©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Involvement of a Putative Protein-tyrosine Phosphatase and IB- Serine Phosphorylation in Nuclear Factor B Activation by Tumor Necrosis Factor (*)

(Received for publication, December 19, 1994; and in revised form, April 6, 1995)

Sree Devi Menon Graeme R. Guy Y. H. Tan (§)

From theInstitute of Molecular and Cell Biology, National University of Singapore, 10 Kent Ridge Crescent, Singapore 0511

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Inhibitors of phosphotyrosyl protein phosphatases, pervanadate and phenylarsine oxide, abrogate tumor necrosis factor (TNF)-induced nuclear factor kappaB (NF-kappaB) nuclear translocation in transformed cell lines (U-937 and Jurkat) and primary fibroblasts (MRC-5 and REF). The inhibitors also abrogate NF-kappaB activation by the phosphoseryl/threonyl protein phosphatase inhibitor okadaic acid in U-937 cells. Inhibition of NF-kappaB activation is not due to a general inhibitory effect since neither pervanadate nor phenylarsine oxide treatment affected the constitutive DNA-binding activity of the transcription factors octamer-1 and cAMP response element-binding protein in U-937 cells, nor did these compounds inhibit the TNF-induced phosphorylation of proteins, viz. hsp-27, eukaryotic initiation factor 4e, and pp19, in MRC-5 fibroblasts. Overexpression of the protein-tyrosine phosphatase HPTPalpha resulted in a constitutive nuclear NF-kappaB-like DNA-binding activity in REF cells. Conversely, treatment of human protein-tyrosine phosphatase alpha-overexpressing cells with phenylarsine oxide led to a loss of the constitutive NF-kappaB activity. The presence of a tyrosine phosphorylation site on the inhibitor of NF-kappaB (IkappaB-alpha) suggested that it could be a target for TNF/okadaic acid-induced tyrosine dephosphorylation. However, no tyrosine phosphorylation was detected on IkappaB-alpha from unstimulated cells, while TNF/ okadaic acid-treated cells showed increased phosphorylation of IkappaB-alpha exclusively at serine residue(s). Treatment of cells with pervanadate inhibited TNF-induced IkappaB-alpha phosphorylation and degradation, whereas the serine protease inhibitors tosylphenylalanyl chloromethyl ketone and N-p-tosyl-L-lysine chloromethyl ketone prevented TNF-induced IkappaB-alpha degradation and NF-kappaB nuclear translocation, but not the TNF-induced phosphorylation of IkappaB-alpha. The data suggest that TNF and okadaic acid induce the activation of a putative protein-tyrosine phosphatase(s), leading to IkappaB-alpha serine phosphorylation and degradation and NF-kappaB nuclear translocation.


INTRODUCTION

Reversible protein tyrosine phosphorylation is a common feature in early transmembrane signaling, affecting cellular metabolism, proliferation, and differentiation(1, 2) . Although the activation of growth factor receptor and non-receptor tyrosine kinases was previously thought to initiate many cellular processes, with PTPases (^1)playing a crucial but more passive role as signal terminators, it is increasingly clear that PTPases cannot be only regarded as antagonists of the tyrosine kinase activity. Indeed, several PTPases are now known to act as positive regulators of a variety of cellular processes, including proliferation, T-cell activation, and hematopoiesis(3, 4) .

TNF is a primary mediator of the immune and inflammatory responses(5) . It has also been implicated in the pathogenesis of acquired immune deficiency syndrome, the endotoxic shock response, and cachexia. Two TNF receptors have been identified(6) , both of which lack intrinsic phosphotransferase activity. Even so, the binding of TNF to its receptor(s) rapidly induces the phosphorylation of several proteins (7) . The lack of receptor phosphotransferase activity and the stringent requirement for receptor trimerization in signal initiation (6) suggest that the signal may be transduced through association of the receptor(s) with signaling molecules. The positive regulatory role of PTPases in the signaling systems of other cytokines, viz. IL-3, IL-4, granulocyte/macrophage colony-stimulating factor, and interferon types 1 and 2(4, 8) , the receptors of which are also devoid of phosphotransferase activity, resulted in the initiation of this study to determine whether PTPases were similarly involved in the early post-receptor events induced by TNF. For this, the activation of the transcription factor NF-kappaB was chosen as an end point assay for TNF signal transduction since TNF is a potent and rapid inducer of NF-kappaB (9) . Many of the TNF-induced genes, viz. human immunodeficiency virus type 1, major histocompatibility complex class 1 and 2 antigens, IL-2 receptor alpha-chain, IL-6, and IL-8, appear to require NF-kappaB for transcriptional activation(10) .

NF-kappaB is a heterodimer comprising a 48-55-kDa DNA-binding subunit (p50 or NF-kappaB1) and a 65-68-kDa transactivator (p65 or RelA)(10) . It is sequestered within the cytosol by association with an inhibitory protein known as IkappaB-alpha. Activation is post-translational and results from the dissociation of the IkappaB-alphabulletNF-kappaB complex followed by translocation of the released NF-kappaB into the nucleus. Several other agents are known to activate NF-kappaB, including the cytokines IL-1 and IL-2, viruses, bacterial lipopolysaccharides, and T-cell activators(10) . Although modification of IkappaB-alpha by phosphorylation and/or proteolysis appears to be involved in NF-kappaB activation(11, 12, 13, 14) , none of the signaling pathways leading to activation has been fully elucidated. Several signaling events have been implicated in the TNF-induced phosphorylation of IkappaB-alpha, viz. activation of phosphatidylcholine-specific phospholipase C/sphingomyelinase/ceramide, Ras/c-Raf, and phosphatidylcholine-specific phospholipase C/-protein kinase C pathways(13, 15, 16) , while recent findings suggest that TNF-induced IkappaB-alpha degradation occurs via the ubiquitin-proteasome pathway(17, 18) .

In this study, we report that PTPase inhibitors, pervanadate and phenylarsine oxide (PAO), abrogate the TNF-induced nuclear translocation of NF-kappaB in all transformed and primary cells tested. The inhibitors also abrogate NF-kappaB activation by okadaic acid, a phosphoseryl/threonyl phosphatase inhibitor that mimics the early phosphorylation events induced by TNF(19) . Correspondingly, a constitutive NF-kappaB-like DNA-binding activity was present in nuclear extracts of REF cells overexpressing the receptor-like PTPase HPTPalpha. Although a tyrosine phosphorylation site is present on IkappaB-alpha(20) , it was not phosphorylated at tyrosine and was found to undergo only serine phosphorylation in TNF- or okadaic acid-treated cells. Pervanadate treatment inhibited both the TNF-induced phosphorylation and degradation of IkappaB-alpha, suggesting that PTPase activity is required for the TNF/okadaic acid-induced phosphorylation and proteolysis of IkappaB-alpha and NF-kappaB activation. Although IkappaB-alpha phosphorylation is sufficient to induce NF-kappaB DNA-binding activity in vitro(11, 12, 13) , this is not the case in vivo as the protease inhibitors TPCK and TLCK, which do not affect the TNF-induced phosphorylation of IkappaB-alpha, effectively prevent the TNF-induced proteolysis of IkappaB-alpha with a concomitant loss of NF-kappaB nuclear translocation.


