Relative to ~75, this 66 kd protein displayed virtually no DNA binding activity in mobility shift assays (Figure 3A, lane 2) consistent with the absence of an appropriately sized UV cross-linked adduct in crude or fractionated extracts (Figure 1B). However, this protein also promoted Bl complex formation when renatured with ~55, albeit with lower efficiency than p75 (Figure 3A, lane 5). It seems likely that this 66 kd protein is identical to the previously described 65 kd &ens-modulator protein that lacks DNA binding activity and interacts with the 50 kd subunit of NF-KB (Baeuerle and Baltimore, 1969). The Entire NF-KB Family of Enhancer Proteins Appears Structurally Related we/ Oncogene Product As noted above, the murine 50 kd NF-KB human KBFl protein have been shown to

Binding to the protein and the share extensive

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Inhibits NF-KB Action

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Figure 4. A Family of Cellular KB Enhancer Binding Proteins Is Specifically lmmunoprecipitated by Anti-v-Rel Antibodies Radiolabeled DNA-protein adducts corresponding to ~50, ~55, ~75, and p85 (lanes l-4) or a control DNA-protein adduct (lane 13) were immunoprecipitated with either preimmune or specific anti-v-Rel antisera (lanes 5-12) and analyzed on 7.5% SDS-polyacrylamide gels. Fluorographic exposure times for the ~501~55, ~75, and p85 immunoprecipitations were 39, 6, and 48 hr, respectively. The electrophoretic doublet specifically immunoprecipitated by anti-v-Rel in lane 8 probably reflects partial degradation of the ~55 adduct that occurred during the immunoprecipitation reaction (see lane 2). The control adduct (lane 13) contained oligonucleotide sequences corresponding to an Spl motif located downstream of the IL-2Ra KB site and a presumed 45-50 kd proteolytic product of the full-length Spl protein (Ballard et al., 1989).

amino acid sequence homology with the MeI oncoprotein (Ghosh et al., 1990; Kieran et al., 1990). To explore whether other members of the NF-KB family are also related to v-Rel, we tested the ability of v-Rel-specific antisera to immunoprecipitate the ~50, ~55, ~75, and ~85 proteins (Figure 4). To facilitate this analysis, each of these proteins was 32P-radiolabeled by preparative UV crosslinking and electrophoretically purified (lanes l-4). As shown in lanes 5-8, radiolabeled nucleoprotein adducts corresponding to ~50 and ~55 were specifically immunoprecipitated by the anti-v-Rel antisera as compared to the corresponding preimmune serum. These results are consistent with the proposed identity between p55 and the 50 kd subunit of NF-KB (see Figure 2) and the likely precursor/product relationship of p55 and ~50 established in partial proteolytic mapping studies (Molitor et al., 1990). The anti-v-Rel antisera also specifically immunoprecipitated ~75 and ~85 (lanes 9-12). In fact, when combinations of these four UV cross-linked adducts were immunoprecipitated with anti-v-Rel antisera in a single reaction, greater quantities of the ~75 and ~85 adducts were recovered than ~50 and ~55, suggesting preferential antibody reactivity with these higher molecular weight species (data not shown). The failure of these v-Rel antibodies to immunoprecipitate an irrelevant 52 kd DNA-protein adduct generated with an Spl DNAsequence (Figure 4, lane

13-15) confirmed the specificity of these anti-v-Rel immunoprecipitations. The we/ Oncogene Encodes a Polypeptide That Directly Binds to the KB Enhancer In view of the reactivity of each of the KB enhancer binding proteins with the anti-v-Rel antisera, we next examined whether the v-Rel oncoprotein was capable of specifically binding to the KB enhancer. In this regard, prior studies have provided evidence for KB binding activity by a C-terminally truncated form of v-Rel that retains the region of homology shared with the 50 kd DNA binding subunit of NF-KB (Kieran et al., 1990). However, it has also been reported that the isolated cDNA for this 50 kd NF-KB subunit actually encodes a 105-107 kd precursor protein that displays no detectable DNA binding activity. Rather, a proteolytic processing event appears required for removal of an inhibitory C-terminal domain that then allows DNA binding by the truncated product (Ghosh et al., 1990; Kieran et al., 1990). In light of these findings, we first analyzed the DNA binding activity of the full-length v-Rel protein translated in a rabbit reticulocyte lysate system in the presence of [sS]cysteine (Figure 5A). As expected, the full-length radiolabeled v-Rel translation product immunoprecipitated with anti-v-Rel antisera migrated on SDS-polyacrylamide gels as a 58-60 kd polypeptide (lanes 1 and 4). When lysates containing the v-Rel protein were incubated with a 32P-radiolabeled KB-wt probe followed by UV irradiation, a DNA-protein adduct approximately 5 kd larger than v-Rel was formed that was also specifically immunoprecipitated by the anti-v-Rel antisera (lanes 2 and 5). In contrast, this nucleoprotein complex was not detected in immunoprecipitations of DNA binding reactions employing the nonfunctional kB-mu probe (lanes 3 and 6) nor in unprogrammed reticulocyte lysates (data not shown). To assess whether v-Rel itself directly engages the KB enhancer rather than associating with other KB binding proteins that are endogenously expressed in the translation mix, UV cross-linking studies (Figure 58) were performed on size-fractionated and renatured proteins derived from v-ml RNA-programmed (lanes l-3) or mock-programmed (lanes 4-6) reticulocyte lysates. These studies demonstrated that a renatured 57-59 kd protein unique to v-mlprogrammed lysates gave rise to a nucleoprotein complex (lane 2) that comigrated with the 65 kd adduct formed with the unfractionated lysates (Figure 5A, lane 5). These results thus demonstrate that the full-length v-Rel protein itself is capable of directly and specifically binding to the KB enhancer in the absence of other lysate factors. Additionally, recent studies with extracts from REV-T-infected Cells similarly support an intrinsic xB-specific DNA binding activity for in vivo synthesized v-Rel (data not shown). To delineate the carboxy-terminal boundary of the DNA binding domain within the 503 amino acid v-Rel oncoprotein, a nested series of carboxy-terminal truncations of the v-Rel protein was synthesized from run-off transcripts in the presence of [aaS]cysteine (Figure 5C, lanes 1, 4, 7, 10, and 13). Each of these truncated v-Rel proteins

