©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Activation of NF-B by Phosphatase Inhibitors Involves the Phosphorylation of IB at Phosphatase 2A-sensitive Sites (*)

(Received for publication, March 7, 1995; and in revised form, May 8, 1995)

Shao-Cong Sun (§) Sanjay B. Maggirwar Edward Harhaj

From the Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, Hershey Medical Center, P. O. Box 850, Hershey, Pennsylvania 17033

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Activation of NF-kappaB by various cellular stimuli involves the phosphorylation and subsequent degradation of its inhibitor, IkappaBalpha, although the underlying mechanism remains unclear. In the present study, the role of serine/threonine phosphatases in the regulation of IkappaBalpha phosphorylation was investigated. Our studies demonstrate that incubation of human T cells with low concentrations (1-5 nM) of calyculin A or okadaic acid, potent inhibitors of protein phosphatase type 1 (PP-1) and type 2A (PP-2A), induces the phosphorylation of IkappaBalpha even in the absence of any cellular stimulus. This action of the phosphatase inhibitors, which is associated with the activation of the RelAbulletp50 NF-kappaB heterodimer, is not affected by agents that block the induction of IkappaBalpha phosphorylation by tumor necrosis factor alpha (TNF-alpha). Furthermore, the phosphorylated IkappaBalpha from calyculin A-treated cells, but not that from TNF-alpha-stimulated cells, is sensitive to PP-2A in vitro, suggesting the existence of fundamental differences in the phosphorylation of IkappaBalpha induced by the two different NF-kappaB inducers. However, induction of IkappaBalpha phosphorylation by both TNF-alpha and the phosphatase inhibitors is associated with the subsequent degradation of IkappaBalpha. We further demonstrate that TNF-alpha- and calyculin A-induced IkappaBalpha degradation exhibits similar but not identical sensitivities to a proteasome inhibitor. Together, these results suggest that phosphorylation of IkappaBalpha, mediated through both the TNF-alpha-inducible and the PP-2A-opposing kinases, may serve to target IkappaBalpha for proteasome-mediated degradation.


INTRODUCTION

The NF-kappaB transcription factor plays a pivotal role in the regulation of various cellular genes involved in the immediate early processes of immune, acute phase, and inflammatory responses(1, 2) . In addition, NF-kappaB has also been implicated in the transcriptional activation of human viruses, most notably the type 1 human immune deficiency virus (HIV-1)(^1)(3, 4, 5, 6, 7) . NF-kappaB corresponds to a set of hetero- or homodimeric complexes composed of a family of related polypeptides. In higher vertebrates, this family includes p50, p52, RelA (previously termed p65), RelB, and c-Rel, all of which contain an N-terminal domain of homology (Rel homology domain, 300 amino acids; (8, 9, 10, 11, 12, 13, 14, 15) , reviewed in Refs. 16 and 17). In most cell types, the predominant form of NF-kappaB is a heterodimer composed of p50 and RelA. This form of NF-kappaB is normally sequestered in the cytoplasmic compartment by physical association with an inhibitory protein, termed IkappaBalpha(18, 19) . Recent studies have revealed that IkappaBalpha specifically binds to and masks the nuclear localization signal of RelA, thereby preventing the nuclear translocation of the RelAbulletp50 NF-kappaB heterodimer(20, 21, 22, 23) .

The latent cytoplasmic NF-kappaB complex can be posttranslationaly activated by a variety of cellular stimuli, including mitogens like phorbol esters, cytokines such as tumor necrosis factor alpha (TNF-alpha) and interleukin-1, and the Tax protein from the type I human T-cell leukemia virus (for a recent review, see (17) ). Activation of NF-kappaB by these various inducers involves the proteolytic degradation of the inhibitor IkappaBalpha, concomitant with the nuclear translocation of the liberated NF-kappaB heterodimer(24, 25, 26, 27, 28, 29, 30) . The biologically active nuclear NF-kappaB complex in turn activates the transcription of a large set of cellular genes encoding not only various factors involved in immune stimulation, inflammation, and cell growth but also the NF-kappaB inhibitor IkappaBalpha(24, 25, 31, 32, 33, 34) . Thus the nuclear expression of NF-kappaB is subject to tight control by an autoregulatory feedback mechanism.

