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J Biol Chem, Vol. 275, Issue 12, 8549-8555, March 24, 2000

Pervanadate-induced Nuclear Factor-B Activation Requires Tyrosine Phosphorylation and Degradation of IB
COMPARISON WITH TUMOR NECROSIS FACTOR-^*

Asok Mukhopadhyay, Sunil K. Manna, and Bharat B. Aggarwal

From the Cytokine Research Laboratory, Department of Bioimmunotherapy, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Tumor necrosis factor activates nuclear transcription factor B (NF-B) by inducing serine phosphorylation of the inhibitory subunit of NF-B (IB), which leads to its ubiquitination and degradation. In contrast, pervanadate (PV) activates NF-B and induces tyrosine phosphorylation of IB (Singh, S., Darney, B. G., and Aggarwal, B. B. (1996) J. Biol. Chem. 271, 31049-31054; Imbert, V., Rupec, R. A., Antonia, L., Pahl, H. L., Traenckner, E. B.-M., Mueller-Dieckmann, C., Farahifar, D., Rossi, B., Auderger, P., Baeuerle, P. A., and Peyron, J.-F. (1996) Cell 86, 787-798). Whether PV also induces IB degradation and whether degradation is required for NF-B activation are not understood. We investigated the effect of PV-induced tyrosine phosphorylation on IB degradation and NF-B activation. PV activated NF-B, as determined by DNA binding, NF-B-dependent reporter gene expression, and phosphorylation and degradation of IB. Maximum degradation of IB occurred at 180 min, followed by NF-B-dependent IB resynthesis. N-Acetylleucylleucylnorlucinal, a proteasome inhibitor, blocked both IB degradation and NF-B activation, suggesting that the IB degradation is required for NF-B activation. PV did not induce serine phosphorylation of IB but induced phosphorylation at tyrosine residue 42. Unlike tumor necrosis factor (TNF), PV did not induce ubiquitination of IB. Like TNF, however, PV induced phosphorylation and degradation of IB, and subsequent NF-B activation, which could be blocked by N-tosyl-L-phenylalanine chloromethyl ketone, calpeptin, and pyrrolidine dithiocarbomate, suggesting a close link between PV-induced NF-B activation and IB degradation. Overall, our studies demonstrate that PV activates NF-B, which, unlike TNF, requires tyrosine phosphorylation of IB and its degradation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Nuclear transcription factor-B (NF-B)¹ plays a pivotal role in expression of various inducible target genes related to immune and inflammatory responses, including the type I human immunodeficiency virus (1-3). NF-B is a dimer of members of the Rel family of proteins (1, 4). In nonstimulated cells, the heterodimeric NF-B complexes are restricted to the cytoplasm, where they are associated with an inhibitory molecule of the IB family (5). In mammalian species, six structural homologs of IB have been identified (6), but only one of them, the IB form, has been extensively studied.

IB regulates NF-B activity by masking the nuclear localization signal located on the p50-p65 heterodimer of NF-B (7, 8). In response to stimulation by various agents, among them phorbol esters (e.g. phorbol 12-myristate 13-acetate), tumor necrosis factor (TNF), interleukin-1 (IL-1), -radiation, and lipopolysaccharide, IB undergoes degradation, allowing the p50-p65 heterodimer to migrate to the nucleus (1, 9-12). A protein kinase complex, consisting of IB kinase , , and  subunits, stimulated by TNF or IL-1 phosphorylates Ser-32 and Ser-36 of IB; these steps are essential for its degradation and the consequent nuclear translocation of NF-B (13, 14). It has been demonstrated by using specific proteasome inhibitors that inducible phosphorylation of IB is needed but not sufficient for its degradation by proteasome (15). Before being degraded by 26 S proteasome (8, 9), serine-phosphorylated IB is polyubiquitinated at the Lys-21 and Lys-22 positions (16, 17).

Tyrosine phosphorylation also plays a role in NF-B activation, although that role is not fully understood. Inhibitors of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (e.g. PTPase) suppress NF-B activation (18-23). Recently, it was shown that hypoxia, reoxygenation, and the PTPase inhibitor pervanadate (PV) induce tyrosine phosphorylation of IB (23-27) and activate NF-B (23-25). Whether tyrosine phosphorylation leads to IB degradation and whether degradation is required for NF-B activation are not known. In this report, we demonstrate that PV-induced tyrosine phosphorylation leads to degradation of IB and that this degrdation is required for NF-B activation.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