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of Full-Length

and Truncated Forms of v-Rel

(A) Enhancer binding activity of the full-length oncoprotein. Complete v-reel transcripts were translated in a rabbit reticulocyte lysate in the presence of [%S]cysteine. The resultant 59 kd v-Rel translation product was incubated with either no DNA probe (lanes 1 and 4) or 32P-radiolabeled KB-wi (lanes 2 and 5) and KB-mu (lanes 3 and 6) probes followed by immunoprecipitation with either preimmune (lanes l-3) or anti-v-Rel-specific antisera (lanes 4-6). The arrow indicates the sequence-specific nucleoprotein complex containing MeI and the KB-wt enhancer. (B) Renaturation studies on in vitro translated v-Rel. Reticulocyte lysates programmed either with v-rel (lanes l-3) or no (mock) (lanes 4-6) RNA were size fractionated by preparative SDS-PAGE. Electrophoretically purified and renatured samples within the indicated molecular weight ranges were then tested for DNA binding activity with the KB-pd probe in UV cross-linking assays. The 57-59 kd fraction (lane 2), which corresponded precisely to the migration position for authentic %-labeled v-Rel, gave rise to a 65 kd DNA-protein adduct identical in size to that detected with unfrac-

tionated lysates (see [A], lane 5). (C)C-terminal deletion mapping of the v-Rel DNA binding domain. A nested series of 35S-radiolabeled v-Rel mutants deleted of the indicated number of C-terminal residues was tested for DNA binding activity by incubation with buffer alone (lanes 1, 4, 7, 10, and 13), the KB-mu probe (lanes 2, 5, 8, 11, and 14), or the KB-pd probe (lanes 3, 6, 9, 12, and 15) followed by UV cross-linking and immunoprecipitation with anti-v-Rel antisera. The arrows indicate the cross-linked nucleoprotein complex formed with the full-length and various mutant v-Rel proteins.

was tested for specific KB-binding activity by incubation with either the &-mu or KB-pd radiolabeled probes followed by UV cross-linking and immunoprecipitation with anti-Mel antisera. As shown in lanes 5, 8, 11, and 14, none of these truncated proteins complexed with the nonfunctional KB-mu probe. In contrast, a nuCleOprOtein adduct was readily detected with the functional KB-pd probe using the carboxy-terminal truncations of v-Rel lacking 74 (lane 8), 130 (lane 9), or 172 (lane 12) amino acids. However, removal of 230 carboxy-terminal amino acids abrogated all detectable DNA binding by the residual 273 amino acid v-Rel polypeptide (lane 15). These domain mapping studies suggest that the carboxy-terminal one-third of v-Rel is fully dispensable for DNA binding activity and, in contrast to the 105-107 kd precursor of p50/NF-KB, does not inhibit DNA binding by the wild-type v-Rel protein. The Human c-rel Proto-Oncogene Encodes a KB Enhancer Binding Protein These findings with MeI prompted us to examine whether its human cellular homolog, hc-Rel, also corresponds to a KB enhancer binding protein. Notably, Brownell et al. (1989) have identified an 82 kd protein in Daudi human B cells that is specifically immunoprecipitated by antisera generated against a C-terminal peptide deduced from the

hc-rel cDNA sequence. In the present study, a complete hc-rel cDNA was independently derived using specific primers for PCR amplification from Daudi cytoplasmic RNA (Veres et al., 1987). In contrast to the previously published clone isolated from a Daudi cDNA library (Brownell et al., 1989), this amplified hc-rel cDNA clone did not contain an in-frame 98 bp Alu fragment. For DNA binding assays, the hc-rel cDNA was transcribed in vitro and the resultant hc-rel RNA was used to program a wheat germ extract system in the presence of [35S]cysteine. Consistent with the molecular size of hcRel expressed in Daudi B cells (Brownell et al., 1989), the major radiolabeled translation product migrated as an 80-82 kd polypeptide (data not shown). When extracts containing this radiolabeled hc-Rel protein were incubated with a 32P-labeled KB probe followed by UV irradiation, a cross-linked DNA-protein adduct 3-5 kd larger than hcRel was detected (Figure 8A, lane 2). The formation of this cross-linked complex was inhibited by the addition of unlabeled oligonucleotides corresponding to related KB enhancer elements present in several cellular and viral transcription units (lanes 4-iO), but not by the addition of KB-deleted oligonucleotides (lane 3). These direct binding and cross-competition studies thus demonstrate that the 82 kd hc-Rel protein represents a cellular KB enhancer

v-Rel Inhibits NF-KB Action 809

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Figure 6. Human c-Rel Corresponds

to a KB Enhancer

“II

Binding Protein Identical to ~85

(A) DNA binding properties of hc-Rel. Full-length hc-rel transcripts were translated in wheat germ extracts containing [35S]cysteine. Programmed (lanes 2-10) and unprogrammed (lane 1) extracts were mixed with 32P-labeled rcB-pd probe (lOscpm), cross-linked by UV irradiation, and analyzed on a 7.5% SDS-polyacrylamide gel. Competitive DNA binding reactions contained a lOO-fold molar excess of unlabeled oligonucleotides comprising KB enhancer elements (27 bp) from the IL-2Ra (lane 4) and IL-2 (lane 5) promoter (Hoyos et al., 1989), H-2Kb class I MHC (lane 8; Israel et al., 1987) and 8s-microglobulin (lane 7; Kimura et al., 1986) genes, the HIV-1 LTR (lanes 8 and 9; Nabel and Baltimore, 1987) the SV40 promoter (lane 10; Gruss et al., IgEl), or a rcB-deleted variant of the IL-2Ra enhancer (lane 3; Hoyos et al., 1989). The positions of the hc-Rel-KB adduct. 35Sradiolabeled hc-Rel, and an uncharacterized 68-70 kd adduct endogenous to the wheat germ extract (denoted WG) are indicated with arrows. (B) Relative molecular sizes of ~85 and hc-Rel DNA-protein adducts. isolated 3zP-labeled adducts corresponding to ~75 (lane 1) and ~85 (lane 2) from HeLa cytosolic extracts, ~85 from nuclear extracts of an HTLV-l-infected human T cell line (ATL, lane 3), and hc-Rel from programmed wheat germ extracts (lane 4) were analyzed on a 7.5% SDS-polyacrylamide gel under reducing conditions. Adducts comigrating at 85 kd are indicated by the arrow. (C) Peptide mapping of ~85 and hc-Rel. Radiolabeled adducts corresponding to ~85 (lanes 1, 3. 5, and 7) and hc-Rel (lanes 2, 4. 6, and 8) were subjected to partial proteolysis with the indicated endoproteases or cyanogen bromide (CNBr). Cleavage products retaining covalently bound 32Plabeled DNA were separated on 15% SDS-polyacrylamide gels and detected by autoradiography.