Recent studies have suggested that degradation of IkappaBalpha appears to be mediated through the proteasome complex(35, 36) . Furthermore, it is likely that the degrading enzymes within the proteasome are constitutively active and that the inducible degradation of IkappaBalpha is regulated by the posttranslational modification of IkappaBalpha. Interestingly, the degradation of IkappaBalpha, induced by various NF-kappaB inducers, is preceded by the transient phosphorylation of this cytoplasmic inhibitor(30, 36, 37, 38, 39) , thus raising the possibility that phosphorylation may serve to target IkappaBalpha for subsequent degradation. In support of this proposal, kinase activators like phorbol 12-myristate 13-acetate and phosphatase inhibitors such as calyculin A and okadaic acid have been shown to induce the nuclear expression of NF-kappaB(40, 41, 42, 43) . In the present study, we demonstrate that incubation of the cells with calyculin A or okadaic acid leads to the phosphorylation of IkappaBalpha. Furthermore, unlike TNF-alpha, calyculin A induces phosphorylation of IkappaBalpha at sites that are sensitive to PP-2A. However, as observed with TNF-alpha, calyculin A-induced phosphorylation of IkappaBalpha is also associated with the subsequent proteasome-mediated degradation of this inhibitory protein. These findings suggest that differential phosphorylation of IkappaBalpha may target this inhibitor for degradation.


MATERIALS AND METHODS

Cell Culture and Reagents

Human Jurkat leukemic T cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mML-glutamine, and antibiotics. Calyculin A and okadaic acid were purchased from LC Laboratories (Woburn, MA). Tosylphenylalanyl chloromethyl ketone (TPCK) and pyrrolidinedithiocarbamate (PDTC) were from Sigma. TNF-alpha and the purified serine/threonine phosphatases, PP-1, PP-2A, and PP-2B, were obtained from Upstate Biotechnology, Inc. The proteasome inhibitor MG132 was a gift from MyoGenics Inc. (Cambridge, MA).

Immunoblotting and Electrophoresis Mobility Shift Assay (EMSA)

Jurkat T cells were collected by centrifugation and subjected to preparation of cytoplasmic and nuclear extracts(44, 49) . Cytoplasmic extracts (15 µg) were fractionated by reducing 10% SDS-polyacrylamide gel electrophoresis, electrophoretically transferred to nitrocellulose membranes, and then analyzed for immunoreactivity with a peptide-specific antiserum recognizing the C terminus of human IkappaBalpha using an enhanced chemiluminescence detection system (ECL; Amersham Corp.). For phosphatase treatment, the extracts were incubated with 0.5 units of the indicated specific serine/threonine phosphatases or 20 units of calf intestinal alkaline phosphatase at 30 °C for 30 min prior to immunoblotting analysis.

EMSA was performed by incubating the nuclear extracts (3 µg) with a P-radiolabeled high affinity palindromic kappaB probe, kappaB-pd (45) followed by resolving the DNA-protein complexes on native 5% polyacrylamide gels. For antibody ``super shift'' assays, 1 µl of anti-peptide antiserum specifically recognizing each of the NF-kappaB subunits was added to the EMSA reaction 10 min prior to electrophoresis.


RESULTS

Activation of NF-kappaB by Phosphatase Inhibitors Involves Phosphorylation and Degradation of IkappaBalpha

As described above, the phosphatase inhibitor calyculin A induces the nuclear expression of NF-kappaB in human T cells(43) . Using antibody super shift assays, we analyzed the NF-kappaB species in the kappaB-binding protein complex. As shown in Fig. 1A, a major DNA-protein complex was detected by EMSA in the nucleus of calyculin A-treated cells (Fig. 1A, lane2). More importantly, incubation of the DNA binding mixtures with either a p50-specific (lane4) or a RelA-specific (lane5) antiserum resulted in the supershift of the kappaB binding complex (Fig. 1A, arrowheads), suggesting the presence of both p50 and RelA in this complex. In contrast, this NF-kappaB/DNA complex only slightly immunoreacted with anti-c-Rel (lane6) and had no immunoreactivity with either anti-p52 (lane7) or a control preimmune serum (lane3). These results suggest that calyculin A-induced nuclear NF-kappaB complex contains predominantly the p50bulletRelA heterodimer.