MaterialsHuman myeloid U937 and epithelial HeLa cells were obtained from the American Type Culture Collection (Manassas, VA). Rabbit polyclonal antibodies anti-IB, anti-p50, and anti-p65 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal antibodies against the serine-32-phosphorylated form of IB was purchased from New England BioLabs, Inc. (Beverly, MA). Sodium orthovanadate, pyrrolidine dithiocarbomate (PDTC), N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), cycloheximide, biotinylated anti-phosphotyrosine monoclonal antibody, horseradish peroxidase-conjugated anti-biotin monoclonal antibody, anti-Flag monoclonal antibody, and the alkaline phosphatase fluorescent substrate 4-methyl-umbelliferyl phosphate were obtained from Sigma. Bacterium-derived recombinant human TNF, purified to homogeneity with a specific activity of 5 × 10⁷ units/mg, was kindly provided by Genentech Inc. (South San Francisco, CA). Calf intestine alkaline phosphatase (CIP), RPMI 1640 medium, minimum Eagle's medium, and fetal bovine serum were obtained from Life Technologies Inc. Genistein, calpeptin, and N-acetylleucylleucylnorlucinal (ALLN) were procured from Calbiochem-Novabiochem Corp. (San Diego, CA). Protein A/G-Sepharose beads were obtained from Pierce. The plasmids pCMV4-FIB and pCMV4-FIB/Y42F, which encode epitope-tagged derivatives of IB were kindly provided by Dr. Dean Ballard (Vanderbilt University School of Medicine, Nashville, TN).

PV was freshly prepared in each experiment as follows: 20 µl of 1 M sodium orthovanadate, placed in an Eppendorf tube containing 270 µl of phosphate-buffered saline, was treated with 10 µl of 33% H₂O₂ for 5 min at room temperature. The pH of the solution was neutralized with 1 N HCl, and excess H₂O₂ was deactivated with catalase (200 µg/ml). The concentration of pervanadate generated is denoted by the vanadate concentration taken in the reaction mixture. U-937 cells were maintained in RPMI 1640 medium containing 10% fetal bovine serum and a 1× antibiotic-antimycotic solution. The culture was split every 3 days. HeLa cells were maintained in MEM containing 10% fetal bovine serum.

Electrophoretic Mobility Shift Assay-- NF-B activation was analyzed by electrophoretic mobility shift assay as described previously (28). In brief, 8-µg nuclear extracts prepared from TNF- or PV-treated cells were incubated with ³²P end-labeled 45-mer double-stranded NF-B oligonucleotide for 15 min at 37 °C, and the DNA-protein complex resolved in 6.6% native polyacrylamide gel. The specificity of binding was examined by competition with unlabeled 100-fold excess oligonucleotide and with mutant oligonucleotide. The composition and specificity of binding was also determined by supershift of the DNA-protein complex using specific and irrelevant antibodies. The antibody-treated samples of NF-B were resolved on a 5.5% native gel. The radioactive bands from the dried gels were visualized and quantitated by PhosphorImager (Molecular Dynamics, Sunnyvale, CA) using ImageQuant software.

Western Blot of IB-- Thirty-microgram cytoplasmic protein extracts, prepared as described (28), were resolved on 9% SDS-PAGE gel. After electrophoresis, the proteins were electrotransferred to a nitrocellulose membrane, blocked with 5% nonfat milk, and probed with anti-IB polyclonal antibodies (1:3000) for 1 h. The blot was washed, exposed to horseradish peroxidase-conjugated secondary antibodies for 1 h, and finally detected by chemiluminescence (ECL, Amersham Pharmacia Biotech). The bands obtained were quantitated using Personal Densitometer Scan version 1.30 using ImageQuant software version 3.3 (Molecular Dynamics, Sunnyvale, CA).

To examine the dephosphorylation of IB, we exposed 30 µg of PV-treated cytoplasmic cell extracts to CIP (0.1-5 units) for 10 min at 37 °C. The reaction was stopped by boiling with reducing sample buffer, and the samples were subjected to electrophoresis and Western blot for IB.

Identification of Tyrosine-phosphorylated IB-- After treatment with PV, cells were washed with phosphate-buffered saline, and whole cell lysates were prepared in lysis buffer (20 mM HEPES, 250 mM NaCl, 1 mM dithiothreitol, 1% Nonidet P-40, 2 mM EDTA, 0.5 mM EGTA, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml phenylmethylsulfonyl fluoride, 0.5 µg/ml benzamidine, 3 mM sodium orthovanadate). One milligram of lysate protein was treated with 1 µg of anti-IB antibodies in 800 µl of lysate buffer at 4 °C for 2 h, and the immune complexes were precipitated with protein A/G-Sepharose beads. The beads were thoroughly washed and boiled for 5 min with 1× reducing sample buffer. After boiling, the proteins were resolved on 9% SDS-PAGE gel, electrotransferred to a nitrocellulose membrane, and probed with anti-phosphotyrosine biotin monoclonal antibody (1:2000). The blot was then treated with anti-biotin-horseradish peroxidase conjugate and finally detected by ECL reagent.