binding protein that is clearly distinct from the 50 kd subunit of NF-KB. The hc-rel Proto-Oncogene Product Is Identical to p85 Given these DNA binding properties and the molecular size of hc-Rel, we were intrigued by the possibility that this proto-oncogene product might be identical to the factor giving rise to the ~85 adduct previously detected in our UV cross-linking studies. As shown in Figure 6B, adducts corresponding to ~85 derived from HeLa cytosolic extracts (lane 2) as well as crude nuclear extracts from HTLV-Iinfected ATL cells (lane 3) precisely comigrated on SDSpolyacrylamide gels with cross-linked DNA-protein complexes generated with in vitro translated hc-Rel (lane 4). Furthermore, these adducts were conclusively shown to contain the same KB binding protein as indicated by proteolytic digestion with four different agents including Arg-C (Figure 6C, lanes l-2) Asp-N (lanes 3-4), Lys-C (lanes 5-6), and cyanogen bromide (lanes 7-8). Thus, the hc-Rel polypeptide mediates the formation of the ~85 cross-linked adduct and corresponds to one of the two KB binding proteins expressed late in the nucleus following T cell activation (Molitor et al., 1990). This 82 kd polypeptide may also be related to HIVEN86A, an inducible 86 kd

cellular protein previously shown to bind the HIV-1 and IL2Ra KB enhancers (Franza et al., 1987; Bijhnlein et al., 1988; Ballard et al., 1988). The we/ Oncogene Encodes a Repressor of &-Directed Transcription The finding that the full-length v-Rel oncoprotein itself specifically binds to the KB enhancer prompted us to investigate the functional effects of this oncoprotein on the expression of cellular and viral transcription units controlled by NF-KB. As shown in Figure 7A, both the IL-2Ra promoter and the HIV-1 long terminal repeat (LTR), when linked to the bacterial chloramphenicol acetyltransferase (CAT) reporter gene, are markedly induced by the Tax protein of HTLV-I (22- and %-fold relative to media alone) and to a lesser extent by PMA (6- and 4.6-fold) when transfected into human Jurkat T cells. Prior studies have documented that this activation involves both the induction of NF-KB by Tax and PMA and its subsequent binding to functional KB enhancers present in these transcription units (Nabel and Baltimore, 1987; Ballard et al., 1988; Bijhnlein et al., 1988; Leung and Nabel, 1988; Ruben et al., 1988). Cotransfection of v-rel with these promoter-CAT constructs in the absence of Tax or PMA produced no signifi-

Cell 610

B

Fold Stimulation 8 12 I6 20

4

cant increases in the basal activity of either of these promoters (0.6- to 0.8-fold induction), suggesting that v-Rel in Jurkat T cells fails to function as a transcriptional activator through the KB enhancer. However, coexpression of Tax and v-Rel resulted in marked inhibition of Tax-induced activation of the IL-2Ra promoter (93% inhibition) and HIV-1 LTR (86% inhibition). Similarly, the PMA-induced response of these two NFKB-regulated promoters was significantly inhibited (63% and 60% inhibition, respectively). In contrast, v-Rel failed to inhibit Tax-induced activation of the HTLV-I LTR, a response that is mediated through a set of host transcription factors distinct from NF-KB (Paskalis et al., 1986; Shimotohno et al., 1986; Brady et al., 1987). These functional results suggest that the wild-type v-Rel protein, which specifically binds to the KB enhancer, is also capable of inhibiting the expression of a select set of cellular genes whose transcriptional activation is under NF-KB control.

24

aw.....l

43.0-

Figure 7. Functional Mutants

Analyses

of v-&l

and Nontransforming

v-Rel

(A) Inhibition of NF-rcB-dependent activation byv-Rel. Jurkat T cells (5 x 106) were cotransfected with -317 IL-ZRa-CAT, HIV-l LTR-CAT, or HTLV-I LTR-CAT reporter plasmids in the presence and absence of CM/-driven expression vectors encoding the HTLV-I Tax and v-Rel proteins and assayed for CAT activity after 46 hr of culture. Selected cultures were stimulated with PMA (50 nglml) 24 hr after transfection. Results are normalized for protein recovery (Bradford, 1976) and expressed as the mean fold increase in CAT activity f SEM relative to media alone (0.64% f 0.16% for -317 IL-2Ra-CAT, 1.43% f 0.25% for HIV-LTR-CAT, and 2.75% f 0.94% for HTLV-I LTR-CAT) obtained in three to eight independent experiments. (B) Functional effects of nontransforming v-m/ mutants. Jurkat T cells were cotransfected with the -317 IL-2RaCAT reporter plasmid (5 pg), an HTLV-I tax expression vector (5 ng), and SNV-based expression vectors encoding the wild-type v-m/ gene product (pSD214-v-ml) or various nontransforming mutants of v-m/ (pASXre/, pSDPlCCV-rel, and pdStul-Hincll). The various deletions present in these mutants are schematically represented. The wavy line in the pASX-re/ plasmid indicates the carboxy-terminal substitution of chicken c-rel sequences. CAT assay results are presented as fold stimulation relative to media controls. (C) DNA binding activities of nontransforming v-re/ mutants. Wild-type (lanes 1-3) and mutant v-Rel proteins expressed from pSD214-cv-nsl (lanes 4-6), pdStul-Hincll (lanes 7-9) or pASX-rel (lanes 10-12) were