Figure 1: Activation of RelAbulletp50 NF-kappaB heterodimer by calyculin A and okadaic acid involves the phosphorylation and degradation of IkappaBalpha. A, antibody super shift analysis of calyculin A-induced nuclear NF-kappaB complex. Nuclear extracts from either untreated (NT) (lane1) or calyculin A-treated (25 nM for 1 h, lanes2-7) Jurkat T cells were subjected to EMSA using a P-radiolabeled kappaB probe. To determine the NF-kappaB species in the DNA-protein complex, specific antisera recognizing different NF-kappaB subunits (lanes4-7) or a preimmune rabbit serum (PI) (lane3) were included in the EMSA. The major super shifted bands are indicated by arrowheads. B, the time course analysis of IkappaBalpha phosphorylation and degradation, as well as NF-kappaB DNA binding activity. Jurkat cells were incubated with either 25 nM calyculin A (lanes2-5) or 25 nM okadaic acid (lanes6 and 7) for the indicated time periods. Cytoplasmic and nuclear extracts, isolated from these cells, were subjected to immunoblotting with an IkappaBalpha-specific antiserum (upper panel) and EMSA using the P-labeled kappaB probe (lower panel), respectively. IkappaBalpha* is a modified form of IkappaBalpha.



To investigate the underlying mechanism of this induction event, studies were performed to test the effect of calyculin A on the fate of IkappaBalpha, a major cytoplasmic inhibitor regulating the nuclear expression of the p50bulletRelA NF-kappaB heterodimer. In untreated Jurkat cells, a single 37-kDa form of IkappaBalpha was detected with an IkappaBalpha-specific antiserum (Fig. 1B, upperpanel, lane1). Incubation of the cells with calyculin A (25 nM) for 5 min led to the appearance of a more slowly migrating IkappaBalpha species (IkappaBalpha*, lane2), which became the predominant form of IkappaBalpha after 15 min of incubation (lanes3-5). The slower mobility of this IkappaBalpha species was apparently the result of phosphorylation, since this form could be completely converted to the fast migrating 37-kDa species by in vitro incubation with calf intestinal alkaline phosphatase (data not shown). Moreover, calyculin A-induced phosphorylation of IkappaBalpha led to the subsequent degradation of this cytoplasmic inhibitor. Indeed, the preexisting IkappaBalpha was almost depleted at 60 min posttreatment (Fig. 1B, upperpanel, lane5). Okadaic acid (25 nM) also induced the phosphorylation of IkappaBalpha, albeit with delayed kinetics (3-8 h, lanes7 and 8). These results are consistent with the previous finding that calyculin A and okadaic acid exhibit differential kinetics in the inhibition of cellular phosphatases(43) .

Parallel EMSA studies revealed that the kinetics of phosphorylation and degradation of IkappaBalpha correlated with that of NF-kappaB nuclear expression in the cells treated with the phosphatase inhibitors (Fig. 1B, lower panel). Thus, activation of NF-kappaB by the phosphatase inhibitors is likely mediated through the induction of IkappaBalpha phosphorylation and subsequent degradation.

Calyculin A-induced Phosphorylation of IkappaBalpha Is Not Affected by Inhibitors of TNF-alpha Signaling

Prior studies have demonstrated that phosphorylation of IkappaBalpha by various NF-kappaB inducers, such as TNF-alpha, can be blocked by the chymotrypsin type of protease inhibitors, such as TPCK, and reducing agents like PDTC(30, 36, 47) , suggesting the role of chymotrypsin-like proteases and reactive oxygen species in this signaling pathway. To explore the signaling pathway involved in the induction of IkappaBalpha phosphorylation by the phosphatase inhibitors, the effect of TPCK and PDTC on calyculin A-induced phosphorylation of IkappaBalpha was investigated. As expected, in the absence of these agents, calyculin A induced both the phosphorylation and degradation of IkappaBalpha (Fig. 2, lanes2-4). However, in contrast to that observed with TNF-alpha(30) , preincubation of the cells with TPCK did not affect calyculin A-induced phosphorylation of IkappaBalpha (lanes5-7), although this treatment blocked the subsequent degradation of the IkappaBalpha-P (lanes5-7). Furthermore, neither phosphorylation nor degradation of IkappaBalpha induced by calyculin A was affected by preincubating the cells with PDTC (lanes8 and 9). These results suggest that induction of IkappaBalpha phosphorylation by calyculin A does not involve the signaling steps that require the action of reactive oxygen species or chymotrypsin-like proteases.