Transient Transfection with IB Gene-- Transfection was performed as described earlier (26). In brief, the FLAG-tagged IB genes cloned into the eukaryotic expression vector pCMV4 (pCMV4-FIB, pCMV4-FIB/42F) were transiently transfected into 50% confluent HeLa cells by calcium-phosphate method as described by the manufacturer (Life Technologies, Inc.). After 9 h of transfection, cells were washed and incubated with complete medium without addition or containing 1 nM TNF or 200 µM PV for 24 h. Thirty micrograms of whole-cell lysate protein, prepared as mentioned earlier, was resolved on 9% SDS-PAGE gel, electrotransferred on to a nitrocellulose membrane, and probed with anti-FLAG antibody.

NF-B SEAP Reporter Assay-- The NF-B-SEAP reporter gene expression assay was based on our earlier report (29). In brief, 0.5 × 10⁶ HeLa cells/1.5 ml were plated in each well of a 6-well plate and incubated for 16-18 h. Cells were transiently transfected with pNF-B-SEAP2 (0.5 µg) and the expression vector (2.5 µg of pCMV) by the calcium-phosphate method for 9 h. After transfection, cells (duplicate wells) were washed and incubated with medium or with medium containing 1 nM TNF or 200 µM PV for 24 h. The culture supernatant was removed and assayed for SEAP activity. The culture supernatant (25 µl) was mixed with 30 µl of 5× buffer (500 mM Tris·Cl, pH 9, and 0.5% bovine serum albumin) in a total volume of 100 µl in a 96-well plate, and the heat labile endogenous alkaline phosphatase was deactivated by heating the mixture at 65 °C for 30 min. The plate was chilled on ice for 2 min, 50 µl of 1 mM 4-methylumbelliferyl phosphate was added to each well, the plate was incubated at 37 °C for 2 h, and fluorescence was read on a 96-well fluorescent plate reader (Fluoroscan II, Lab Systems, Needham Heights, MA) with excitation set at 360 nm and emission at 460 nm. The average (± S.E.) number of relative fluorescent light units for each transfection was reported.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We examined the effects of PV on NF-B and IB in U-937 cells because the effects on these cells are well characterized in our laboratory. The concentration and time of exposure to PV used in our experiments had no significant effect on cell viability (data not shown).

Pervanadate-induced NF-B Activation Correlates with IB Degradation-- We first examined the kinetics of PV-induced NF-B activation as detected by electrophoretic mobility shift assay in U-937 cells. For this, we treated cells with 100 µM PV for different times, prepared the nuclear extracts, and analyzed them by electrophoretic mobility shift assay. Time course analysis revealed that NF-B/DNA binding activity was first detected at 120 min, reached a maximum at 180 min, and declined thereafter (Fig. 1A). The activated form of NF-B was evident even at 480 min. Under similar conditions, TNF-induced NF-B activation could be noted as early as 5 min (data not shown). Thus the kinetics of NF-B activation by PV appears to be much slower than activation by TNF.

View larger version (34K): [in this window] [in a new window]   Fig. 1.   Pervanadate-induced NF-B activation correlates with IB degradation. A, kinetics of PV-induced NF-B activation in U937 cells. Two million cells per ml were incubated with freshly prepared 100 µM pervanadate for the indicated times, and nuclear extracts were prepared and assayed for NF-B. B, supershift and specificity of NF-B. Nuclear extracts, prepared by treating cells with 100 µM PV for 180 min, were incubated for 15 min with different antibodies, unlabeled oligo, or mutant oligo and then assayed for NF-B as described. C, kinetics of degradation of IB. Cytoplasmic extracts from PV-treated cells were analyzed by Western blot using IB-specific antibodies. s, slowly migrating band; n, normally migrating band. D, PV-induced NF-B is transcriptionally active. HeLa cells were transiently transfected with 3 µg of total plasmid DNA in duplicate with pNF-B-SEAP (0.5 µg) as described under "Experimental Procedures." Cells were induced with none or 100 µM PV or 1 nM TNF for 24 h. The culture supernatants were assayed for SEAP activity as described under "Experimental Procedures."

Incubation of nuclear extracts with anti-p65 (Fig. 1B, Anti-p65) or anti-p50 antibodies (Anti-p50) resulted in the abrogation in NF-B/DNA complex, whereas irrelevant anti-Cyclin D1 antibodies (Anti-Cyclin D1) or preimmune sera (PIS) had no effect. Thus, NF-B induced with PV contained both the p65 (RelA) and p50 subunits. The specificity of the PV-induced NF-B/DNA complex was further confirmed by demonstrating that the binding was disrupted in the presence of a 100-fold excess of unlabeled B-oligonucleotide (Fig. 1B, Cold oligo) but not by mutant oligonucleotide (Mutant oligo).