Nontransforming Mutants of v-Rel Neither Inhibit NF-KB Function nor Associate with the KB Enhancer To explore the possible relationship between the known transforming properties of v-Rel and its inhibitory effects on NF-KB-activated gene expression, we examined the functional activity of three nontransforming mutants of v-Rel containing the various deletions or substitutions depicted in Figure 78. Both the wild-type and mutant v-rel DNAs were expressed using a spleen necrosis virus @NV) LTR-based vector. The expression plasmid, pASXrel, which encodes a mutant v-Rel protein lacking amino acids 102-273 of the wild-type v-Rel protein and contains chicken c-n?/ sequences downstream of residue 431, failed to significantly inhibit Tax-induced activation of the -317 IL-2Ra promoter. Similarly, the pSD21CWrel plasmid, which encodes an N-terminally deleted (29 amino acids) v-Rel protein, also did not inhibit the Tax-induced response. Finally, the pdStul-Hincll plasmid, which encodes a v-Rel deletion mutant lacking amino acids 274-331, also failed to inhibit the Tax response. The SNV LTR-based rel expression vectors used in these transfections all produced comparable quantities of protein when introduced into chicken embryo fibroblasts (M. Hannink, unpublished data), suggesting that these differential inhibitory effects cannot be attributed to inefficient expression of the nontransforming v-Rel mutants. Each of these nontransforming mutants of v-Rel was also tested for KB binding activity in UV cross-linking assays (Figure 7C). In contrast to the wild-type v-Rel protein (lane 3) none of these nontransforming mutants appeared capable of engaging the KB enhancer (lanes 6, 9, and 12). These findings thus support the intriguing possibility that v-Rel-mediated transformation and the inhibitory translated in a reticulocyte lysate system in the presence of [35S]cysteine. Lysates (2 11) were incubated with the indicated 32Pradiolabeled KB enhancer probes, UV cross-linked, immunoprecipitated with anti+Rel, and analyzed on a 7.5% SDS-polyacrylamide gel. Molecular size markers (in kilodaltons) are denoted on the left. The arrow indicates the position of nucleoprotein adducts formed exclusively with wild-type v-Rel.

v-Rel Inhibits NF-KB Action 811

effects of this oncoprotein on KB-directed transcription may be functionally linked.

Discussion Identification of a Novel 72 kd KB Enhancer Binding Protein In these experiments, we have identified an apparently novel 72 kd KB binding protein that is present in the cytoplasm of unstimulated human HeLa and Jurkat T cells. This protein binds specifically to and coelutes from KB-Sepharose affinity columns with the prototypical 50 kd DNA binding subunit of NF-KB at relatively high salt concentrations. UV cross-linking assays performed with electrophoretically purified preparations of this 72 kd factor have confirmed that it corresponds to the protein component present in the ~75 cross-linked nucleoprotein complex that we have previously detected in the crude nuclear extracts of PMA-activated Jurkat T cells (Molitor et al., 1990). This property, as well as its larger molecular size, altered mobility in gel retardation assays, and a distinct partial proteolytic cleavage pattern clearly distinguishes p75 from the previously identified 50 kd DNA binding subunit of NF-KB. However, we have found that ~75 binds DNA with lower apparent affinity than this 50 kd protein, a property that may have hampered its identification in prior renaturation studies. Prior studies have identified a 65 kd trans-modulator protein (~65) that associates with the 50 kd DNA binding subunit of NF-KB but exhibits no detectable independent DNA binding activity (Baeuerle and Baltimore, 1989). The relationship between ~75 and ~65 remains puzzling. Like ~65, ~75 can associate with the 50 kd NF-KB subunit (~55, identified in our UV cross-linking assays) to form a distinct nucleoprotein complex based on its altered migration in mobility shift assays. Interestingly, this intermediate mobility complex is not obtained unless these purified proteins are renatured together. Thus, the unique mobility of the resultant complex appears to reflect the association of ~55 and ~75 rather than simultaneous but independent binding of these proteins to the DNA probe. Therefore, as reported for ~65 (Baeuerle and Baltimore, 1989), it seems likely that ~75 and ~55 together form a heterodimer or higher order multimer. However, in sharp contrast to ~65, our in vitro DNA binding studies conclusively demonstrate that purified and renatured ~75 is itself a KB-specific DNA binding protein. These findings do not exclude the possibility that ~75 is a precursor of ~85 that retains DNA binding activity; molecular cloning of the corresponding cDNAs for these two proteins will be required to resolve this issue. Of note, we have recently found that ~75 exists as an inactive precursor in the cytoplasm of unstimulated Jurkat T cells and appears in the nuclear compartment within minutes following cellular activation with PMA (Molitor et al., 1990). Both of these properties are reminiscent of those ascribed to the rapidly induced 50 kd DNA binding subunit of NF-KB, suggesting that the nuclear expression of ~75 may also be regulated, perhaps coordinately, by its association with an IrcB-like inhibitory factor,

The hc-rel Proto-Oncogene Product Is a Member of the NF-KB Family of DNA Binding Proteins Data presented in this manuscript coupled with prior reports (Ghosh et al., 1990; Kieran et al., 1990) have established a functional and structural link between the v-ml oncogene product and the entire NF-KB family of enhancer binding proteins. First, renaturation experiments with the transforming v-Rel protein have confirmed that the full-length oncoprotein is a functional enhancer binding protein that engages the KB motif via N-terminal residues sharing homology with the 50 kd subunit of NFKB (Ghosh et al., 1990; Kieran et al., 1990). In contrast to the inactive 105-107 kd precursor of this 50 kd NF-KB polypeptide, the C-terminal 172 amino acids of the full-length v-Rel protein do not impair its intrinsic DNA binding activity. Second, we have found that anti-v-Rel antibodies immunoprecipitate each of the four KB-specific nucleoprotein complexes (~50, ~55, ~75, and ~85) that we have previously identified in UV cross-linking assays (Ballard et al., 1988; Molitor et al., 1990). Finally, results from DNA binding and peptide mapping studies demonstrate that the full-length hc-Rel protein also interacts with the KB enhancer in a sequence-specific manner and appears identical to the cellular protein present in the ~85 UV crosslinked adduct. Together with our evidence for a novel 72 kd KB enhancer binding protein, it seems likely that not one but multiple Rel-related DNA binding proteins contribute to the pleiotropic regulatory act’wities of NF-KB. The v-Rel Oncoprotein Functions As a TranscriptIonal Repmssor Transient gene expression studies with human Jurkat T cells have revealed that the v-rel oncogene product functions as an inhibitor of NF-KB-dependent activation of the IL-2Ra promoter and HIV-1 LTR induced by the HTLV-1 Tax protein or PMA. A relatively selective inhibitory action of v-Rel on the NF-KB family of transcription factors is suggested by the finding that this oncoprotein fails to inhibit Tax-induced activation of the HTLV-I LTR, a response that proceeds independent of NF-KB (Paskalis et al., 1986; Shimotohno et al., 1986; Brady et al., 1987). However, it is certainly possible that v-Rel may produce inhibitory effects on promoters not solely under NF-KB control. Furthermore, our finding that three nontransforming mutants of v-&fail to inhibit xB-directed transcription raises the intriguing possibility of a mechanistic link between this KB inhibitory activity and cellular transformation mediated by v-rel. While additional mutants must be tested to define precisely the v-Rel structural determinants involved in this inhibition, removal of only the N-terminal 29 amino acids (pSD214-cv-mI) or 58 central amino acids (pdStul-Hincll) from the v-Rel protein appears sufficient to disrupt entirely both its KB inhibitory function (Figure 78) and its transforming activity (Sylla and Temin, 1986; Gilmore and Temin, 1988). Of note, both of these deletions affect v-Rel sequences that share homology with the ~50 subunit of NF-KB (Ghosh et al., 1990; Kieran et al., 1990). Given the KB binding activity identified for v-Rel, one attractive mechanism to explain its inhibitory effects would