Figure 2: Effect of the chymotrypsin type of TPCK protease inhibitor and the reducing agent PDTC on calyculin A-induced phosphorylation and degradation of IkappaBalpha. Jurkat cells were either untreated (NT) (lane1) or incubated for the indicated time periods with 25 nM calyculin A in the absence (lanes2-4) or presence of either 50 µM TPCK (lanes5-7) or 200 µM PDTC (lanes 8 and 9). For the double treatments, cells were preincubated with either TPCK for 30 min or PDTC for 1 h before adding calyculin A (Cal A) to the culture. Whole-cell extracts were isolated and subjected to immunoblotting analysis with the IkappaBalpha-specific antiserum.



A PP-2A-opposing Kinase Appears to Mediate the Phosphorylation of IkappaBalpha Induced by Calyculin A but Not That Induced by TNF-alpha

To further explore the biochemical mechanism underlying the induction of IkappaBalpha phosphorylation by the phosphatase inhibitors, the molecular nature of the opposing phosphatases was investigated. In this regard, both calyculin A and okadaic acid have been shown to potently inhibit the PP-2A class of phosphatases at low concentrations (IC is 0.5-1.0 nM) (50) . However, inhibition of PP-1 and PP-2B by okadaic acid requires much higher concentrations (IC values are 60-200 nM and 10 µM, respectively). To explore the phosphatases negatively regulating IkappaBalpha phosphorylation, concentration-dependent induction of IkappaBalpha phosphorylation was performed using okadaic acid as inducer. As shown in Fig. 3A, incubation of the cells with as low as 1 nM okadaic acid led to the appearance of the IkappaBalpha-P (lane2). The level of IkappaBalpha phosphorylation and subsequent degradation was enhanced in a dose-dependent manner between 1 and 50 nM (lanes2-5). A similar concentration range was observed with calyculin A although the induction of IkappaBalpha phosphorylation by this drug was achieved within a shorter time period (30 min, data not shown) as compared with okadaic acid (12 h, Fig. 3A). These results indicate that induction of IkappaBalpha phosphorylation by these phosphatase inhibitors is probably a result of the inhibition of PP-2A but not PP-1 or PP-2B type phosphatase(s). To further explore this possibility, protein extracts isolated from calyculin A-treated cells were incubated in vitro with various purified protein phosphatases and then subjected to immunoblotting with the IkappaBalpha-specific antiserum (Fig. 3B). In support of our in vivo results, incubation of the extract with PP-2A (lane2) but not PP-1 (lane3), PP-2B (lane5), or a control buffer (lane1) resulted in the complete conversion of the IkappaBalpha-P to its basal form. A similar result of IkappaBalpha dephosphorylation was obtained with okadaic acid-treated cell extract (data not shown). Together, these in vivo and in vitro results suggest that phosphorylation of IkappaBalpha in calyculin A- or okadaic acid-treated cells is likely mediated by a protein kinase(s) opposing the action of the PP-2A type of phosphatases.


Figure 3: Phosphorylation of IkappaBalpha induced by calyculin A, but not that induced by TNF-alpha, appears to be negatively regulated by PP-2A-like phosphatases. A, concentration dependence of induction of IkappaBalpha phosphorylation by okadaic acid. Jurkat cells were incubated with okadaic acid at the indicated concentrations for 12 h. Whole-cell extracts were isolated from these cells and then subjected to immunoblotting with the IkappaBalpha-specific antiserum. IkappaBalpha and its phosphorylated form are indicated. B and C, in vitro dephosphorylation analyses of IkappaBalpha-P. A cytoplasmic extract isolated from either calyculin A-treated (A, 25 nM, 15 min) or TNF-alpha-treated (C, 10 ng/ml, 5 min) cells was incubated in vitro with either a control buffer or the indicated phosphatases at 30 °C for 30 min and then subjected to immunoblotting using the IkappaBalpha-specific antiserum. CIP, calf intestinal alkaline phosphatase.