Activation of NF-B by TNF is achieved through Ser-32 and Ser-36 phosphorylation of IB followed by polyubiquitination and degradation, which results in the nuclear translocation of NF-B. Whether PV-induced NF-B activation is associated with the degradation of IB is not clear. To investigate this, cytoplasmic extracts from cells treated with PV for different times were subjected to Western blot analysis using IB-specific polyclonal antibodies. Within 30 min of PV treatment, all IB appeared as slow migrating species from 37 to 39 kDa, which then gradually was degraded (Fig. 1C). Maximum degradation of the slowly migrating species was noted at 240 min. Beyond 240 min, no further degradation was observed, but rather new synthesis of IB began. The resynthesis of IB, which is dependent on NF-B activation, started at 180 min (normally migrating 37-kDa size) and reached a maximum at 480 min (Fig. 1C). The kinetics of PV-induced IB degradation correlated well with the kinetics of activation of NF-B (Fig. 1A). The synthesis of newly formed IB indicates that IB had been transcribed by the activated NF-B.

NF-B Induced by PV Is Transcriptionally Active-- Due to the additional steps involved, induction of binding of NF-kB to the DNA does not always indicate transcriptional activation. Therefore, we examined the ability of PV to activate NF-B-dependent SEAP reporter gene expression. The relative activities of SEAP induced either by 100 µM PV or 1 nM TNF are shown in Fig. 1D. Both PV and TNF increased SEAP activity by 2.5-3-fold over control. These results indicate that NF-B activated by PV was transcriptionally active and comparable with TNF.

Proteasome Inhibitor Blocks PV-induced IB Degradation and NF-B Activation-- ALLN, a peptide-aldehyde inhibitor that blocks the activity of the enzyme calpain I, is reported to inhibit the proteolytic activity of the proteasome (30) and reduce the degradation of ubiquitin-conjugated proteins (31). ALLN also blocks the TNF-induced degradation of IB without inhibiting its hyperphosphorylation, and this causes suppression of NF-B activation (15). Because activation of NF-B in PV-induced U937 cells is associated with the degradation of IB, we examined the effects of the proteasome inhibitor on the degradation of IB and subsequent NF-B activation. As shown in Fig. 2A, ALLN blocked PV-induced degradation of IB (Fig. 2A, upper right panel) and attenuated NF-B activation (Fig. 2A, lower panels). Furthermore, as indicated by the appearance of a slowly migrating band, ALLN treatment did not inhibit PV-induced hyperphosphorylation of IB. Similar results were obtained in case of TNF-treated cells (Fig. 2B). These ALLN results indicate that a proteasome is involved in PV-induced NF-B activation and IB degradation and suggest that degradation of IB protein is a prerequisite for NF-B activation.

View larger version (56K): [in this window] [in a new window]   Fig. 2.   Proteasome inhibitor (ALLN) blocks PV- and TNF-induced degradation of IB and activation of NF-B. Kinetics for degradation of IB and activation of NF-B by PV (A) and TNF (B) in the presence and absence of ALLN. U937 cells (2 × 106/ml) were preincubated at 37 °C with 100 µg/ml ALLN for 1 h. Untreated and treated cells were either induced with 0.1 nM TNF or 100 µM PV for the indicated times. Cytoplasmic extracts were analyzed by Western blot using IB-specific antibodies, and nuclear extracts assayed for NF-B. s, slowly migrating band; n, normally migrating band. C, inhibition of the synthesis of IB by cycloheximide. U937 cells were treated with either none or 10 µg/ml cycloheximide for 1 h, followed by induction with 100 µM PV for the indicated times. Cytoplasmic extracts were analyzed for IB by Western blot.

PV Induces Synthesis of New IB-- Cycloheximide, a protein synthesis inhibitor, completely blocks the IL-1-induced resynthesis of IB (32). In the previous subsection (Fig. 1C), we have shown that upon PV treatment, IB was first degraded and then resynthesized in U937 cells. To reconfirm this, we treated the cells with PV for different times in the presence of cycloheximide and then examined the cellular IB levels. Cycloheximide caused complete cessation of IB synthesis, as no IB was detected after 60 min (Fig. 2C). The inhibition of degradation by proteasome inhibitor and blockage of resynthesis by cycloheximide indicates that IB resynthesis is mediated through the activation of NF-B (Fig. 1C).