MATERIALS AND METHODS

Reagents

Recombinant human TNF (Cellular Products Inc., Buffalo, NY) and recombinant murine TNF (Genzyme Corp., Cambridge, MA) had a specific activity of 4 10^7 units/mg. Okadaic acid (BIOMOL Research Laboratories Inc., Plymouth Meeting, PA) was prepared as a 0.4 mM stock solution in dimethyl sulfoxide. All other reagents were purchased from Sigma. Stock solutions of H(2)O(2) (200 mM) and orthovanadate (10 mM) were prepared fresh in distilled water and added directly to the culture medium to produce pervanadate. Phenylarsine oxide was prepared at 120 mM in dimethyl sulfoxide. 100 mM stock solutions of TPCK and TLCK were stored in aliquots at -20 °C in dimethyl sulfoxide and distilled water, respectively.

Cell Cultures

The transformed human cell lines (U-937 monocytes (American Type Culture Collection, Rockville, MD) and Jurkat T lymphoblasts (provided by Kazuo Sugamura, Tohoku University School of Medicine)) and primary human fibroblasts (MRC-5; European Collection of Animal Cell Cultures, Salisbury, United Kingdom) were maintained as described previously(21) . Nontransfected, vector-transfected, and HPTPalpha-transfected rat embryo fibroblasts (REF; provided by C. J. Pallen, Institute of Molecular and Cell Biology, National University of Singapore) were maintained in Dulbecco's modified Eagle's medium containing 4.5 mg/liter glucose and 10% fetal bovine serum. 48 h prior to treatment of cells with various reagents, the growth media were replaced with media containing 1% serum.

Antibodies

Affinity-purified rabbit polyclonal antibody (Multiple Peptide Systems, San Diego, CA) was raised against the synthetic peptide sequence EFTEDELPYDDCVFGGQRLTL, corresponding to the C terminus of human monocytic IkappaB-alpha(20) . This antibody recognized both the 37- and 43-kDa forms of in vitro synthesized HA-tagged IkappaB-alpha (HA-IkappaB-alpha), while neither was detected using preimmune serum. Specificity of the antibody is further suggested by the absence of either form of HA-IkappaB-alpha when immunoprecipitations were conducted in the presence of the IkappaB-alpha peptide antigen and by its inability to immunoprecipitate a heterologous protein, such as in vitro synthesized mammalian transcription factor E2F-1 (provided by Wan Jin Hong, Institute of Molecular and Cell Biology, National University of Singapore). The rabbit polyclonal anti-HA antibody, raised against the peptide YPYDVPDYA from influenza HA, and the peptide antigen were purchased from Berkeley Antibody Co. (Richmond, CA).

Electrophoretic Mobility Shift Assay

Nuclear or cytoplasmic extracts were prepared and examined for NF-kappaB DNA-binding activity using NF-kappaB binding sequence from the human immunodeficiency virus type 1 enhancer or its nonbinding mutant as described previously(21) . All protein-DNA complexes in extracts from human cells were immunologically characterized using antibodies against members of the Rel/NF-kappaB family(21) . Electrophoretic mobility shift assays using nuclear extracts from REF cells were performed with a probe comprising only the NF-kappaB core binding sequence or its mutant. The oligonucleotides for the octamer-1 (22) and CREBP (23) binding sequences were provided by the Institute of Molecular and Cell Biology DNA synthesis service.

Two-dimensional Gel Electrophoresis

Radiolabeling of cells, extract preparation, and two-dimensional gel electrophoresis were done according to Guy et al.(7) .

Immunofluorescent Staining

Subcellular localization of NF-kappaB was performed by indirect immunofluorescence using an antibody against the p65 subunit of NF-kappaB as described previously(21) .

Immunoblotting

Cells were lysed by freeze-thawing in buffer containing the phosphatase inhibitors NaF (50 mM), NaPP(i) (30 mM), orthovanadate (1 mM), and microcystin-LR (4 µM) (Life Technologies Inc.) and the protease inhibitors phenylmethylsulfonyl fluoride (1 mM), 10 µg/ml each antipain, benzamidine, chymostatin, leupeptin, and pepstatin, and 5 µg/ml aprotinin. Lysates (10 µg of total protein) were resolved by electrophoresis on 10% SDS-polyacrylamide gels and electroblotted onto polyvinylidene difluoride membranes. IkappaB-alpha was detected using the anti-IkappaB-alpha antibody followed by horseradish peroxidase-preconjugated donkey anti-rabbit antibody (Amersham, Buckinghamshire, United Kingdom). Tyrosine-phosphorylated proteins were detected using horseradish peroxidase-preconjugated anti-phosphotyrosine antibody (PY20, Transduction Labs, Lexington, KY). Blots were developed by enhanced chemiluminescence (Amersham Corp.) according to the manufacturer's instructions.

Orthophosphate Labeling of Cells

U-937 cells were placed for 3.5 h in 60 ml of phosphate-free medium (Life Technologies, Inc.) containing 1% bovine serum albumin and then incubated for 3 h in 5 ml of the same medium containing 1 mCi/ml H(3)PO(4) (DuPont NEN). The cells were resuspended in HEPES-buffered saline containing CaCl(2) and MgCl(2) at a density of 1.4 10^7 cells/ml/reaction. After treatment, cells were instantaneously lysed with 5 stop solution (1 stop solution = 25 mM Tris-Cl (pH 7.8), 5 mM EDTA, 100 mM NaCl, 1% Triton X-100, 1% bovine serum albumin, and 0.005% sodium azide containing phosphatase and protease inhibitors). Confluent cultures of MRC-5 fibroblasts grown in 150-mm diameter Petri dishes were radiolabeled as described for U-937 cells. After treatment, cells were washed with ice-cold phosphate-buffered saline (pH 7.4) and then incubated for 10 min at 4 °C in hypotonic buffer (10 mM HEPES (pH 7.0), 10 mM KCl, 10 mM NaCl, and 2.4 mM MgCl(2)). Cells were harvested and lysed by homogenization in 600 µl of buffer (10 mM HEPES (pH 7.9), 1.5 mM MgCl(2), and 10 mM KCl) containing protease and phosphatase inhibitors. Lysates were clarified by centrifugation, and this extract is referred to as the whole cell extract.