To examine whether TNF-alpha-induced phosphorylation of IkappaBalpha is also opposed by the action of the PP-2A type of phosphatases, in vitro dephosphorylation assays were performed using a protein extract isolated from TNF-alpha-stimulated cells and the various serine/threonine phosphatases as well as a control nonspecific alkaline phosphatase, calf intestinal alkaline phosphatase (CIP, Fig. 3C, lane2). As previously observed (49) , the more slowly migrating phosphorylated form of IkappaBalpha (IkappaBalpha-P) (Fig. 3C) was readily dephosphorylated by calf intestinal alkaline phosphatase (lane6). However, incubation of the extracts with each of the serine/threonine protein phosphatases tested had no effect on this form of IkappaBalpha-P (lanes3-5), which was in sharp contrast to the dephosphorylation result obtained with calyculin A-induced IkappaBalpha-P (see Fig. 3B). While the molecular nature of the physiological phosphatases opposing the action of TNF-alpha-induced protein kinases remains unknown, these findings indicate that induction of IkappaBalpha phosphorylation by calyculin A and TNF-alpha may involve different protein kinases.

A Proteasome Inhibitor Exhibits a Differential Inhibitory Effect on Calyculin A- and TNF-alpha-stimulated IkappaBalpha Degradation

Proteasome inhibitors, including MG132 (MyoGenics), have been recently shown to inhibit TNF-alpha-induced degradation of IkappaBalpha, suggesting the involvement of the multicatalytic proteasome complex in this degradation event(35) . To investigate whether the same type of proteases is also involved in calyculin A-induced degradation of IkappaBalpha, the effect of MG132 on IkappaBalpha degradation was examined by immunoblotting (Fig. 4, upper panel) in cells treated with either calyculin A (lanes2-5) or TNF-alpha (lanes7-11). Preincubation of the cells for 1 h with 25 µM MG132 completely blocked calyculin A-induced degradation of IkappaBalpha (Fig. 4, upper panel; compare lanes2 and 3 with lanes4 and 5) as well as the nuclear expression of NF-kappaB, as determined by a parallel EMSA (lower panel). Under the same conditions, MG132 also strongly inhibited the degradation of IkappaBalpha in cells treated with TNF-alpha (Fig. 4, upper panel; compare lanes7 and 8 with lanes9 and 10). However, in contrast to the observation with calyculin A, MG132 failed to completely block the degradation of IkappaBalpha induced by TNF-alpha. Indeed, even in the presence of MG132, TNF-alpha still stimulated slow but significant loss of the IkappaBalpha-P (Fig. 4, upper panel; compare lane9 with lane10). Remarkably, along with the degradation of IkappaBalpha-P, a small peptide (20 kDa) appeared in the cells and was detected in the immunoblotting with the anti-peptide antiserum reacting with the C terminus of IkappaBalpha (lane10, IkappaBalpha-C). This peptide was apparently the C-terminal degradation product of IkappaBalpha since it could also be detected by a different antiserum specific for the C-terminal peptide of IkappaBalpha (data not shown). Moreover, the generation of this degradation product was likely mediated through a protease insensitive to MG132, since the appearance of this small peptide was not affected even in the presence of higher amounts of MG132 (50 µM, lane11, and 75 µM, data not shown). Parallel EMSA studies revealed that MG132 also failed to completely block TNF-alpha induction of the nuclear expression of NF-kappaB (Fig. 4, lower panel; compare lanes7 and 8 and lanes9-11).


Figure 4: Effect of the proteasome inhibitor MG132 on the degradation of IkappaBalpha. Jurkat T cells were either untreated (lanes1 and 6) or incubated with 50 nM calyculin A (Cal A) (lanes2 and 3) or 10 ng/ml TNF-alpha (lanes7 and 8) for the indicated time intervals. For the double treatments, cells were preincubated for 1 h with the indicated amount of MG132 and then further incubated for the indicated time periods in the presence of calyculin A (lanes4 and 5) or TNF-alpha (lanes9-13). Whole-cell extracts were analyzed by immunoblotting with the peptide-specific antiserum recognizing the C terminus of IkappaBalpha. IkappaBalpha-C is likely a degradation product containing the C terminus of IkappaBalpha. This small fragment was also detected with a different antiserum recognizing the C terminus of IkappaBalpha (data not shown).



Calyculin A and TNF-alpha Exhibit Synergistic Action in the Induction of IkappaBalpha Degradation and Activation of NF-kappaB

In view of the differential induction of IkappaBalpha phosphorylation by TNF-alpha and calyculin A, we next investigated whether these two inducers had synergistic action on the induction of IkappaBalpha degradation and nuclear expression of NF-kappaB. For these studies, Jurkat T cells were stimulated with either TNF-alpha alone (Fig. 5, lanes2-6) or TNF-alpha together with calyculin A (lanes8-12). Degradation of IkappaBalpha and nuclear expression of NF-kappaB were detected by immunoblotting (upper panel) and EMSA (lower panel), respectively. As expected, incubation of the cells with TNF-alpha led to the transient appearance of the IkappaBalpha-P and the gradual degradation of this inhibitor (Fig. 5, upper panel, lanes2-6), which was concomitant with the nuclear expression of NF-kappaB (lower panel). Under these conditions (10 ng of TNF-alpha/7 10^5 cells/ml), TNF-alpha stimulation for 30 min led to the degradation of only about 50% of the IkappaBalpha molecules (compare lane1 with lane6). Remarkably, costimulation of the cells with both TNF-alpha and calyculin A markedly enhanced the rate of IkappaBalpha degradation (upper panel, lanes8-12). Significant loss of IkappaBalpha was detected as early as 10 min after stimulation (lane10), and the entire intracellular pool of IkappaBalpha was almost depleted at 30 min (lane12). Furthermore, the enhanced rate of IkappaBalpha degradation was associated with the more rapid and remarkably higher level of NF-kappaB nuclear expression (Fig. 5, lower panel, compare lanes2-6 with lanes8-12). Of note, under the same conditions, depletion of IkappaBalpha by calyculin A alone required at least 60 min (see Fig. 1B, upper panel). Thus, the phosphatase inhibitor calyculin A and the cytokine TNF-alpha have synergistic activity in the induction of IkappaBalpha degradation and NF-kappaB nuclear expression.


Figure 5: Synergy between calyculin A (Cal A) and TNF-alpha in the induction of IkappaBalpha degradation and NF-kappaB nuclear expression. Jurkat cells were incubated with either TNF-alpha (10 ng/ml) alone (lanes2-6) or TNF-alpha together with 25 nM calyculin A (lanes8-12) for the indicated time periods. Cytoplasmic and nuclear extracts were subjected to immunoblotting with the IkappaBalpha-specific antiserum (upper panel) and EMSA using the P-labeled kappaB probe (lower panel), respectively.




DISCUSSION

The nuclear expression and biological function of the NF-kappaB transcription factor are tightly regulated through its cytoplasmic retention by the ankyrin-rich inhibitor IkappaBalpha(20, 21, 22, 23) . Activation of NF-kappaB by various cellular stimuli involves the proteolytic degradation of IkappaBalpha and the concomitant nuclear translocation of the liberated NF-kappaB heterodimer(24, 25, 26, 27, 28, 29, 30) . Although the biochemical mechanism underlying the degradation of IkappaBalpha remains unclear, it appears that degradation of IkappaBalpha induced by various mitogens and cytokines occurs in association with the transient phosphorylation of IkappaBalpha(30, 36, 37, 38, 39) . In the present study, we have demonstrated that the serine/threonine phosphatase inhibitors, calyculin A and okadaic acid, also induce the phosphorylation and subsequent degradation of IkappaBalpha. Induction of IkappaBalpha phosphorylation by okadaic acid can be achieved at low concentrations (1-5 nM), at which PP-2A, but not other phosphatases including PP-1 and PP-2B, is able to be inhibited(48) . These findings suggest that the action of okadaic acid, and probably also calyculin A, is mediated through the inhibition of the cellular PP-2A type of phosphatases. In support of this notion, the IkappaBalpha-P produced in both calyculin A- and okadaic acid-treated cells can be readily dephosphorylated in vitro by PP-2A but not by PP-1 or PP-2B. This result further suggests that phosphorylation of IkappaBalpha may be directly mediated by a PP-2A-opposing protein kinase. However, from our current studies, we are not certain whether the activation of the IkappaBalpha kinase is a direct result of PP-2A inhibition or is mediated through an indirect mechanism that, for example, may involve the activation of the upstream signaling molecules.

A previous study has shown that activation of NF-kappaB by okadaic acid can be partially inhibited by the antioxidant cysteine, suggesting the involvement of reactive oxygen species in this induction process(42) . However, our present study is not in full agreement with these previous findings. We have demonstrated that induction of the phosphorylation as well as subsequent degradation of IkappaBalpha by calyculin A (25 nM) is not appreciably inhibited by PDTC, a potent antioxidant that blocks TNF-alpha-induced degradation of IkappaBalpha and nuclear expression of NF-kappaB(25, 50) . This discrepancy is likely due to the much higher concentration (2 µM) of the phosphatase inhibitors used in the previous study. At this concentration, both calyculin A and okadaic acid will nonspecifically inhibit most types of the serine/threonine phosphatases(48) , which may in turn trigger additional, perhaps antioxidant-sensitive, signaling pathways. In support of this proposal, okadaic acid has been shown to synergize the action of H(2)O(2) only at high concentrations (2 µM)(42) . Furthermore, a more recent study using a low concentration of calyculin A and okadaic acid has revealed that activation of NF-kappaB by these phosphatase inhibitors is indeed insensitive to antioxidants(43) .

We have also shown that induction of IkappaBalpha phosphorylation by calyculin A is not inhibited by TPCK, a potent inhibitor of the chymotrypsin type of proteases that blocks TNF-alpha-induced IkappaBalpha phosphorylation(30, 36) . Together, these findings suggest that induction of IkappaBalpha phosphorylation by the phosphatase inhibitors may either bypass the signaling steps that are sensitive to TPCK and antioxidants or involve a signaling pathway that is different from that triggered by TNF-alpha. Our data favor the latter possibility. We have shown that the IkappaBalpha-P produced in TNF-alpha-treated cells can not be dephosphorylated in vitro by PP-2A, although under the same conditions this phosphatase proves to be efficient in removing the phosphate from calyculin A-induced IkappaBalpha-P. Thus, phosphorylation of IkappaBalpha induced by these two different NF-kappaB inducers may be mediated by distinct kinases. However, a more conclusive answer to this question awaits the precise determination of the phosphorylation sites within these two types of IkappaBalpha-P.

Recent studies have demonstrated that inhibition of IkappaBalpha degradation by certain proteasome inhibitors leads to the accumulation of the IkappaBalpha-P(36, 37, 38, 39) , thus raising the possibility that phosphorylation of IkappaBalpha may target this inhibitor for degradation by the proteasome. In this regard, one potential model may involve phosphorylation serving as a molecular trigger to alter the conformation of IkappaBalpha, thus facilitating the action of the constitutively active proteases. According to this scenario, phosphorylation of IkappaBalpha on different sites can cause distinct conformational changes, which may in turn lead to the differential sensitivities of IkappaBalpha to the cellular proteases. Indeed, this outcome has been observed in our experiments. Specifically, calyculin A-induced degradation of IkappaBalpha can be completely blocked in the presence of 25 µM MG132, suggesting the involvement of solely the MG132-sensitive proteases in this degradation process. In contrast, TNF-alpha-induced IkappaBalpha degradation appears to involve additional proteases that are insensitive to MG132. In the presence of up to 75 µM of MG132, partial degradation of IkappaBalpha still occurs in TNF-alpha-stimulated cells, which leads to the accumulation of a small degradation product (20 kDa) containing the C terminus of IkappaBalpha. Of course, it remains a possibility that calyculin A and TNF-alpha both induce IkappaBalpha kinases and up-regulate cellular proteases, which act coordinately to eliminate IkappaBalpha. Nevertheless, from our current studies, we cannot conclude that phosphorylation targets IkappaBalpha for degradation. Studies are in progress to precisely map the phosphorylation sites within the IkappaBalpha-P, which will allow us to determine the biological importance of phosphorylation in the degradation of IkappaBalpha.


FOOTNOTES

*
This study was supported in part by American Cancer Society Grant IRG-196. 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.

§
Scholar of the American Foundation for AIDS Research. To whom correspondence should be addressed. Tel.: 717-531-4164; Fax: 717-531-6522.

^1
The abbreviations used are: HIV-1, type 1 human immune deficiency virus; TNF-alpha, tumor necrosis factor alpha; PP-1, PP-2A, and PP-2B, phosphatase type 1, 2A, and 2B, respectively; TPCK, tosylphenylalanyl chloromethyl ketone; PDTC, pyrrolidinedithiocarbamate; EMSA, electrophoresis mobility shift assay; IkappaBalpha-P, phosphorylated IkappaBalpha.


ACKNOWLEDGEMENTS

We thank Dr. Warner Greene for the IkappaBalpha-specific antisera and MyoGenics, Inc. for providing us with the proteasome inhibitor MG132.


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