PV-induced Slowly Migrating Band Is Due to Tyrosine Phosphorylation of IB-- To verify that the slowly migrating species of IB that appeared after PV treatment was due to its phosphorylation, cytoplasmic extracts from PV-treated cells were incubated with different concentrations of CIP. To facilitate the dephosphorylation reaction, a vanadate-free lysis buffer was used. As shown in Fig. 3A, incubation with CIP did not affect the normally migrating band (37 kDa) of IB, but the slowly migrating band was completely converted to the 37-kDa IB noted in control untreated cells. This demonstrates that IB was phosphorylated by PV.

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Pervanadate causes Tyr-42 phosphorylation of IB. A, dephosphorylation of IB from PV-treated cells by protein phosphatase. Thirty micrograms of lysates prepared from untreated or PV-treated cells (100 µM, 30 min) was incubated with various concentrations of CIP for 10 min at 37 °C and then assayed for IB. B, effect of protein tyrosine kinase inhibitor (genistein) on PV-induced degradation of IB and activation of NF-B. U937 cells (2 × 106 cells/ml) were treated with none or 80 µg/ml genistein for 1 h, followed by induction with 100 µM PV for the indicated times. Cytoplasmic extracts were analyzed for IB (upper panels) and nuclear extracts for NF-B (lower panels). s, slowly migrating band; n, normally migrating band. C, anti-phosphotyrosine Western blot (WB) analysis of IB immunoprecipitated from PV-treated cells. IB was immunoprecipitated (IP) from untreated (control) or PV-treated cells either by preimmune sera (PIS) or anti-IB antibodies and protein A/G-Sepharose and immunoblotted with biotinylated anti-phosphotyrosine antibody (left two panels) as described under "Experimental Procedures." The same blot was reprobed with anti-IB antibodies (right two panels). D, cell transfection of FLAG-tagged IB implicates Tyr-42 as the phosphorylation site induced by PV in vivo. HeLa cells were transfected with pCDNA3, epitope-tagged Y42F mutant IB, or wild type (WT) IB as described under "Experimental Procedures." Cells were left untreated or treated with 100 µM PV for 2 h, whole cell lysates were prepared, and 30 µg of protein was resolved on 10% SDS-PAGE gel and immunoblotted with anti-FLAG antibody as described under "Experimental Procedures." E, pervanadate does not phosphorylate IB at the Ser-32 position. U937 cells were treated with 100 µg/ml ALLN for 1 h at 37 °C and then induced (ALLN-treated cells) either with 0.1 nM TNF for 5 or 10 min or with 100 µM PV for 30 or 60 min. Cytoplasmic extracts were resolved on 9% SDS-PAGE gel and immunoblotted with antibodies against Ser32-phosphorylated IB (left panel). The same blot was reprobed with anti-IB antibodies (right panel). F, pervanadate does not induce ubiquitination of IB. U937 cells were treated with 100 µg/ml ALLN for 1 h at 37 °C, followed by induction either with 0.1 nM TNF for 3, 7, or 10 min or with 100 µM PV for 15, 30, or 60 min. Sixty micrograms of cytoplasmic extracts was resolved on 9% SDS-PAGE gel and immunoblotted with anti-IB antibodies. NSB, nonspecific band.

To determine whether slow migration was due to phosphorylation of, IB U937 cells were incubated with genistein, a PTK inhibitor, for 1 h and then exposed to PV. Genistein completely blocked PV-induced IB phosphorylation and degradation, and this correlated with total suppression of NF-B activation (Fig. 3B). These data suggest that a genistein-sensitive PTK is activated by PV to phosphorylate IB and this phosphorylation is needed for IB degradation and subsequent NF-B activation.

PV Induces Phosphorylation of IB at Tyrosine 42-- Metabolic inhibitors such as genistein are not always specific. We and other groups have shown that PV induces phosphorylation of IB at tyrosine 42 (25-27). To confirm this in our system, we first investigated tyrosine phosphorylation of IB by immunoprecipitation and Western blot analysis. Untreated and PV-induced whole cell lysates were immunoprecipitated either with preimmune sera or with anti-IB antibodies followed by Western blot with the anti-phosphotyrosine antibody. As shown in Fig. 3C (left two panels), IB immunoprecipitated from PV-treated cells, but not from control untreated cells, was indeed tyrosine-phosphorylated. The tyrosine-phosphorylated band was confirmed to be the slower migrating species (39 kDa) of IB by Western blot analysis using anti-IB antibodies (Fig. 3C, right two panels).

Next, we transfected FLAG-tagged wild type and mutant (Y42F) IB genes into HeLa cells and analyzed the expression of IB protein in control (untreated) and PV-treated cells by Western blot with anti-FLAG antibody. The FLAG-tagged Y42F-IB did not become phosphorylated and migrated faster than the wild type IB in SDS-PAGE gel (Fig. 3D). Furthermore, FLAG-tagged wild type IB decreased in mobility upon PV treatment, providing evidence that in our system, PV induced phosphorylation of IB at Tyr-42 in vivo .