Immunoprecipitation of IkappaB-alpha or HA-IkappaB-alpha

Immunoprecipitation was performed at 4 °C. Whole cell extracts from P-labeled cells or an aliquot of in vitro synthesized HA-IkappaB-alpha (3 ng) was added to preimmune rabbit serum (17 µl) conjugated to protein A-agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA) and mixed for 15 h. Precleared lysates were mixed for 2 h with anti-IkappaB-alpha antibody (17 µl) or anti-HA antibody (130 µl) preconjugated to protein A-agarose beads. For competition reactions, immunoprecipitations were conducted in the presence of the IkappaB-alpha peptide antigen at 1 mg/ml. For double immunoprecipitation reactions, IkappaB-alpha or HA-IkappaB-alpha was eluted from the beads after the first immunoprecipitation by incubation with either the IkappaB-alpha or HA peptide antigen, depending on the order of the immunoprecipitations. Immunoprecipitates were washed with 1 ml of radioimmune precipitation buffer (50 mM Tris-Cl (pH 7.4), 150 mM NaCl, 1% sodium deoxycholate, 1% Triton X-100, and 0.1% SDS) followed by radioimmune precipitation/urea buffer (radioimmune precipitation buffer with 1 M urea) and then were resuspended in 2 SDS sample buffer. Samples were resolved by electrophoresis on 10% SDS-polyacrylamide gels.

Phosphoamino Acid Analysis

After electroblotting and autoradiography, the piece of the membrane containing IkappaB-alpha was excised and subjected to acid hydrolysis (105 °C for 70 min in 6 N HCl) and one-dimensional thin-layer chromatography as described(24) .

Construction of HA-IkappaB-alpha Expression Vector

The IkappaB-alpha cDNA (20) was a gift from Drs. Steve Haskill and Albert Baldwin Jr. (Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC). The epitope-tagged IkappaB-alpha molecule was created by inserting an epitope from the viral hemagglutinin, YPYDVPDYA(25) , between the first and second amino acid residues of IkappaB-alpha. This was obtained through polymerase chain reaction-mediated mutagenesis with the 5`-primer HA-IkappaB-alpha-N, which carries an EcoRI site (underlined), TCT CTC TGC GGC CGC GAA TTC GCC ATG TAC CCC-TAC GAC GTG CCC GACTAC GCC TTC CAG CCG GCC GAG-CGC CCC, and the 3`-primer IkappaB-alpha-C, which contains a BamHI site (underlined), CTC TCT GGA TTC TCA TAA CGT CAG ACG CTG GCC TCC AAA CAC. The polymerase chain reaction-amplified 980-base pair DNA fragment was digested sequentially with EcoRI and BamHI and ligated with the mammalian expression vector pXJ41(26) . The full length of the cDNA was sequenced to ensure that no unintended sequence changes had occurred. This placed the HA-IkappaB-alpha cDNA under the influence of the strong constitutive cytomegalovirus promoter.

In Vitro Transcription and Translation of HA-tagged IkappaB-alpha

In vitro transcription and translation of HA-IkappaB-alpha were achieved with the T7 transcription/translation coupled reticulocyte lysate system (Promega). Using the construct described above, 2 µg of DNA was used in a 100-µl reaction containing [S]methionine according to the manufacturer's instructions.


RESULTS

Inhibitors of PTPase Activity Abrogate the Induction of NF-kappaB DNA-binding Activity by TNF and Okadaic Acid

The combination of H(2)O(2) and orthovanadate results in the formation of aqueous peroxovanadium compounds (collectively referred to as pervanadate) that inhibit PTPase activity(27, 28) . To assess if tyrosine dephosphorylation is involved in TNF signal transduction leading to NF-kappaB activation, U-937 cells were treated with pervanadate prior to TNF. Pervanadate markedly inhibited the TNF-induced nuclear NF-kappaB DNA-binding activity (Fig.1A, compare lanes1 and 2 with lanes7 and 8), whereas treatment with either H(2)O(2) or orthovanadate alone had no significant effect (lanes 1-6). Treatment of cells with another PTPase inhibitor, PAO(29, 30) , also abrogated NF-kappaB activation by TNF (Fig.1A, compare lanes1 and 2 with lanes9 and 10).


Figure 1: PTPase inhibitors abrogate the TNF- or okadaic acid-induced NF-kappaB DNA-binding activity. Cells were treated with H(2)O(2) (1 mM), orthovanadate (Van) (100 µM), pervanadate (pV) (100 µM; prepared by mixing H(2)O(2) at 1 mM with orthovanadate at 100 µM), or PAO (2.5 µM) in the absence or presence of TNF at 1.2 nM for 15 min (A, C, and D) or with okadaic acid (OA) at 2 µM for 30 min (E). Nuclear extracts (5 µg) were analyzed for NF-kappaB DNA-binding activity using P-labeled wild-type NF-kappaB sequence. The protein-DNA complexes were immunologically identified as NF-kappaB or homodimers of p50(11) . Nonspecific DNA-binding complexes (n.s.) and the unbound probe (open arrowheads) are indicated. B, U-937 cells were treated with various compounds as described above. Whole cell extracts (10 µg of protein) were analyzed for phosphotyrosine by immunoblotting.



Immunoblotting with an anti-phosphotyrosine antibody showed that pervanadate and PAO treatment resulted in a marked increase in the phosphotyrosine content of cellular proteins, an observation consistent with the expected inhibitory effect of these compounds on PTPase activity (Fig.1B, compare lane1 with lanes4 and 5). However, differences were noted between the pervanadate- and PAO-induced patterns of protein phosphorylation, suggesting differences in the sensitivity of cellular PTPases to the two agents. Although inhibition of PTPase activity by H(2)O(2) or orthovanadate alone has been reported (28, 31) , this was not observed here (Fig.1B, lanes 1-3). The effect of pervanadate, H(2)O(2), and orthovanadate on PTPases was verified by PTPase activity assays using extracts from pervanadate-treated cells, which possessed no detectable PTPase activity against the tyrosine-phosphorylated hirudin C-terminal peptide (residues 53-65) or gastrin N-terminal peptide (residues 1-17) as substrate, while the activity in H(2)O(2)- or orthovanadate-treated cells was comparable to that in untreated controls (data not shown). This indicates that H(2)O(2) or orthovanadate alone is significantly less effective than pervanadate as a PTPase inhibitor when used on intact cells. The inhibition of NF-kappaB activation by pervanadate was dose-dependent (25-100 µM) and correlated with the inhibition of cellular PTPase activity (data not shown).

Pervanadate and PAO also inhibited the TNF-induced NF-kappaB DNA-binding activity in Jurkat T-cells (Fig.1C) and primary MRC-5 fibroblasts (Fig.1D). Both inhibitors also abrogated NF-kappaB activation by okadaic acid (Fig.1E), a compound reported to mimic the early phosphorylation events induced by TNF (19) .

Pervanadate and PAO Do Not Inhibit Octamer-1 or CREBP DNA-binding Activity or the TNF-induced Phosphorylation of hsp-27, eIF-4e, and pp19

To determine if the loss of TNF/okadaic acid-induced NF-kappaB activation was specific, the effect of pervanadate and PAO on the DNA-binding activity of two constitutive transcription factors, octamer-1 and CREBP, was examined. Neither pervanadate nor PAO had any effect on octamer-1 or CREBP DNA-binding activity in U-937 cells (Fig.2, A and B). In addition, the effect of the PTPase inhibitors on other TNF-inducible events, like the phosphorylation of key cellular substrates, was examined. Treatment of MRC-5 fibroblasts with either pervanadate or PAO did not inhibit the TNF-induced phosphorylation of hsp-27, eIF-4e, and the currently unidentified phosphoprotein pp19 (Fig.2, C-F).


Figure 2: PTPase inhibitors do not inhibit octamer-1 and CREBP DNA-binding activities or the TNF-induced phosphorylation of cellular proteins. A and B, nuclear extracts from the U-937 cells used in Fig.1were analyzed for octamer-1 (Oct-1) or CREBP DNA-binding activity using P-labeled oligonucleotides encompassing the binding site for either transcription factor. C-F, shown is the two-dimensional gel electrophoresis of cytosolic extracts from P-labeled MRC-5 fibroblasts treated with TNF alone or together with pervanadate (pV) or PAO as described in the legend to Fig.1. The major substrates for TNF-induced protein phosphorylation (hsp-27, eIF-4e, and pp19) are indicated.



PTPase Inhibitors Block the Release of NF-kappaB from IkappaB-alpha

Indirect immunofluorescence using an antibody against the p65 subunit of NF-kappaB showed that the staining pattern in pervanadate- or PAO-treated MRC-5 cells was similar to that in untreated controls, with NF-kappaB being predominantly in the cytoplasm (Fig.3, compare A with C and D). No nuclear staining was detectable when inhibitor-treated cells were exposed to TNF, whereas a distinctly nuclear fluorescence was seen in cells treated with TNF alone (Fig.3, compare B with C and D), suggesting that NF-kappaB does not translocate from the cytosol to the nucleus in inhibitor-treated cells. Furthermore, the cytosolic NF-kappaB did not bind to its cognate DNA sequence (Fig.3E, lanes 1-4), but could be induced to bind DNA upon treatment of cytosolic extracts with sodium deoxycholate, a compound known to disrupt the IkappaB-alphabulletNF-kappaB complex (Fig.3E, lanes 5-9)(32) . The sodium deoxycholate-induced activities from untreated and inhibitor-treated cells were comparable and were significantly greater than in cells treated with TNF alone, indicating that the PTPase inhibitors prevent the release of NF-kappaB from the latent cytosolic pool.


Figure 3: PTPase inhibitors abrogate the TNF-induced nuclear translocation of NF-kappaB. A-D, MRC-5 fibroblasts were immunostained with peptide antigen affinity-purified anti-p65 antibody. The bound antibody was visualized with biotinylated goat anti-rabbit IgG and fluorescein isothiocyanate-conjugated avidin. E, cytosolic extracts (20 µg of protein) from U-937 cells were incubated with labeled probe for 10 min, after which sodium deoxycholate (DOC) was added to a final concentration of 0.5%, followed by Nonidet P-40 at 1.2% (lanes 5-9). The wild-type NF-kappaB sequence was used as the probe in all lanes except lane9, where the mutant sequence was used instead. Nonspecific protein-DNA complexes (n.s.) and the unbound probe (open arrowhead) are indicated. The time and dosage of the treatments were as described in the legend to Fig.1. pV, pervanadate.



Constitutive Nuclear NF-kappaB-like DNA-binding Activity in Cells Overexpressing HPTPalpha

The findings described above led us to examine the status of NF-kappaB activation in rat embryo fibroblasts (REF) overexpressing the receptor-like PTPase HPTPalpha(26, 33) . In nontransfected REF cells, pervanadate and PAO inhibited the NF-kappaB-like DNA-binding activity induced by TNF (Fig.4A, compare lanes 1-4). Nuclear extracts from HPTPalpha-transfected cells contained three DNA-binding complexes, designated bands 1-3, that specifically bound the NF-kappaB probe (Fig.4A, lanes6 and 7). Bands 1 and 2 were absent in vector-transfected controls, while the band 3 complex was already present in vector-transfected cells and was induced further upon HPTPalpha transfection (Fig.4A, compare lanes5 and 6). The similar electrophoretic mobility of band 1 compared with that of the TNF-induced protein-DNA complex in nontransfected REF cells suggests that this complex is likely to comprise NF-kappaB. Complexes corresponding to bands 2 and 3 were not TNF-inducible. Treatment of HPTPalpha-transfected cells with PAO resulted in the loss of bands 1 and 2, suggesting that the constitutive nuclear NF-kappaB-like DNA-binding activity requires PTPase activity (Fig.4B).


Figure 4: NF-kappaB-like DNA-binding activity in cells overexpressing HPTPalpha and inhibition of the DNA-binding activity by PAO. A, nuclear extracts from REF cells treated with TNF alone (lane2) or in combination with pervanadate (pV) (lane3) or PAO (lane4) were analyzed for NF-kappaB-like DNA-binding activity together with nuclear extracts from REF cells transfected with the vector pXJ41 (lane5) or with the vector encoding the PTPase HPTPalpha (lanes6 and 7). The labeled wild-type NF-kappaB oligonucleotide was used as the probe in all lanes except lane7, where the mutant sequence served as the probe. Specific protein-DNA complexes (Bands 1-3), the nonspecific protein-DNA complex (n.s.), and the unbound probe (openarrowhead) are indicated. B, HPTPalpha-expressing cells were incubated with PAO for the times indicated, after which nuclear extracts were prepared and analyzed for NF-kappaB-like DNA-binding activity.