Cell 812

involve interference with nuclear NF-KB action by competition at the level of DNA binding. Indeed, dynamic competitive interactions between constitutive and inducible KB binding factors for enhancer occupancy have been proposed to regulate tumor necrosis factor a-induced expression of MHC class I genes (Israg et al., 1989). Consistent with this model, in vitro DNA binding experiments indicate that all of the nontransforming v-Flel mutants that lack functional inhibitory effects also fail to bind the KB enhancer in vitro. While nuclear expression of v-Rel would appear required for this form of inhibition, it should be noted that v-Rel is capable of mediating transformation of primary chicken lymphocytes from either the cytoplasm or the nucleus (Gilmore and Temin, 1988). A potential alternative mechanism for v-Rel inhibition mediated from either the cytoplasm or nucleus is suggested by the recent finding that v-Rel can dimerize with the 50 kd DNA binding subunit of NF-KB in vitro (Kieran et al., 1990). Similarly, it has been previously shown that this oncoprotein can also associate in vivo with c-Rel as well as a variety of other host proteins (38-40 kd, 115 kd, and 124 kd) whose functions remain unknown (Simek and Rice, 1988; Tung et al., 1988; Davis et al., 1990). The size and predominant cytoplasmic location of the 36-40 kd v-Rel-associated protein is particularly reminiscent of IKB (Ghosh and Baltimore, 1990; Zabel and Baeuerle, 1990); however, this protein remains untested for IKB activity. Thus, through protein-protein interactions, v-Rel could uncouple the normal NF-KB signal transduction pathway by altering the subunit structure of the cytoplasmic or nuclear NF-KB complex. For example, v-Rel could function either as an active IKB analog, or, alternatively, as an inactive p75 or p65 analog should these proteins prove necessary for the formation of a transcriptionally active nuclear NF-KB complex. Experimental Procedures Purification and Renaturation of p55 and p75 Cytosolic extracts were prepared from human HeLa cells (-50 liters) as described by Dignam et al. (1983). Proteins precipitated from these extracts with 25% (v/v) saturated ammonium sulfate were resuspended in 10 ml of modified buffer D (Dignam et al., 1983), containing 25 mM KCI and 15% (v/v) lauryldimethylamine oxide (Calbiochem), and consecutively applied to two 7 ml DNA-Sepharose columns equilibrated in the same buffer. The first column consisted of the alternating copolymer poly(d[iC]) covalently coupled to cyanogen bromideactivated Sepharose CL-48 (Pharmacia) according to Wu et al. (1987). Flowthrough fractions from this nonspecific DNA column were reap plied to a second specific DNA-Sepharose column coupled with oligonucleotide duplexes corresponding to a tandem repeat of a fully functional palindromic derivative (designated xB-pd) of the IL-2Ra KB enhancer (Lowenthal et al., 1989) (coding strand: Y-CAACGGCAGGGGAATT&CCTCCAGGGGAATTCCCCTCCA-3’; noncoding strand: 5’~TGGAGGGGAATTCCCCTGGAGGGGAATTCCCCTG-3’, where the underlined nucleotides represent the two It&pd core sequences). After extensive washing with modified buffer D, proteins were eluted by serial washes (68 column volumes) with detergent-depleted buffer D containing 100 mM increments in KCI concentration. Chromatography buffer solutions were supplemented with 5 mM dithiothreitol, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), and a protease inhibitor cocktail containing antipain, aprotinin, ieupeptin, pepstatin, and soybean trypsin inhibitor (5 pglml each). Eluted protein fractions containing peak KB binding activity, as defined in mobility shift assays, were pooled, concentrated by precipita-

tion with 80% (v/v) acetone at -2oOC, and subjected to preparative SDS-PAGE on 7.5% gels (Laemmli, 1970). Resolved polypeptides were visualized by KCI shadowing and passively eluted from the gel in the presence of SDS (Hager and Burgess, 1980). Proteins were concentrated and depleted of detergent by acetone precipitation and then renatured either alone or in combination as previously described (Baeuerie and Baltimore, 1989).

DNA Binding Studies Photoreactive 32P-radiolabeled DNA probes were prepared by annealing coding strand templates (27 bases) to a complementary 10 base primer (5’-AAGGAGAGGG-3’) and filling in the overhang with the Kienow fragment of DNA polymerase I in the presence of [a-aP]dATP, [a-32P]dCTP, dGTP, and equimoiar amounts of TTP and 5bromo-2’deoxyuridine 5’-triphosphate (BrdU) &Vu et al., 1987). The following coding strand templates and probe designations were used throughout this study: 5”CAACGGCAGGGGAATCTCCCTCTCCTT-3’, KB-wt; 5’-CAACGGCAGGGGAATTCCCCTClCCTT-3’, KB-pd; S”CAACGGCAGATCTATCTCCCTCTCCTT-3’, KbNI. DNA binding reactions were performed by mixing renatured proteins (05-10 pmoi) or in vitro translation products (2 ui of &sate) with these BrdU-substituted probes (l-5 x 10’ cpm/pmoi) in the presence or absence of the nonspecific competitors, poiy(d[i-C]) and p(dN)6, as previously described (Ballard et al., 1989). in some experiments, preformed DNA-protein complexes were subjected to proteolysis with 1 pg of trypsin (Worthington Biochemicais) for ~5 min at room temperature. The resultant nucleoprotein complexes were detected by gel retardation on native 5% polyacrylamide gels using a Tris-borate-EDTA buffer system (BOhnlein et al., 1988). To analyze the protein component of these complexes, DNA binding reactions were UV irradiated for 15 min at 300 nm using a Fotodyne transiiiuminator and analyzed directly on SDS-poiyacryiamide gels (Ballard et al., 1989). Alternatively, nucleoprotein complexes were first resolved by gel retardation prior to UV irradiation in situ, and excised gel slices containing select complexes were analyzed by SDS-PAGE (Molitor et al., 1990).

Antibody Bindlng Studies Rabbit anti-v-Rel antisera were prepared against a bacterially expressed P-galactosidase fusion protein containing 370 amino acids from the central region of v-Rel and shown to specifically immunoprecipitate a 59 kd polypeptide corresponding to the predicted size of v-Rel from extracts of REV-T-transformed spleen ceils (Gilmore and Temin, 1988). For immunoprecipitation studies, radiolabeled adducts (~50, ~55, ~75, and ~85) were generated from preparative-scale (20fold) UV cross-linking reactions performed with ammonium suifatefractionated cytosolic extracts (see above). Each adduct was electrophoreticaiiy purified by SDS-PAGE and then electroeluted onto DEAE membranes (NA45; Schleicher and Schuell). isolated adducts were eluted from these membranes at room temperature in RIPA buffer (10 mM Tris-HCI [pH 8.0],150 mM NaCI, 0.5% SDS, 1% Nonidet P-40, 1% deoxycholate) containing bovine serum albumin (200 pg./ml), yeast tRNA(10 @ml), and PMSF(l mM). in immunoprecipitation reactions, adducts (50 pl, 4500 cpm) were mixed with 1 ~1 of undiluted sera and 20 pl (packed volume) of protein A-Sepharose beads, and the resultant immune complexes were washed with RIPA buffer and analyzed by SDS-PAGE (Laemmli, 1970). In other experiments, the four cross-linked adducts were combined prior to immunoprecipitation.

Expression Vectors The v-rel expression plasmid, pCMV-v-m/ (Hannihk and Temin, 1989), contains the complete v-&coding sequence (Wilhemsen et al., 1984) under the transcriptional control of the cytomegalovirus (CMV) immediate early promoter (Boshart et al., 1985). The SNV-derived v-rel expression vectors contain either the transforming, wild-type v-re/ gene (pSD214+re/) or nontransforming v-rel mutant genes (plasmids pSD214-cv-ml, pdStui-Hincii, and pASX-re/)in SNV-derived retroviral vectore (Dougherty and Temin, 1986; Giimore and Temin. 1988). The v-rel gene in-pSD214-cv-re/ is a recombinant between v-rel and the turkey c-rel gene, resulting in deletion of the first 29 codons in v-re/ and the introduction of a methionine initiation codon. Piasmid pdStul-Hincll contains an internally deleted v-fel gene that encodes a 50 kd protein lacking amino acids 274-332. Piasmid pASX-rwl contains a chimeric

v&l 613

Inliibits NF-KB Action

v&-chicken c-rel gene encoding a v-Ftel mutant deleted of amino acids 102-273 and containing 172 amino acids from the c-Rel C-terminus fused to v-Rel following residue 431 (see Figure 6C). The nontransforming phenotype of the proteins encoded by these various v-rel mutants in primary chicken spleen lymphocytes have been previously described (Sylla and Temin, 1966; Gilmore and Temin, 1966; Hannink and Temin, 1969).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with USC Section 1734 solely to indicate this fact.

In Vitro TranscrIption and Wanslatlon of v-Rel and hc-Rel A Bglll-Sal1 DNA fragment from pCMV-v-re/ containing the entire v-rel coding sequence was subcloned into pGEM-4. To produce the fulllength v-Rel protein and a nested set of C-terminal deletions of this polypeptide, recombinant POEM-v-rel expression vectors (5 pg) were completely digested with Sall, BstXI, Fspl, Hincll, or Stul. These fulllength or truncated DNA templates were then transcribed with SP6 polymerase and nucleoside triphosphates as described (Melton et al., 1964). Run-off RNA transcripts (~2 pg) were translated by programming rabbit reticulocyte lysates (Promega) in the presence or absence of IssS]cysteine according to the manufacturer’s instructions. A 1.65 kb cDNA containing the entire hc-re/ coding sequence (Brownell et al., 1969) was derived by specifically priming reverse transcription of cytoplasmic RNA from Daudi B cells (a human Burkitt lymphoma line) and subsequent product amplification by PCR (Veres et al., 1967). The oligonucleotide primers were synthesized based on published N- and C-terminal coding sequences deduced from two overlapping hc-rel cDNAs (Brownell et al., 1969). These primers also contained artificial BamHl and EcoRl sites to facilitate subsequent directional subcloning into the pGEM-3 expression vector (Promega). The full-length hc-Rel protein was translated in wheat germ extracts (Promega) using [35S]cysteine, and hc-re/ run-off RNA transcripts were prepared as described above.

Refemnces

Peptlde Mapping DNA-protein adducts were generated from preparative-scale (20-fold) UV cross-linking reactions containing BrdU-substituted KB-pd probe (5 x 10’ cpm) and ~150 l.tg of protein from cellular extracts (Dignam et al., 1963) or programmed translation lysates. These adducts were separated on 7.5% SDS-polyacrylamide gels, electroblotted onto DEAE membranes, and eluted at 55°C into a minimal volume of 50 m M Tris (pH 6.0), 1 m M EDlA, 2% SDS, 1 m M PMSF, bovine serum albumin (200 &ml), and tRNA (100 us/ml). Eluted adducts were precipitated with 4 vol of acetone, dissolved in 0.4 M ammonium bicarbonate-6 M urea, reduced with dithiothreitol, and alkylated as described previously (Stone et al., 1969). Samples were subjected to partial cleavage with endoproteases Lys-C (1 hr, PC), Asp-N (3 hr, 3PC), or Arg-C (13 hr, room temperature) (Boehringer Mannheim Biochemicals) at an enzyme to protein ratio of 1:lO. Alternatively, eluted adducts were chemically digested with cyanogen bromide (40 &ml; Sigma) in 70% formic acid (Charbonneau, 1969) for 4 hr at room temperature. Following digestion, adduct cleavage products retaining covalently bound 32Plabeled DNA were analyzed on 15% SDS-polyacrylamide gels.

Received September

5, 1990; revised October 2, 1990.

Baeuerle, F?, and Baltimore, D. (1966a). Activation of DNA-binding activity in an apparently cytoplasmic precursor of the NF-KB transcription factor. Cell 53, 21 l-217. Baeuerle, P., and Baltimore, D. (1966b). IKE: a specific inhibitor of the NF-KB transcription factor. Science 242, 540-546. Baeuerle, P., and Baltimore, D. (1969). A 65kD subunit of active NF-KB is required for inhibition of NF-KB by IxB. Genes Dev. 3, 1669-1696. Ballard, D. W., B6hnlein, E., Lowenthal, J. W., Wano, Y., Franza, B. R., and Greene, W. C. (1966). HTLV-I Tax induces cellular proteins that activate the KB element in the IL-2 receptor a gene. Science 241, 1652-1655. Ballard, D. W., Bdhnlein, E., Hoffman, J., Bogerd, H., Dixon, E., Franza, 8. R., and Greene, W. C. (1969). Activation of the interleukin-2 receptor cz gene: regulatory role for DNA-protein interactions flanking the KB enhancer. New Biologist 7, 63-92. Bijhnlein, B. R., and regulates gene and

E., Lowenthal, J. W., Siekevitz, M., Ballard, D. W., Franza, Greene, W. C. (1966). The same inducible nuclear protein(s) mitogen activation of both the interleukin-2 receptor-alpha type 1 HIV. Cell 53, 627-636.

Boshart, M., Weber, F., Jahn, G., Dorsch-Hasler, K., Fleckenstein, B., and Schaffner, W. (1965). A very strong enhancer is located upstream of an immediate early gene of human cytomegalovirus. Cell 41, 521-530. Bradford, M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 246-254. Brady, J., Jeang, K.-T., Duvall, J., and Khoury, G. (1967). Identification of p40x-responsive regulatory sequences within the HTLV-I LTR. J. Virol. 67, 2175-2161. Brownell, E., Mittereder, N., and Rice, N. (1969). A human rel protooncogene cDNA containing an Alu fragment as a potential coding exon. Oncogene 4, 935-942. Charbonneau, H. (1969). Strategies for obtaining partial amino acid sequence data from small quantities of pure or partially purified protein. In A Partial Guide to Protein and Peptide Purification for Microsequencing, P Matsudiera, ed. (San Diego: Academic Press), pp. 17-30. Davis, N., Bargmann, W., Lim, M.-Y., and Bose, H. R., Jr. (1990). Avian reticuloendotheliosis virus-transformed lymphoid cells contain multiple pp59v-rsl complexes. J. Virol. 64, 564-591. Dignam, J., Lebovitz, R., and Roeder, R. G. (1963). Accurate transcrip tion initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucl. Acids Res. 17, 1475-1469.

Cell l’bansfections and CAT Assays The Jurkat human T cell line was cultured in RPM1 1640 media supplemented with 10% fetal calf serum, 2 m M glutamine, and antibiotics. Reporter plasmids containing either the IL-2Ra promoter (-317 to +109; Lowenthal et al., 1966) or full-length LTRs of HIV-1 and HTLV-I (Sodroski et al., 1965) linked to the CAT gene and the CMV-based tax cDNA expression vector (Rimsky et al., 1966) have been previously described. Jurkat human T cells (5 x 10s) were transfected with plasmid DNA using DEAE-dextran (Holbrook et al., 1967). Some cell cultures were stimulated after 24 hr with PMA (50 nglml). Cell extracts were assayed for CAT activity 46 hr posttransfection as described by Neumann et al. (1967).

Gelinas, C., and Temin, H. M. (1966). The v-rel oncogene cell-specific transcriptional activator of certain promoters. 3, 349-355.

Acknowledgments

Ghosh, S., and Baltimore, D. (1990). Activation in vitro of NF-KB by phosphorylation of its inhibitor IKB. Nature 344, 676-662.

We thank R. Randall for oligonucleotides, R. Sorg, J. Enczmann, and J. Ostrowski for advice on RNA amplification by PCR, J. Nevins for HeLa extracts, R. Puranam for advice on protein purification, T. Gilmore for anti-v-Rel antisera and some v-re/ mutants, N. Arima for F6T cell ATL extract, H. Temin for critical comments, and 8. Kissell for manuscript preparation.

Dougherty, J., and Temin, H. M. (1966). High mutation rate of a spleen necrosis virus-based retrovirus vector. Mol. Cell. Biol. 6, 43674395. Franza, B. R., Josephs, S., Gilman, M., Ryan, W., and Clarkson, B. (1967). Characterization of cellular proteins recognizing the HIV enhancer using a microscale DNA-affinity precipitation assay. Nature 331, 391-395. encodes a Oncogene

Ghosh, S., Gifford, A. M., Riviere, L. R., Tempst, P.. Nolan, G. P., and Baltimore, D. (1990). Cloning of the p50 DNA binding subunit of NF-KB: homology to rel and dqrsal. Cell 62. 1019-1029. Gilmore, T D. (1990). NF-KB, KBFl, dorsal, and related matters. Cell 62, 641-643. Gilmore, T D., and Temin, H. M. (1966). Different localization

of the

Cell 014

product of the v-rel oncogene correlates with transformation

in chicken fibroblasts and spleen cells by REV-T. Cell 44, 791-600.

Gilmore, T. D., and Temin, H. M. (1968). v-f& oncoproteins in the nucleus and in the cytoplasm transform chicken spleen cells. J. Virol. 82,

703-714. Greene, W. C., B8hnlein, E., and Ballard, D. W. (1989). HIV-l, HTLV-I and normal T-ceil growth: transcriptional strategies and surprises. Immunol. Today 10, 272-276. Gruss, P., Dhar, R., and Khoury, G. (1961). Simian virus 40 tandem repeated sequences as an element of the early promoter. Proc. Natl. Acad. Sci. USA 78, 943-947. Hager, D., and Burgess, Ft. (1960). Elution of proteins from sodium dodecyl sulfate-polyacrylamide gels, removal of sodium dodecyl sulfate, and renaturation of enzymatic activity: results with sigma subunit of Escherichie co/i RNA polymerase, wheat germ DNA topoisomerase, and other enzymes. Anal. Biochem. 109, 76-66. Hannink, M., and Temin, H. M. (1989). Transactivation of gene expression by nuclear and cytoplasmic re/ proteins. Mol. Cell. Biol. 9,

4323-4338. Holbrook, N., Gulino, A., and Ruscetti, F (1967). Cis-acting transcriptional regulatory sequences in the gibbon ape leukemia virus (GALV) long terminal repeat. Virology 757; 211-219.

activates expression ture 328, 7ll-7l3.

of human immunodeficiency

virus in T cells. Na-

Neumann, J. F., Morency, C., and Russian, K. (1967). A novel rapid assay for chloramphenicol acetyltransferase gene expression. Biotechniques 5, 444-447. Osborn, L., Kunkel, S., and Nabel, G. (1989). Tumor necrosis factor a and interleukin 1 stimulate the human immunodeficiency virus enhancer by activation of the nuclear factor KB. Proc. Natl. Acad. Sci. USA 88, 2336-2340. Paskalis, H., Felber, B., and Pavlakis, G. (1966). Cis-acting sequences responsible for the transcriptional activation of human T-cell leukemia virus type I constitute a conditional enhancer. Proc. Natl. Acad. Sci. USA 83, 8556-6562. Rimsky, L., Hauber, J., Dukovich, M., Malim, M., Langlois, A., Cullen, B., and Greene, W. C. (1966). Functional replacement of the HIV-l rev protein by the HTLV-I rex protein. Nature 335, 736-740. Ruben, W., and tion of Science

S., Poteat, H., Tan, T-H., Kawakami, K., Roeder, R., Haseltine, Rosen, C. A. (1966). Cellular transcription factors and regulaIL-2 receptor gene expression by HTLV-I tax gene product. 241, 69-91.

Sen, R., and Baltimore, with the immunoglobulin

D. (1986a). Multiple nuclear factors interact enhancer sequences. Cell 48, 705-7l6.

Hoyos, B., Ballard, D. W., BOhnlein, E., Siekevitz, M., and Greene, W. C. (1969). kB-specific DNA binding proteins: role in the regulation of human interleukin-2 gene expression. Science 244, 457-460.

Sen, R., and Baltimore, D. (1966b). lnducibility of K immunoglobulin enhancer-binding protein NF-KB by a posttranslational mechanism. Cell 47, 921-926.

Israel, A., Kimura, A., Kieran, M., Yano, O., Kanellopoulos, J., Le Bail, O., and Kourilsky, P (1967). A common positive trans-acting factor binds to enhancer sequences in the promoters of mouse H-2 and f32microglobulin genes. Proc. Natl. Acad. Sci. USA 84, 2653-2657.

Shimotohno, K., Takano, M., Teruuchi, T., and Miwa, M. (1966). Requirement of multiple copies of a 21nucleotide sequence in the U3 regions of human T-cell leukemia virus type I and type II long terminal repeats for trans-acting activation of transcription. Proc. Natl. Acad. Sci. USA 83, 6112-6116.

IsraOl, A., Le Bail, 0. Hatat. D., Piette, J., Kieran, M., Logeat, F, Wallath, D., Fellous, M., and Kourilsky, P (1989). TNF stimulates expression of mouse MHC class I genes by inducing an NF-KB-like enhancer binding activity which displaces constitutive factors. EMBO J. 8,

3793-3900. Kadonaga, J., and Tjian, R. (1966). Affinity purification of sequencespecific DNA binding proteins. Proc. Natl. Acad. Sci. USA 83, 5009-5093. Kawakami, K., Scheidereit, C., and Roeder, R. (1968). Identification and purification of a human immunoglobulin enhancer-binding protein (NF-KB) that activates transcription from a human immunodeficiency virus type I promoter in vitro. Proc. Natl. Acad. Sci. USA 854700-4704. Kieran, M., Blank, V, Logeat, F., Vandekerckhove, J., Lottspeich, F., Le Bail, O., Urban, M. B., Kourilsky, P., Baeuerle, l? A., and Israel, A. (1990). The DNA binding subunit of NF-KB is identical to factor KBFl and homologous to the reI oncogene product. Cell 82, 1007-1016. Kimura, A., Israel, A., Le Bail, O., and Kourilsky, P. (1966). Detailed analysis of the mouse H-2kb promoter: enhancer-like sequences and their role in the regulation of class I gene expression. Cell 44,261-272. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 860-665. Lenardo, M. J., and Baltimore, D. (1989). NF-KB: a pleiotropic mediator of inducible and tissue-specific gene control. Cell 58, 227-229. Leung, K., and Nabel, G. (1966). HTLV-I transactivator induces interleukin-2 receptor expression through an NF-kB-like factor. Nature

333, 776-770. Lowenthal, J. W., Ballard, D. W., Bogerd, H., Btihnlein, E., and Greene, W. C. (1969). Tumor necrosis factor-alpha activation of the IL-2 receptor-alpha gene involves the induction of kB-specific DNA binding proteins. J. Immunol. 742, 3121-3126. Melton, D., Krieg, P., Rebagliati, M., Maniatis, T, Zinn, K., and Green, M. (1964). Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucl. Acids Res. 72, 7036-7056. Molitor, J., Walker, W., Doerre, S., Ballard, D. W., and Greene, W. C. (1990). NF-KB: a family of inducible and differentially expressed enhancer-binding proteins in human T cells. Proc. Natl. Acad. Sci. USA, in Dress. Nabel, G., and Baltimore,

D. (1967). An inducible transcription

factor

Simek, S., and Rice, N. (1966). pp59 v rBr, the transforming protein of reticuloendotheliosis virus, is complexed with at least four other proteins in transformed chicken lymphoid cells. J. Virol. 82, 4730-4736. Sodroski, J., Rosen, C., Goh, W. C., and Ha&tine, W. (1965). A transcriptional activator protein encoded by the x-lor region of the human T cell leukemia virus. Science 228, 1430-1434. Stone, K., LoPresti, M., Crawford, L., DeAngelis, R., and Williams, K. (1969). Enzymatic digestion of proteins and HPLC peptide isolation. In A Practical Guide to Protein and F’eptide Purification for Microsequencing, P Matsudeira, ed. (San Diego: Academic Press), pp. 31-47. Sylla, B., and Temin, H. M. (1966). Activation of oncogenicityof proto-oncogene. Mol. Cell. Biol. 8, 4709-4716.

the c-rel

Tung, H. L., Bargmann, W., Lim, M., and Bose, H. R., Jr. (1966). The v-rel oncogene product is complexed to a 40-kDa phosphoprotein in transformed lymphoid cells. Proc. Natl. Acad. Sci. USA 85,2479-2463. Veres, G., Gibbs, R., Scherer, S., and Caskey, T. (1967). The molecular basis of the sparse fur mouse mutation. Science 237, 415-417. Wilhemsen, K., Eggleton, K., and Temin, H. M. (1984). Nucleic acid sequences of the oncogene v-rel in reticuloendotheliosis virus strain T and its cellular homolog, the proto-oncogene c-rel. J. Virol. 52, 172-162. Wu, C., Wilson, S., Walker, B., David, L., Paisley, T., Zimarino, V, and Ueda, H. (1967). Purification and properties of Drosophila heat shock activator protein. Science 238, 1247-1253. Zabel., V, and Baeuerle. P A. (1990). Purified human IKE can rapidly dissociate the complex of the NF-KB transcription factor with its cognate DNA. Cell 87, 255-265.