PV Does Not Phosphorylate at Ser-32 of IB-- TNF-induced phosphorylation of IB at Ser-32 and Ser-36 is essential for its degradation and subsequent NF-B activation (14, 33). To study whether IB phosphorylation at these sites was required for degradation induced by PV, ALLN-pretreated U937 cells were induced with either TNF or PV. The cytoplasmic extracts were probed with Ser-32 phosphospecific IB antibody. As shown in Fig. 3E, neither untreated nor ALLN-treated cells showed any Ser-32-phosphorylated IB species. Upon TNF treatment, however, phosphorylated IB began to appear as early as 5 min (Fig. 3E, left panel). No Ser-32-phosphorylated IB was detected in PV-treated cells. Reprobing the same blot with anti-IB antibodies verified the presence of IB (Fig. 3E, right panel). These data suggest that unlike cytokine induction, PV-induction of IB degradation is not due to serine phosphorylation.

PV Does Not Induce Ubiquitination of IB-- TNF/IL-1-induced degradation of IB requires phosphorylation at Ser-32 and Ser-36 followed by polyubiquitination at Lys-21 and Lys-22 (16, 17). To examine whether PV induces ubiquitination of phosphorylated IB before degradation, we treated U937 cells with ALLN and then with either 0.1 nM TNF for 3, 7, and 10 min or 100 µM PV for 15, 30, and 60 min. Cytoplasmic extracts (60 µg of protein) were resolved and probed with anti-IB antibodies. As shown in Fig. 3F, a ladder of high molecular mass proteins appeared following stimulation with TNF, and the signals intensified at 10 min. The molecular mass increments of this ladder were ~8.5 kDa, which is the size of ubiquitin. In contrast, PV-treated samples did not show any such ladder. This result suggests that PV does not induce IB ubiquitination. To enhance the detection limit for ubiquitinated IB protein, the blot was exposed for a longer duration, resulting in broader bands of IB. Equally intensified nonspecific band in each lane signifies equal loading of the samples.

PDTC Blocks Tyrosine Phosphorylation and Degradation of IB-- Numerous reports have demonstrated that cytokine-induced NF-B activation is sensitive to intracellular redox changes. The oxygen radical scavenger PDTC has been reported to block signal-induced phosphorylation of IB and its degradation, leading to suppression of NF-B activation (34, 35). PDTC was also reported to prevent PV-induced cleavage of ErbB-4 (36). In addition, tyrosine phosphorylation of IB by peroxovanadium compound (phen) was prevented by PDTC (37). We therefore examined the effect of PDTC on PV-induced degradation of IB and on activation of NF-B. U937 cells were treated with PDTC prior to induction with either TNF or PV. As shown in Fig. 4A, PDTC effectively blocked both TNF- and PV-induced IB degradation and NF-B activation. In addition, PDTC also blocked both PV- and TNF-induced phosphorylation of IB.

View larger version (61K): [in this window] [in a new window]   Fig. 4.   Protease inhibitors block TNF- and PV-induced IB degradation and NF-B activation. A, PDTC inhibits TNF- and PV-induced degradation of IB and activation of NF-B. U937 cells (2 × 106 cells/ml) were pretreated with 100 µM PDTC for 1 h, followed by induction with 0.1 nM TNF or with 100 µM PV for the indicated times. Cytoplasmic and nuclear extracts were analyzed for IB (upper panels) and NF-B (lower panels), respectively. s, slowly migrating band; n, normally migrating band. B, TPCK inhibits TNF- and PV-induced degradation of IB and activation of NF-B. U937 cells (2 × 106 cells/ml) were pretreated with 100 µM TPCK for 1 h, followed by induction with 0.1 nM TNF or with 100 µM PV for the indicated times. Cytoplasmic and nuclear extracts were analyzed for IB (upper panels) and NF-B (lower panels), respectively. s, slowly migrating band; n, normally migrating band. C, calpeptin inhibits TNF- and PV-induced degradation of IB and activation of NF-B. U937 cells (2 × 106 cells/ml) were pretreated with 100 µM calpeptin for 1 h, followed by induction with 0.1 nM TNF or with 100 µM PV for the indicated times. Cytoplasmic and nuclear extracts were analyzed for IB (upper panels) and NF-B (lower panels), respectively. s, slowly migrating band; n, normally migrating bands.