Immunoprecipitation and Phosphoamino Acid Analysis of IkappaB-alpha from TNF/Okadaic Acid-treated Cells

The involvement of tyrosine dephosphorylation in NF-kappaB activation and the presence of a conserved tyrosine phosphorylation site on human, chicken, rat, and porcine IkappaB-alpha (34) led us to examine if IkappaB-alpha was the direct target for TNF-induced tyrosine dephosphorylation. A rabbit polyclonal antibody raised against a synthetic peptide corresponding to the C-terminal residues of human IkappaB-alpha was used to immunoprecipitate IkappaB-alpha from P-labeled cells. Immunoprecipitation yielded a single phosphorylated protein of 43 kDa (Fig.5A, lanes 1-6 and 9-12) that was absent in experiments performed in the presence of excess IkappaB-alpha peptide antigen (lane7) or when preimmune serum was used instead of the anti-IkappaB-alpha antibody (lane8), indicating specificity in the interaction between the phosphoprotein and the anti-IkappaB-alpha antibody. In vitro transcription and translation of recombinant IkappaB-alpha tagged at its N terminus with an epitope from viral HA (25) yielded two products of 43 and 37 kDa (Fig.5B, lane2). Both were immunoprecipitated using antibodies against the HA epitope or IkappaB-alpha, suggesting that they are full-length translation products (data not shown). Treatment of the fusion proteins with protein phosphatase 1, but not protein phosphatase 2A, resulted in an apparent size shift from 43 to 37 kDa, suggesting that the difference in size is due to the phosphorylation of the 37-kDa IkappaB-alpha (Fig.5B, compare lanes 2-4). Immunoblots of cell extracts detect only the 37-kDa species, indicating that most of IkappaB-alpha exists in the nonphosphorylated form in vivo (Fig.5C; see last paragraph under ``Results'').


Figure 5: Kinetics of ligand-induced IkappaB-alpha phosphorylation and proteolysis and NF-kappaB activation. A, immunoprecipitation of IkappaB-alpha from P-labeled cells. U-937 cells were treated with TNF (1.2 nM) for various lengths of time (lanes 2-8) or with okadaic acid (OA) (2 µM) for 15 min (lane10). Orthophosphate-labeled MRC-5 fibroblasts were incubated with TNF at 1.2 nM for 2 min (lane12). Cells were lysed, and IkappaB-alpha was immunoprecipitated with an antibody raised against its C terminus (lanes 1-6 and 9-12) or with the same antibody in the presence of a 1 mg/ml concentration of the peptide against which it was generated (lane7). Preimmune serum was used instead of the anti-IkappaB-alpha antibody in lane8. Immunoprecipitates were analyzed by SDS-PAGE followed by autoradiography. B, in vitro transcription and translation of HA-tagged IkappaB-alpha. Expression vector pXJ41 (lane1) or the vector carrying the HA-IkappaB-alpha insert (lanes 2-4) was transcribed and translated in vitro in the presence of 2.5 mCi/ml [S]methionine. An aliquot (3 ng) of the synthesized product was treated with 40 units/ml protein phosphatase 1 or 2A (PP1 and PP2A, respectively) at 30 °C for 2 h in buffer (50 mM Tris-HCl (pH 7.0), 1 mM EDTA, 0.5 mM dithiothreitol, 0.1% beta-mercaptoethanol, and 0.2 mg/ml bovine serum albumin), after which samples were resolved by SDS-PAGE. C, levels of IkappaB-alpha in TNF-treated cells. U-937 cells were treated with TNF, and whole cell lysates were analyzed for IkappaB-alpha by immunoblotting using the anti-IkappaB-alpha antibody. D, NF-kappaB activation in TNF-treated cells. Nuclear extracts of TNF-treated U-937 cells were analyzed for NF-kappaB DNA-binding activity using the P-labeled wild-type NF-kappaB sequence as the probe. E, phosphoamino acid analysis of IkappaB-alpha from TNF- or okadaic acid-treated cells. Immunoprecipitates of IkappaB-alpha from P-labeled U-937 cells were resolved by SDS-PAGE and electroblotted onto polyvinylidene difluoride membranes. Membranes were autoradiographed, and the portion of the membrane containing IkappaB-alpha was cut out and processed as described under ``Materials and Methods.''



TNF treatment of U-937 cells resulted in an overall increase in IkappaB-alpha phosphorylation (Fig.5A, lanes 1-6). The induced phosphorylation was rapid and transient, peaking at 1 min after stimulation. The kinetics of IkappaB-alpha phosphorylation paralleled its degradation (Fig.5C), and both events preceded NF-kappaB nuclear translocation (Fig.5D). Treatment of U-937 cells with the TNF mimetic, okadaic acid(19) , also led to enhanced phosphorylation of IkappaB-alpha (Fig.5A, lanes 9 and 10). Similar results were obtained with TNF-treated MRC-5 fibroblasts, although the increase in phosphorylation was less marked (Fig.5A, lanes11 and 12). Phosphoamino acid analysis of IkappaB-alpha revealed that the basal and TNF/okadaic acid-induced phosphorylations were exclusively due to phosphorylation of serine residue(s) (Fig.5E). No phosphorylation was detected at either tyrosine or threonine, even upon prolonged autoradiography. Although a conserved tyrosine phosphorylation site is present on IkappaB-alpha, it is not phosphorylated at tyrosine and cannot be the direct target for the putative PTPase(s) involved in NF-kappaB activation.

Effect of Pervanadate on TNF-induced IkappaB-alpha Phosphorylation and Proteolysis

Pervanadate treatment resulted in inhibition of the induced phosphorylation, suggesting involvement of PTPase(s) in signaling events upstream of the serine phosphorylation of IkappaB-alpha (Fig.6A, compare lanes1 and 2 with lanes7 and 8). Pervanadate treatment also prevented the TNF-induced proteolysis of IkappaB-alpha (Fig.6B). Peroxide and orthovanadate, which failed to inhibit PTPase activity when added alone to cells (Fig.1B), had no effect on the TNF-induced phosphorylation of IkappaB-alpha (Fig.6A, lanes 1-6).


Figure 6: PTPase inhibitors abrogate the TNF-induced phosphorylation and proteolysis of IkappaB-alpha. A, immunoprecipitation of IkappaB-alpha from P-labeled cells. Orthophosphate-labeled U-937 cells were treated with TNF for 2 min (lane2) or with H(2)O(2), orthovanadate (Van), or pervanadate (pV) for 15 min (lanes3, 5, and 7, respectively). Treatment with a combination of H(2)O(2), orthovanadate, or pervanadate and TNF was performed by preincubating cells with either reagent for 13 min followed by TNF for an additional 2 min (lanes4, 6, and 8). IkappaB-alpha was immunoprecipitated, and samples were resolved by SDS-PAGE. B, immunoblot of IkappaB-alpha. U-937 cells were treated with TNF for various intervals of time in the absence or presence of pervanadate. Pervanadate treatments were for a total of 20 min. Whole cell extracts were prepared, and the level of IkappaB-alpha was determined by immunoblotting using the anti-IkappaB-alpha antibody.



Effect of Serine Protease Inhibitors on TNF-induced IkappaB-alpha Phosphorylation

The serine protease inhibitors TPCK and TLCK block the TNF-induced proteolysis of IkappaB-alpha and NF-kappaB activation (14, 35) . Since the activity of kinases, viz. protein kinase C, and the maturation promoter factor can be affected by proteolysis (36, 37) , it remained possible that the abrogation of NF-kappaB activation in TPCK/TLCK-treated cells was due to inhibition of IkappaB-alpha phosphorylation rather than IkappaB-alpha proteolysis itself. We therefore examined the effect of the protease inhibitors on the TNF-induced phosphorylation of IkappaB-alpha.

Treatment of U-937 cells with TPCK inhibited the TNF-induced proteolysis of IkappaB-alpha (Fig.7A) and nuclear NF-kappaB DNA-binding activity (data not shown) without affecting IkappaB-alpha phosphorylation (Fig.7B, compare lanes1 and 2 with lanes3 and 4). Similarly, treatment of cells with TLCK did not affect the TNF-induced phosphorylation of IkappaB-alpha (Fig.7B, lanes5 and 6). Although the 43-kDa IkappaB-alpha was not detected in immunoblots of whole cell extracts, it was observed when extracts from cells cotreated with TPCK and TNF were immunoprecipitated for IkappaB-alpha prior to immunoblotting (data not shown). Hence, the TNF-induced phosphorylation of IkappaB-alpha can occur under conditions that inhibit IkappaB-alpha proteolysis, and in vivo, the phosphorylation of IkappaB-alpha may not be sufficient to induce NF-kappaB activation.


Figure 7: Serine protease inhibitors prevent the TNF-induced proteolysis of IkappaB-alpha without affecting IkappaB-alpha phosphorylation. A, immunoblot of IkappaB-alpha. U-937 cells were incubated with TNF, for the times indicated, in the absence or presence of TPCK. Cells were exposed to TPCK at 25 µM for a total of 1 h. IkappaB-alpha was detected in whole cell lysates by immunoblotting using the anti-IkappaB-alpha antibody. B, immunoprecipitation of IkappaB-alpha from P-labeled cells. Orthophosphate-labeled U-937 cells were treated with TNF for 2 min or pretreated with TPCK (25 µM) or TLCK (200 µM) for 1 h prior to the addition of TNF for 2 min. IkappaB-alpha was immunoprecipitated, and samples were analyzed by SDS-PAGE.




DISCUSSION

TNF signal transduction leads to the rapid phosphorylation of several proteins at serine, threonine, or tyrosine residues(7, 38) . A number of the serine/threonine-phosphorylated proteins have been identified, and the increase in their phosphorylation appears to result from the activation of multiple serine/threonine kinases(4, 7, 39, 40) and the simultaneous inhibition of the opposing phosphatases, particularly protein phosphatase 2A(4, 41) . Little is known of the tyrosine kinases and phosphatases that are involved in TNF signal transduction. Here, we investigated the role of PTPases in the activation of the transcription factor NF-kappaB by TNF. Inhibition of PTPase activity leads to non-dissociation of the IkappaB-alphabulletNF-kappaB complex and abrogation of NF-kappaB nuclear translocation in TNF-treated cells ( Fig.1(A, C, and D), 3, and 4A). Notably, neither of the PTPase inhibitors used in this study affected the TNF-induced phosphorylation of hsp-27 or eIF-4e, indicating selectivity in the action of the inhibitors and an early divergence in the cytokine-induced signals leading to the phosphorylation of these proteins from signals effecting the activation of NF-kappaB (Fig.2, C-F). The constitutive NF-kappaB-like DNA-binding activity in cells with elevated HPTPalpha activity (Fig.4A) and inhibition of this DNA-binding activity by treatment of HPTPalpha-expressing cells with PAO (Fig.4B) support a role for PTPases in NF-kappaB activation.

IkappaB-alpha has been shown to undergo an overall increase in its phosphorylation in TNF-treated cells involving serine/threonine residue(s)(18, 42, 43) . However, the evidence to support this is indirect. The suggested activation of PTPase in NF-kappaB induction, together with the presence of a conserved Src-like tyrosine phosphorylation site at the N terminus of IkappaB-alpha(20) , led us to investigate if IkappaB-alpha was dephosphorylated at tyrosine in TNF-treated cells. Phosphoamino acid analysis showed that IkappaB-alpha is not the direct target of the putative TNF-activated PTPase(s) as it was exclusively phosphorylated at serine residue(s) in both unstimulated and TNF-treated U-937 cells (Fig.5E). However, treatment of U-937 cells with the PTPase inhibitor pervanadate abolished the TNF-induced phosphorylation of IkappaB-alpha, indicating that the IkappaB-alpha phosphorylation by its serine kinase/phosphatase is likely to be regulated by an upstream putative TNF-activated PTPase(s) (Fig.6A). Several studies implicate c-Raf as a kinase that phosphorylates IkappaB-alpha in vivo(12, 16) . The putative TNF-induced PTPase may be involved in c-Raf activation, as exemplified by the Drosophila PTPase corkscrew, which in concert with the c-Raf homologue polehole, acts to positively transduce signals initiated by the torso receptor protein-tyrosine kinase(44) . On the other hand, findings by Diaz-Meco et al.(13) suggest that a -protein kinase C-activated 50-kDa kinase rather than c-Raf is responsible for TNF-induced IkappaB-alpha phosphorylation. Since c-Raf and -protein kinase C share common components upstream in the signaling pathway, including Ras and phosphatidylcholine-specific phospholipase C(45, 46) , it is possible that activation of -protein kinase C may also depend on the putative TNF-induced PTPase.

All NF-kappaB activators tested appear to induce the rapid degradation of IkappaB-alpha(14, 35, 43, 47, 48, 49) , indicating that modified/unbound IkappaB-alpha has a short life span. In TNF-treated U-937 cells, the loss of immunoreactive IkappaB-alpha was observed at about the same time as its increase in phosphorylation, but before NF-kappaB nuclear translocation (Fig.5, A, C, and D). Serine protease inhibitors, viz. TPCK and TLCK, were found to inhibit ligand-induced IkappaB-alpha degradation and NF-kappaB DNA-binding activity (14, 35) , suggesting that IkappaB-alpha proteolysis does not merely serve to remove the modified/released protein from the cytosol, but is also essential for NF-kappaB activation. It is also possible that abrogation of NF-kappaB activation is due to inhibition of the protease-dependent phosphorylation of IkappaB-alpha rather than to IkappaB-alpha proteolysis itself. However, neither TPCK nor TLCK affected the TNF-induced phosphorylation of IkappaB-alpha in U-937 cells under conditions where IkappaB-alpha proteolysis and NF-kappaB activation were both effectively abolished (Fig.7). Although the 43-kDa species of IkappaB-alpha was not observed in immunoblots of whole cell lysates (Fig.5C, 6B, and 7A), it was detectable in lysates from cells treated with TNF and TPCK that were enriched for IkappaB-alpha by immunoprecipitation before immunoblotting (data not shown). This suggests that in whole cells, IkappaB-alpha phosphorylation alone does not lead to NF-kappaB activation, as implied by studies in vitro(11, 12, 13) . Our data are in agreement with an earlier report (18) that shows that phosphorylated IkappaB-alpha remains tightly bound to p65. Taken together, these data indicate that IkappaB-alpha proteolysis is essential for NF-kappaB activation. Pervanadate treatment also inhibited the TNF-induced proteolysis of IkappaB-alpha (Fig.6B), indicating that the IkappaB-alpha protease, like the IkappaB-alpha serine kinase/phosphatase, may be regulated by an upstream putative TNF-activated PTPase(s). Alternatively, if IkappaB-alpha phosphorylation targets the protein for subsequent degradation, as previously suggested(14) , the inhibition of IkappaB-alpha proteolysis would be a direct consequence of the inhibition of its phosphorylation. Confirmation of the functional significance of IkappaB-alpha phosphorylation will require site-directed mutagenesis of potential serine phosphorylation sites. Recently, it was reported that site-directed mutagenesis of serine 32 or 36 of IkappaB-alpha resulted in a protein that failed to undergo TNF-induced phosphorylation or degradation, and NF-kappaB activation was not observed in cells expressing the mutant proteins(50) . These findings are consistent with our observation that TNF treatment results in the phosphorylation of IkappaB-alpha at serine residue(s) (Fig.5E). They are also consistent with our findings that inhibition of TNF-induced IkappaB-alpha phosphorylation leads to the concomitant loss of IkappaB-alpha degradation and NF-kappaB activation (Fig.1A and 6).

Although NF-kappaB plays an important role in the TNF-induced transcription of several genes, little is known of the early signaling events involved in its activation. The present findings with PTPase inhibitors suggest that a putative PTPase(s) activity is required for the TNF-induced serine phosphorylation and proteolysis of IkappaB-alpha and NF-kappaB activation. This is consistent with a constitutive NF-kappaB-like DNA-binding activity in cells expressing enhanced HPTPalpha activity (Fig.4A). Signal transduction by other NF-kappaB inducers may also depend on PTPase activation, as suggested by the increased stability of IkappaB-alpha and loss of NF-kappaB activation in pre-B-cells expressing the v-Abl protein-tyrosine kinase(51) . On the other hand, NF-kappaB induction via the T-cell receptor complex appears to be negatively regulated by the PTPase CD45(52) . The present data highlight a potential role for HPTPalpha as a positive regulatory element in TNF signal transduction.


FOOTNOTES

*
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.: 65-777-3496; Fax: 65-777-0402.

^1
The abbreviations used are: PTPases, protein-tyrosine phosphatases; TNF, tumor necrosis factor; IL, interleukin; NF-kappaB, nuclear factor kappaB; IkappaB, inhibitor of NF-kappaB; PAO, phenylarsine oxide; TPCK, tosylphenylalanyl chloromethyl ketone; TLCK, N-p-tosyl-L-lysine chloromethyl ketone; HA, hemagglutinin; HA-IkappaB-alpha, HA-tagged IkappaB-alpha; CREBP, cAMP response element-binding protein; eIF-4e, eukaryotic initiation factor 4e; PAGE, polyacrylamide gel electrophoresis; HPTPalpha, human protein-tyrosine phosphatase alpha.


ACKNOWLEDGEMENTS

We thank Dr. C. J. Pallen for critical reading of the manuscript and for providing the vector- and HPTPalpha-transfected REF cells. We are also grateful to Dr. Paramjeet Singh for assistance with the construction of HA-IkappaB-alpha and Robin Philp for help with two-dimensional gel electrophoresis. We also thank Dr. Wan Jin Hong for in vitro synthesized E2F-1 and Drs. Steve Haskill and Albert Baldwin Jr. for the IkappaB-alpha cDNA.


REFERENCES

  1. Ullrich, A., and Schlessinger, J. (1990) Cell 61,203-212 [Medline] [Order article via Infotrieve]
  2. Bolen, J. B. (1993) Oncogene 8,2025-2031 [Medline] [Order article via Infotrieve]
  3. Pallen, C. J., Tan, Y. H., and Guy, G. R. (1992) Curr. Opin. Cell Biol. 4,1000-1007 [Medline] [Order article via Infotrieve]
  4. Tan, Y. H. (1993) Science 262,376-377 [Medline] [Order article via Infotrieve]
  5. Porter, A. G. (1991) Trends Biotechnol. 9,158-162 [Medline] [Order article via Infotrieve]
  6. Tartaglia, L. A., and Goeddel, D. V. (1992) Immunol. Today 13,151-153 [CrossRef][Medline] [Order article via Infotrieve]
  7. Guy, G. R., Chua, S. P., Wong, N. S., Ng, S. B., and Tan, Y. H. (1991) J. Biol. Chem. 266,14343-14352 [Abstract/Free Full Text]
  8. Broxmeyer, H. E., Lu, L., Hangoc, G., Cooper, S., Hendrie, P. C., Ledbetter, J. A., Xiao, M., Williams, D. E., and Shen, F.-W. (1991) J. Exp. Med. 174,447-458 [Abstract]
  9. Hohmann, H.-P., Remy, R., Poschl, B., and van Loon, A. P. G. M. (1990) J. Biol. Chem. 265,15183-15188 [Abstract/Free Full Text]
  10. Grilli, M., Chiu, J. J.-S., and Lenardo, M. J. (1993) Int. Rev. Cytol. 143,1-62 [Medline] [Order article via Infotrieve]
  11. Ghosh, S., and Baltimore, D. (1990) Nature 344,678-682 [CrossRef][Medline] [Order article via Infotrieve]
  12. Li, S., and Sedivy, J. M. (1993) Proc. Natl. Acad. Sci. U. S. A. 90,9247-9251 [Abstract]
  13. Diaz-Meco, M. T., Dominguez, I., Sanz, L., Dent, P., Lozano, J., Municio, M. M., Berra, E., Hay, R. T., Sturgill, T. W., and Moscat, J. (1994) EMBO J. 13,2842-2848 [Abstract]
  14. Henkel, T., Machleidt, T., Alkalay, I., Kronke, M., Ben-Neriah, Y., and Baeuerle, P. A. (1993) Nature 365,182-185 [CrossRef][Medline] [Order article via Infotrieve]
  15. Schutze, S., Potthoff, K., Machleidt, T., Berkovic, D., Wiegmann, K., and Kronke, M. (1992) Cell 71,765-776 [Medline] [Order article via Infotrieve]
  16. Finco, T. S., and Baldwin, A. S., Jr. (1993) J. Biol. Chem. 268,17676-17679 [Abstract/Free Full Text]
  17. Palombella, V. J., Rando, O. J., Goldberg, A. L., and Maniatis, T. (1994) Cell 78,773-785 [Medline] [Order article via Infotrieve]
  18. Traenckner, E. B.-M., Wilk, S., and Baeuerle, P. A. (1994) EMBO J. 13,5433-5441 [Abstract]
  19. Guy, G. R., Cao, X., Chua, S. P., and Tan, Y. H. (1992) J. Biol. Chem. 267,1846-1852 [Abstract/Free Full Text]
  20. Haskill, S., Beg, A. A., Tompkins, S. M., Morris, J. S., Yurochko, A. D., Sampson-Johannes, A., Mondal, K., Ralph, P., and Baldwin, A. S., Jr. (1991) Cell 65,1281-1289 [Medline] [Order article via Infotrieve]
  21. Menon, S. D., Qin, S., Guy, G. R., and Tan, Y. H. (1993) J. Biol. Chem. 268,26805-26812 [Abstract/Free Full Text]
  22. Fletcher, C., Heintz., N., and Roeder, R. G. (1987) Cell 51,773-781 [Medline] [Order article via Infotrieve]
  23. Lamph, W. W., Dwarki, V. J., Ofir, R., Montminy, M., and Verma, I. M. (1990) Proc. Natl. Acad. Sci. U. S. A. 87,4320-4324 [Abstract]
  24. Hunter, T., and Sefton, B. M. (1980) Proc. Natl. Acad. Sci. U. S. A. 77,1311-1315 [Abstract]
  25. Kolodziej, P. A., and Young, R. A. (1991) Methods Enzymol. 194,508-519 [Medline] [Order article via Infotrieve]
  26. Zheng, X. M., Wang, Y., and Pallen, C. J. (1992) Nature 359,336-339 [CrossRef][Medline] [Order article via Infotrieve]
  27. Fantus, I. G., Kadota, S., Deragon, G., Foster, B., and Posner, B. I. (1989) Biochemistry 28,8864-8871 [Medline] [Order article via Infotrieve]
  28. Heffetz, D., Bushkin, I., Dror, R., and Zick, Y. (1990) J. Biol. Chem. 265,2896-2902 [Abstract/Free Full Text]
  29. Bernier, N., Laird, D. M., and Lane, N. D. (1987) Proc. Natl. Acad. Sci. U. S. A. 84,1844-1848 [Abstract]
  30. Garcia-Morales, P., Minami, Y., Luong, E., Klausner, R. D., and Samelson, L. E. (1990) Proc. Natl. Acad. Sci. U. S. A. 87,9255-9259 [Abstract]
  31. Gordon, J. A. (1991) Methods Enzymol. 201,477-482 [Medline] [Order article via Infotrieve]
  32. Baeuerle, P. A., Lenardo, M., Pierce, J. W., and Baltimore, D. (1988) Cold Spring Harbor Symp. Quant. Biol. 53,789-798 [Medline] [Order article via Infotrieve]
  33. Zheng, X. M., and Pallen, C. J. (1994) J. Biol. Chem. 269,23302-23309 [Abstract/Free Full Text]
  34. de Martin, R., Vanhove, B., Cheng, Q., Hofer, E., Csizmadia, V., Winkler, H., and Bach, F. H. (1993) EMBO J. 12,2773-2779 [Abstract]
  35. Mellits, K. H., Hay, R. T., and Goodbourn, S. (1993) Nucleic Acids Res. 21,5059-5066 [Abstract]
  36. Al, Z., and Cohen, C. M. (1993) Biochem. J. 296,675-683 [Medline] [Order article via Infotrieve]
  37. Maller, J. L. (1991) Curr. Opin. Cell Biol. 3,269-275 [Medline] [Order article via Infotrieve]
  38. Fuortes, M., Jin, W.-W., and Nathan, C. (1993) J. Cell Biol. 120,777-784 [Abstract]
  39. Van Lint, J., Agostinis, P., Vandevoorde, V., Haegeman, G., Fiers, W., Merlevede, W., and Vandenheede, J. R. (1992) J. Biol. Chem. 267,25916-25921 [Abstract/Free Full Text]
  40. Guesdon, F., Freshney, N., Waller, R. J., Rawlinson, L., and Saklatvala, J. (1993) J. Biol. Chem. 268,4236-4243 [Abstract/Free Full Text]
  41. Guy, G. R., Cairns, J., Ng, S. B., and Tan, Y. H. (1993) J. Biol. Chem. 268,2141-2148 [Abstract/Free Full Text]
  42. Beg, A. A., Finco, T. S., Nantermet, P. V., and Baldwin, A. S. (1993) Mol. Cell. Biol. 13,3301-3310 [Abstract]
  43. Koong, A. C., Chen, E. Y., and Giaccia, A. J. (1994) Cancer Res. 54,1425-1430 [Abstract]
  44. Perkins, L. A., Larsen, I., and Perrimon, N. (1992) Cell 70,225-236 [Medline] [Order article via Infotrieve]
  45. Cai, H., Erhardt, P., Troppmair, J., Diaz-Meco, M. T., Rapp, U. R., Moscat, J., and Cooper, G. M. (1993) Mol. Cell. Biol. 13,7645-7651 [Abstract]
  46. Dominguez, I., Diaz-Meco, M. T., Municio, M. M., Berra, E., Garcia de Herreros, A., Cornet, M. E., Sanz, L., and Moscat, J. (1992) Mol. Cell. Biol. 12,3776-3783 [Abstract]
  47. Sun, S.-C., Ganchi, P. A., Ballard, D. W., and Greene, W. C. (1993) Science 259,1912-1915 [Medline] [Order article via Infotrieve]
  48. Brown, K., Park, S., Kanno, T., Franzoso, G., and Siebenlist, U. (1993) Proc. Natl. Acad. Sci. U. S. A. 90,2532-2536 [Abstract]
  49. Cordle, S. R., Donald, R., Read, M. A., and Hawiger, J. (1993) J. Biol. Chem. 268,11803-11810 [Abstract/Free Full Text]
  50. Brown, K., Gerstberger, S., Carlson, L., Franzoso, G., and Siebenlist, U. (1995) Science 267,1485-1488 [Medline] [Order article via Infotrieve]
  51. Klug, C. A., Gerety, S. J., Shah, P. C., Chen, Y.-Y., Rice, N. R., Rosenberg, N., and Singh, H. (1994) Genes & Dev. 8,678-687
  52. Baur, A., Garber, S., and Peterlin, B. M. (1994) J. Immunol. 152,976-983 [Abstract/Free Full Text]

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