Serine Protease Inhibitor Blocks IB Degradation and NF-B Activation-- TNF-induced activation of NF-B is a result of sequential events, such as phosphorylation, polyubiquitination, and finally degradation of IB by the 26 S proteasome (4). We have shown in the previous section that ALLN, a proteasome inhibitor, suppresses PV-induced IB degradation and NF-B activation. Here, we investigated the possibility of the involvement of other proteases in the degradation of IB in U937 cells. The serine protease inhibitor and alkylating agent TPCK, reported to be an effective inhibitor of IB degradation and NF-B activation (38, 39). In our studies, TPCK completely prevented TNF-induced IB degradation and NF-kB activation (Fig. 4B, upper panels). Although TPCK completely blocked PV-induced NF-kB activation, it did not completely protect PV-induced degradation of IB (Fig. 4B, lower panels). Unlike ALLN, however, TPCK also blocked IB phosphorylation induced by both TNF and PV.

Calpain Protease Inhibitor Also Blocks IB Degradation and NF-B Activation-- Next, we tested calpeptin, an inhibitor of calpains, a group of cytosolic Ca²⁺-activated thiol proteases that are implicated in TNF-induced IB degradation (40-42). The result shown in Fig. 4C indicates that IB degradation was induced by both PV and TNF and that calpeptin treatment significantly blocked the degradation. Calpeptin also prevented phosphorylation of IB induced by both TNF and PV. This correlated with suppression of TNF- or PV-induced NF-B activation by calpeptin (Fig. 4C, lower panels). These results suggest that PV induces degradation of IB by activating protease similar to the one activated by TNF. These results also suggest that suppression of IB degradation blocks PV-induced NF-B activation.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In this report, we studied how PV induces NF-B activation in U937 cells. Our results showed that PV-induced NF-B activation was associated with IB degradation. PV-activated NF-B was functional, as it induced IB resynthesis and activated reporter gene expression. Results of genistein sensitivity, immunoprecipitation, Western blot, and site-specific mutagenesis experiments revealed that PV phosphorylated the Tyr-42 residue of IB, the Ser-32 residue remained unphosphorylated, and IB is not ubiquitinated before degradation. Although PV induces tyrosine phosphorylation of IB and TNF induces serine phosphorylation, various inhibitors, such as ALLN, PDTC, TPCK, and calpeptin, which work through different mechanisms, all blocked IB degradation and NF-B activation induced by PV. These results indicate that the degradation of IB is required for NF-kB activation.

Previously, it was reported that PV induces NF-B activation without degrading IB (25). Another report indicated that the p85 subunit of phosphotidylinositol 3-kinase binds to the PV-induced tyrosine-phosphorylated IB and dissociates it from the p50-p65 complex (27). Our results, however, indicate that PV induces tyrosine phosphorylation of IB, which in turn leads to its degradation. Why there is a difference between our results and those reported by Imbert et al. (25) is not clear. The difference in results may be due to the difference in kinetics of NF-B activation: Imbert et al. (25) reported maximum activation of NF-B when Jurkat (T cells) cells are treated with 250 µM PV for 60 min, whereas we found optimum activation when cells were treated with 100 µM PV for 180 min. Similarly, Imbert et al. (25) monitored IB levels maximally up to 150 min after treatment of cells with 200 µM PV and found no significant degradation of IB. We found that 100 µM PV-induced IB degradation begins at 30 min and reaches maximum at 240 min (see Fig. 1C). Besides kinetics, the difference may also be due to cell type (myeloid versus T cells) used.

Another important difference between our results and those of Imbert et al. (25) is that the latter workers noted no resynthesis of IB after PV treatment. Our studies, however, clearly show that the resynthesis of IB began at 180 min and reached its starting level 480 min after PV treatment (see normally migrating band in Fig. 1C). As resynthesis is dependent on NF-B activation, we found that the latter precedes the resynthesis. The effect of cycloheximide on the suppression of resynthesis induced by PV can be noted as early as 120 min after treatment (see Fig. 2C). Imbert et al. (25) found a partial degradation of phosphorylated IB at 150 min only when cycloheximide-pretreated cells were exposed to PV. The normally migrating 37-kDa band of IB was not induced by PV in studies reported by Imbert et al. (25).

In agreement with previous results (25-27), our studies clearly demonstrate that PV induces phosphorylation of IB at tyrosine residue 42. Our studies also suggest that tyrosine phosphorylation is required for IB degradation. We found that treatment of cells with various metabolic inhibitors with diverse mechanisms of action blocked IB phosphorylation and degradation simultaneously. ALLN, a proteosome inhibitor, however, blocked PV-induced degradation without blocking tyrosine phosphorylation of IB, indicating that phosphorylation alone is not sufficient to induce degradation. Genistein (a PTK inhibitor), PDTC (an inhibitor of certain metalloproteases and reactive oxygen intermediate quencher), TPCK (a serine protease inhibitor), and calpeptin (a calpain inhibitor) suppressed both phosphorylation and degradation of IB. These results clearly suggest that phosphorylation of IB is needed but not sufficient for degradation induced by PV. This is consistent with results reported for cytokine-induced IB degradation, although the latter is mediated through phosphorylation of serine at positions 32 and 36. The ability of UV radiation to activate NF-kB correlates with IB degradation but does not require serine phosphorylation at the N-terminal sites (11). These results are similar to ours in that serine phosphorylation of IB was not required for its degradation after PV treatment. We found that although TNF induces ubiquitination of IB, PV does not. This indicates some differences in the mechanism of IB degradation by the two agents.

We found that pretreatment of cells with ALLN, genistein, PDTC, TPCK, or calpeptin blocked both IB degradation and NF-B activation induced by PV. These results suggest that IB degradation is required for NF-B activation by PV. We have also found (data not shown) that lactacystin, another specific inhibitor of proteasome, does not prevent PV-induced hyperphosphorylation of IB but inhibits its degradation and NF-B activation. Although these results are consistent with the mechanism reported for cytokine-induced NF-B activation, they differ from PV-induced NF-B activation reported by Imbert et al. (25). Beraud et al. (27) reported that the p85 subunit of phosphotidylinositol 3-kinase sequesters the tyrosine-phosphorylated IB from p50-p65, thus leading to NF-B activation by PV. Their observation that IB has to be removed to activate NF-B by PV is consistent with our results.

Which PTK phosphorylates IB is not certain, and it is possible that more than one is involved. Several PTKs, including c-Src, Sky, Lck, v-Src, and v-Abl have been implicated, depending on the cell type. A T-cell-specific p56lck was reported to be involved in PV-induced tyrosine phosphorylation of IB and NF-B activation (25). Our results are consistent with those of Imbert et al. (25), who showed that tyrosine phosphorylation of IB is required for NF-B activation. We found that PV-induced NF-B activation and IB phosphorylation is genistein-sensitive, as did Imbert et al. (25). Several PTKs are genistein-sensitive, including v-abl and dual-specific kinases of the mitogen-activated protein kinase family (46). The role of a member of mitogen-activated protein kinase family, MEKK1, in activation of NF-B and IB phosphorylation by TNF and other agents is well established (48).

Phosphorylation of Tyr-42 in IB is in accord with previous findings (25, 26). Our result on anti-Ser³²-phosphorylated IB Western blot confirms that PV did not phosphorylate cytokine-inducible Ser-32/Ser-36 sites of IB. Treatment of Jurkat cells with PDTC prevents tyrosine phosphorylation by peroxovanadium compounds (37), as it did in our observations. Because phosphorylation precedes the degradation of IB, it is apparent that these compounds somehow block the action of PTK rather than inhibit the proteolysis of IB. Because PDTC prevented IB phosphorylation, reactive oxygen intermediates could be inducers of tyrosine phosphorylation of IB. This result supports previous observations of tyrosine phosphorylation of IB upon reoxygenation of Jurkat cells (25). Mammalian cells do not produce large amounts of antioxidant enzymes during hypoxia. As a result, reactive oxygen intermediates are immediately generated upon reoxygenation, which leads to tyrosine phosphorylation of various regulatory proteins. H₂O₂ is reported as a potent inhibitor of PTPase that may lead to the activation of PTKs, and thereby to tyrosine phosphorylation (47). How calpeptin prevents tyrosine phosphorylation is not clear. Our results with calpeptin, the inhibitor of thiol protease (cytosolic calpain), and proteasome inhibitor (ALLN) probably indicate that tyrosine-phosphorylated IB is degraded by both cytosolic calpain-calpastatin and ubiquitin-proteasome pathways, similar to that reported for TNF-induced degradation of IB (42). Overall, our results suggest that PV-induced tyrosine phosphorylation leads to the degradation of IB and the activation of NF-B in U937 cells. These results may be relevant to physiological stimuli, such as anoxia, that activate NF-B through tyrosine phosphorylation.

	FOOTNOTES

^* This research was conducted with support from The Clayton Foundation for Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Cytokine Research Laboratory, Dept. of Bioimmunotherapy, Box 143, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. Tel.: 713-792-3503; Fax: 713-794-1613; E-mail: aggarwal@utmdacc.mda.uth.tmc.edu.

	ABBREVIATIONS

The abbreviations used are: NF-B, nuclear transcription factor-B; ALLN, N-acetylleucylleucylnorlucinal; CIP, calf intestine alkaline phosphatase; IB, inhibitory subunit of NF-B; PAGE, polyacrylamide gel electrophoresis; PV, pervanadate; PTK, phosphotyrosine kinase; PDTC, pyrrolidine dithiocarbomate; SEAP, secretory alkaline phosphatase; TNF, tumor necrosis factor; TPCK, N-tosyl-L-phenylalanine chloromethyl ketone.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES