The Serine/Threonine Phosphatase Inhibitor Calyculin A Induces Rapid Degradation of Ikappa Bbeta
REQUIREMENT OF BOTH THE N- AND C-TERMINAL SEQUENCES*

(Received for publication, November 19, 1996, and in revised form, December 12, 1996)

Edward W. Harhaj and Shao-Cong Sun Dagger

From the Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, Hershey Medical Center, Hershey, Pennsylvania 17033

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgment
REFERENCES


ABSTRACT

Signal-initiated activation of the transcription factor NF-kappa B is mediated through proteolysis of its cytoplasmic inhibitory proteins Ikappa Balpha and Ikappa Bbeta . While most NF-kappa B inducers trigger the degradation of Ikappa Balpha , only certain stimuli are able to induce the degradation of Ikappa Bbeta . The degradation of Ikappa Balpha is targeted by its site-specific phosphorylations, although the mechanism underlying the degradation of Ikappa Bbeta remains elusive. In the present study, we have analyzed the effect of phosphatase inhibitors on the proteolysis of Ikappa Bbeta . We show that the serine/threonine phosphatase inhibitor calyculin A induces the hyperphosphorylation and subsequent degradation of Ikappa Bbeta in both human Jurkat T cells and the murine 70Z-3 preB cells, which is associated with the nuclear expression of active NF-kappa B. The calyculin A-mediated degradation of Ikappa Bbeta is further enhanced by the cytokine tumor necrosis factor-alpha (TNF-alpha ), although TNF-alpha alone is unable to induce the degradation of Ikappa Bbeta . Mutational analyses have revealed that the inducible degradation of Ikappa Bbeta induced by calyculin A, and TNF-alpha requires two N-terminal serines (serines 19 and 23) that are homologous to the inducible phosphorylation sites present in Ikappa Balpha . Furthermore, the C-terminal 51 amino acid residues, which are rich in serines and aspartic acids, are also required for the inducible degradation of Ikappa Bbeta . These results suggest that the degradation signal of Ikappa Bbeta may be controlled by the opposing actions of protein kinases and phosphatases and that both the N- and C-terminal sequences of Ikappa Bbeta are required for the inducible degradation of this NF-kappa B inhibitor.


INTRODUCTION

The NF-kappa B/Rel family of transcription factors play 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, these cellular factors have also been implicated in the transcriptional activation of certain human viruses, most notably the type 1 human immune deficiency virus (3-7). The mammalian NF-kappa B/Rel family is composed of at least five structurally related DNA-binding proteins, including p50, p52, RelA, RelB, and c-Rel, which bind to a target DNA sequence (kappa B) as various heterodimers or homodimers (reviewed in Siebenlist et al. (8)). In most cell types, including resting T cells, the NF-kappa B/Rel proteins are sequestered in the cytoplasmic compartment by physical association with inhibitory proteins that are characteristic of the presence of various numbers of ankyrin-like repeats (reviewed in Verma et al. (9)). The major cytoplasmic inhibitors include Ikappa Balpha (10, 11), Ikappa Bbeta (12), and the precursor proteins of p50 and p52 (9). The Ikappa B molecules appear to bind to and mask the nuclear localization signal of NF-kappa B/Rel, thereby preventing the nuclear translocation of these transcription factors (13-16).

The latent cytoplasmic NF-kappa B/Rel complexes can be activated by a variety of cellular stimuli, including the mitogen phorbol esters, cytokines such as tumor necrosis factor-alpha (TNF-alpha ),1 and interleukin-1, the bacterial component lipopolysaccharide, serine/threonine phosphatase inhibitors such as okadaic acid and calyculin A, and the Tax protein from the type I human T cell leukemia virus (HTLV-I) (8, 17). Activation of NF-kappa B by these various inducers involves phosphorylation of Ikappa Balpha at serines 32 and 36 (18-23), which in turn targets this inhibitory protein for ubiquitination and proteasome-mediated proteolysis (24, 25). Since the Ikappa Balpha gene is positively regulated by the NF-kappa B/Rel factors, the depleted Ikappa Balpha protein pool can be rapidly replenished through de novo protein synthesis following the activation of NF-kappa B/Rel (26-31). Thus, Ikappa Balpha regulates the transient nuclear expression of NF-kappa B/Rel. Unlike Ikappa Balpha , Ikappa Bbeta appears to respond to only certain cellular stimuli, such as lipopolysaccharide, interleukin-1, and Tax, that are known to induce sustained nuclear expression of NF-kappa B/Rel (12, 32, 33). The depleted Ikappa Bbeta protein is not immediately resynthesized, which is likely the molecular basis of persistent activation of NF-kappa B/Rel.

The molecular mechanism underlying the differential signal responses between Ikappa Balpha and Ikappa Bbeta remains elusive. Although Ikappa Bbeta contains two N-terminal serines (serines 19 and 23) that are homologous to the inducible phosphorylation sites of Ikappa Balpha (11, 12), it is unclear whether phosphorylation can target Ikappa Bbeta for degradation. Evidence supporting a role of phosphorylation in Ikappa Bbeta degradation is provided by site-mutagenesis studies which demonstrate that mutation of serines 19 and 23 to alanines abolishes the inducible degration of Ikappa Bbeta (21, 33). In the present study, we have further investigated the role of phosphorylation in the inducible degradation of Ikappa Bbeta by examining the effect of a serine/threonine phosphatase inhibitor, calyculin A, on the fate of Ikappa Bbeta . We demonstrate that incubation of Jurkat T cells or 70Z/3 pre-B cells with calyculin A is sufficient to induce the hyperphosphorylation and subsequent degradation of Ikappa Bbeta .


MATERIALS AND METHODS

Cell Culture and Reagents

Jurkat T cells (ATCC) and Jurkat cells expressing the SV40 large T antigen (Jurkat Tag) (34) were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and antibiotics. Murine 70Z/3 pre-B cells (ATCC) were maintained in the same medium supplemented with 50 µM beta -mercaptoethanol. The serine/threonine phosphatase inhibitor calyculin A was purchased from LC Laboratories (Woburn, MA). The proteasome inhibitor MG132 was purchased from ProScript, Inc. (Cambridge, MA). The antibody against the influenza hemagglutinin (HA) epitope tag (anti-HA) was obtained from Boehringer Mannheim. Anti-Ikappa Bbeta (C-20) was purchased from Santa Cruz Biotechnology, Inc.

Plasmid Constructs and Transient Transfection

The wild type of pCMV4HA-Ikappa Bbeta was constructed by cloning the Ikappa Bbeta cDNA (kindly provided by Dr. Sankar Ghosh, Yale University) (12) into a modified pCMV4 expression vector, pCMV4HA (22), downstream of three copies of the HA epitope tag (YPYDVPDYA). Ikappa Bbeta 19A/23A was generated by substituting serines 19 and 23 with alanines using site-directed mutagenesis (ClonTech, Inc.). Ikappa Bbeta Delta 5-27, which lacks amino acids 5-27, was also generated by site-directed mutagenesis. The C-terminal truncation mutant {Ikappa Bbeta (1-308)} was constructed by introducing a stop codon after codon 308 of the wild type Ikappa Bbeta by restriction digestion (using HindIII), DNA polymerase (Klenow fragment) fill in, and religation. Jurkat Tag cells (5 × 106) were transfected using DEAE-dextran (35) with the indicated amounts of Ikappa Bbeta expression vectors. Between 40 and 48 h post-transfection, the cells were incubated with calyculin A (25 nM) and TNF-alpha (10 ng/ml) for the indicated time periods and then subjected to whole extract preparation and immunoblotting analyses as described below.

Immunoblotting and Electrophoresis Mobility Shift Assay (EMSA)

Jurkat cells, 70Z/3 pre-B cells, or transiently transfected Jurkat-Tag cells were stimulated with the indicated inducers and then collected by centrifugation at 800 × g for 5 min. Whole cell and subcellular extracts were prepared as described previously (36, 37). For immunoblotting analyses, whole cell extracts (~15 µg) were fractionated by reducing 8.75% SDS-PAGE, electrophoretically transferred to nitrocellulose membranes, and then analyzed for immunoreactivity with the indicated primary antibodies using an enhanced chemiluminescence detection system (ECL; Amersham Corp.). For in vitro phosphatase treatment, the extracts were incubated with 20 units of calf intestinal alkaline phosphatase at 35 °C for 30 min prior to immunoblotting analysis. EMSA were performed by incubating the nuclear extracts (~ 3 µg) with a 32P-radiolabeled high-affinity palindromic kappa B probe, kappa B-pd (coding strand sequence was 5'-CAACGGCAGGGGAATTCCCCTCTCCTT-3') followed by resolving the DNA-protein complexes on native 5% polyacrylamide gels (38).


RESULTS

Calyculin A Induces the Rapid Phosphorylation and Degradation of Ikappa Bbeta and the Concurrent Nuclear DNA Binding Activity of NF-kappa B in Both Human Jurkat T Cells and Murine 70Z/3 Pre-B Cells

To investigate the effect of the phosphatase inhibitors on the fate of Ikappa Bbeta , Jurkat T cells were incubated with calyculin A for different time periods followed by analysis of the Ikappa Bbeta protein by immunoblotting (Fig. 1A, upper panel). In untreated cells, a single 45-kDa form of Ikappa Bbeta was detected with an Ikappa Bbeta -specific antiserum (Fig. 1A, upper panel, lane 1). Incubation of the cells with calyculin A (25 nM) for 15 min led to a marked loss of the preexisting Ikappa Bbeta protein (lane 3), which persisted until at least 1 h after calyculin A stimulation (lanes 3-5). The loss of Ikappa Bbeta was apparently due to its proteolysis as this effect of calyculin A was blocked by a potent proteasome inhibitor, MG132 (lane 7), known to inhibit the degradation of Ikappa Balpha (24, 39). Parallel EMSA revealed that degradation of Ikappa Bbeta was associated with the appearance of the NF-kB DNA binding activity in the nucleus (Fig. 1A, lower panel). We noticed that the Ikappa Bbeta isolated from cells treated with calyculin A migrated more slowly on the SDS-polyacrylamide gel compared to the basal form of Ikappa Bbeta (Fig. 1A, compare lanes 1 and 6 with lanes 2-5 and 7). To examine whether the slower migration of Ikappa Bbeta might be due to its phosphorylation, the cell extract was incubated with calf intestine alkaline phosphatase before being subjected to immunoblotting (Fig. 1B). After calf intestine alkaline phosphatase treatment, the more slowly migrating Ikappa Bbeta species from calyculin A-treated cells (lane 2) was completely converted to a faster-migrating Ikappa Bbeta (Fig. 1B, lane 3), thus suggesting that the slower migration of Ikappa Bbeta in calyculin A-treated cells was due to its phosphorylation. Interestingly, the in vitro dephosphorylated form of Ikappa Bbeta (lane 3) migrated slightly faster than the basal form present in untreated cells (lane 1). This result suggests that as seen with murine 70Z/3 pre-B cells (40), Ikappa Bbeta is preexisting in a basally phosphorylated form in untreated Jurkat T cells. This basal form of Ikappa Bbeta seems to become hyperphosphorylated when the cells are treated with calyculin A. Of note, the hyperphosphorylation of Ikappa Bbeta appeared to precede its degradation since inhibition of Ikappa Bbeta degradation by MG132 led to the accumulation of the more slowly migrating hyperphosphorylated Ikappa Bbeta (Fig. 1A, upper panel, lane 7).


Fig. 1. Calyculin A induces the phosphorylation and degradation of Ikappa Bbeta in Jurkat and 70Z/3 cells. A and C, time course analysis of Ikappa Bbeta phosphorylation and degradation and parallel NF-kappa B DNA binding activity. Jurkat (A) or 70Z/3 cells (C) were treated with 25 nM calyculin A for the indicated time periods (lanes 2-5 and 7). The proteasome inhibitor MG132 (25 µm) was added 30 min prior to stimulation with calyculin A (lane 7). Cytoplasmic and nuclear extracts were isolated and subjected to immunoblotting with an Ikappa Bbeta specific antiserum (upper panel) and EMSA with a 32P-labeled kappa B probe (lower panel), respectively. B, in vitro phosphatase treatment of calyculin A-treated extracts. Jurkat cells were treated with 25 nM calyculin A (lanes 2 and 3), and the cytoplasmic extracts were subjected to in vitro calf intestinal alkaline phosphatase (CIP) treatment (20 units) for 30 min at 35 °C (lane 3). The samples were then used for immunoblotting with an Ikappa Bbeta antiserum. Hyper indicates a hyperphosphorylated form of Ikappa Bbeta , and hypo refers to a hypophosphorylated form.
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To examine whether the effect of calyculin A on Ikappa Bbeta could be recapitulated in other cell types, murine 70Z/3 pre-B cells were subjected to the calyculin A treatment. In untreated 70Z/3 cells, Ikappa Bbeta is preexisting in two forms that migrate with slightly different rates on SDS-PAGE (Fig. 1C, upper panel, lane 1). The more slowly migrating band was apparently the phosphorylated form of Ikappa Bbeta since it was converted to the more rapidly migrating form after in vitro incubation with calf intestinal alkaline phosphatase (data not shown) (40). More importantly, incubation of the 70Z/3 cells with calyculin A led to the rapid degradation of the preexisting Ikappa Bbeta proteins (Fig. 1C, lanes 2-5, upper panel). Furthermore, as observed in Jurkat T cells, the degradation of Ikappa Bbeta was preceded by the appearance of the more slowly migrating hyperphosphorylated Ikappa Bbeta (lanes 2-5).

Together, these results suggest that the serine/threonine phosphatase inhibitor calyculin A is able to induce the proteolysis of Ikappa Bbeta in both Jurkat T cells and 70Z/3 pre-B cells and that the degradation of Ikappa Bbeta is preceded by its hyperphosphorylation.

TNF-alpha Promotes Calyculin A-induced Degradation of Ikappa Bbeta

Prior studies have demonstrated that the TNF-alpha -elicited cellular activation signal is insufficient to induce the degradation of Ikappa Bbeta , although this signal induces the degradation of Ikappa Balpha (12). To investigate whether the TNF-alpha -mediated signal could synergize with the phosphatase inhibitor in the degradation of Ikappa Bbeta , we examined the effect of TNF-alpha on calyculin A-mediated degradation of Ikappa Bbeta . As previously reported, incubation of Jurkat T cells with TNF-alpha alone was inefficient in the induction of Ikappa Bbeta degradation (Fig. 2, lanes 4 and 5). However, when the cells were treated with TNF-alpha together with calyculin A, significant Ikappa Bbeta degradation could be detected as early as 5 min after cellular stimulation (lane 6), and the entire intracellular pool of Ikappa Bbeta was almost completely depleted at 30 min poststimulation (lane 7). Consistent with the results shown in Fig. 1A, calyculin A alone induced the degradation of Ikappa Bbeta (lanes 2 and 3); however, the kinetics of Ikappa Bbeta degradation in these cells was slower compared to that detected in cells costimulated with TNF-alpha and calyculin A (compare lanes 2 and 3 with lanes 6 and 7).


Fig. 2. TNF-alpha accelerates calyculin A-mediated degradation of Ikappa Bbeta . Immunoblotting analysis of Ikappa Bbeta in Jurkat cells treated with both calyculin A and TNF-alpha . Jurkat cells were treated with either 25 nM calyculin A (lanes 2 and 3), 10 ng/ml TNF-alpha (lanes 4 and 5), or calyculin A together with TNF-alpha (lanes 6 and 7) for the indicated time periods. The cells were harvested after the treatments and the cytoplasmic extracts were subjected to immunoblotting with an Ikappa Bbeta antiserum. The arrow denotes a nonspecific band cross-reacting with the antiserum.
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Both the N- and C-terminal Sequences Are Required for Degradation of Ikappa Bbeta

To further explore the biochemical mechanism underlying the induction of Ikappa Bbeta degradation by calyculin A and TNF-alpha , studies were performed to examine the sequences required for the inducible degradation of Ikappa Bbeta . For these studies, cDNA expression vectors encoding HA-tagged wild type or mutant Ikappa Bbeta were transfected into Jurkat-Tag cells, and the inducible degradation of these Ikappa Bbeta proteins was analyzed by immunoblotting using an anti-HA antibody. The exogenously transfected wild type Ikappa Bbeta expressed as two forms with slightly different mobilities on SDS-PAGE (Fig. 3A, lane 2). As seen in 70Z/3 cells, the differential mobility of these two forms of Ikappa Bbeta appeared to be due to different levels of phosphorylation as demonstrated by in vitro calf intestinal alkaline phsophatase assays (data not shown). Stimulation of the transfectants with calyculin A and TNF-alpha led to the gradual depletion of the ectopic Ikappa Bbeta (lanes 3-5). Thus, as seen with its endogenous counterpart, the transfected HA-tagged Ikappa Bbeta could be degraded in response to cellular stimulation. Deletion of an N-terminal region (amino acids 5-27) covering two potential phosphorylation sites (serines 19 and 23) did not influence the basal phosphorylation of Ikappa Bbeta , since both the slow and fast migrating bands were detected in cells transfected with this mutant (Fig. 3B, lane 4). However, this Ikappa Bbeta deletion mutant failed to be degraded following cellular stimulation with calyculin A and TNF-alpha (lanes 4-6). To examine whether serines 19 and 23 were important for the degradation of Ikappa Bbeta , an Ikappa Bbeta mutant bearing mutations at these sites was tested in the degradation assay. As expected, mutation of these two serines to alanines significantly inhibited the degradation of Ikappa Bbeta (lanes 1-3). We then examined the potential role of the C-terminal sequences in the degradation of Ikappa Bbeta . In this regard, the C-terminal portion of Ikappa Bbeta is rich in serines and aspartic acids (12) and has recently been shown to contain the sites for constitutive phosphorylation (41). Consistent with this recent study, an Ikappa Bbeta mutant lacking the C-terminal 51 amino acids {Ikappa Bbeta (1-308)} migrated on SDS-PAGE as a single band (lane 7), indicating the lack of constitutive phosphorylation. More importantly, deletion of the C-terminal acidic sequences markedly inhibited the degradation of Ikappa Bbeta (lanes 8 and 9). Thus, degradation of Ikappa Bbeta induced by calyculin A and TNF-alpha requires both the N-terminal potential phosphorylation sites and the C-terminal sequences.


Fig. 3. The N- and C-terminal regions of Ikappa Bbeta are required for degradation by calyculin A. Immunoblotting analysis of various Ikappa Bbeta constructs transiently transfected into Jurkat Tag cells. A. Jurkat Tag cells (5 × 106) were transiently transfected with either the pCMV4 expression vector alone (lane 1) or with an HA-tagged wild type Ikappa Bbeta cDNA construct. Approximately 40 h post-transfection, the Ikappa Bbeta transfectant was split into four equal volumes and was either left unstimulated (lane 2) or stimulated with calyculin A (25 nM) together with TNF-alpha (10 ng/ml) for the indicated times (lanes 2-5). Whole cell extracts were collected and subjected to immunoblotting with a monoclonal HA-specific antiserum (anti-HA). B, Jurkat Tag cells were transiently transfected with HA-tagged cDNA constructs encoding Ikappa Bbeta A19/23, Ikappa Bbeta Delta 5-27, and Ikappa Bbeta 1-308. Each of the transfectants was split into three equal volumes and either left untreated (lanes 1, 4, and 7, respectively) or treated with calyculin A and TNF-alpha for the indicated times. Extract preparation and immunoblotting was performed as mentioned in A.
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DISCUSSION

The nuclear expression and biological function of the NF-kappa B transcription factor is tightly regulated through its cytoplasmic retention by ankyrin-rich inhibitors, including Ikappa Balpha and Ikappa Bbeta (12-16). Activation of NF-kappa B by various cellular stimuli involves the site-specific phosphorylation and subsequent proteolytic degradation of Ikappa Balpha , which is associated with the transient nuclear expression of the liberated NF-kappa B factors (18-21). However, activation of the Ikappa Bbeta -sequestered NF-kappa B pool is triggered by only certain cellular stimuli, which normally induce persistent NF-kappa B activation, such as lipopolysaccharide and interleukin-1 (12) and the HTLV-I Tax protein (32, 33). It remains elusive why the two types of Ikappa B molecules differentially respond to the cellular activation signals. Although Ikappa Bbeta contains two N-terminal serines (Ser-19/Ser-23) that are homologous to the inducible phosphorylation sites of Ikappa Balpha , it is not clear whether these sites are phosphorylated in response to cellular stimulation.

In the present study, we have demonstrated that the serine/threonine phosphatase inhibitor calyculin A is able to induce the phosphorylation and degradation of Ikappa Bbeta . This finding supports a model that phosphorylation may trigger the proteolysis of Ikappa Bbeta . However, from our current study, we cannot conclude that the two N-terminal serines (serines 19 and 23) are phosphorylated. To directly address this question, phosphopeptide analyses are necessary. It is clear, though, that site-directed mutagenesis of these two serines to alanines markedly attenuates degradation by calyculin A (Fig. 3) and other inducers such as Tax, TNF-alpha (in Hela cells), and PMA/anti-CD28 (21, 33, 42). Interestingly, we observed basal phosphorylation of Ikappa Bbeta in both Jurkat and 70Z/3 cells (Fig. 1); however, this appears to involve phosphorylation of sites within the C-terminal region (Fig. 3) (41).

We have also demonstrated that in Jurkat T cells, TNF-alpha in synergy with calyculin A has the capacity to accelerate the degradation of Ikappa Bbeta . However, in agreement with a previous study (12), in these leukemic T cells, TNF-alpha alone is not sufficient to induce the degradation of Ikappa Bbeta (Fig. 2), although it is efficient in the induction of Ikappa Balpha degradation (27). These results suggest that the signals required for triggering the degradation of Ikappa Bbeta are more stringent than those for the degradation of Ikappa Balpha . One possibility is that the N-terminal region of Ikappa Bbeta is not as efficient a substrate for phosphorylation as the homologous region of Ikappa Balpha . Phosphorylation of Ikappa Bbeta thus would require more potent signals which presumably would result in more vigorous kinase activity. Phosphatase inhibitors would act in this manner by leaving kinase activity virtually unopposed by any regulatory cellular phosphatases.

As seen with Ikappa Balpha , the C-terminal region of Ikappa Bbeta is rich in prolines, serines, and acidic amino acids. Such a sequence, known as PEST, has been proposed to be involved in the rapid turnover of certain proteins (43). The PEST sequence appears to be dispensable for the inducible degradation of Ikappa Balpha , although this C-terminal region may regulate the constitutive turnover of Ikappa Balpha (22, 44, 45). However, the PEST sequence is likely required for the inducible degradation of Ikappa Bbeta since deletion of this region renders Ikappa Bbeta nonresponsive to the potent degradation signals initiated by calyculin A together with TNF-alpha (Fig. 3B). A recent study (41) suggests that phosphorylation of two serines within the PEST region is critical for the interaction of Ikappa Bbeta with the c-Rel protooncoprotein. However, it is unclear whether the basal phosphorylation at the C terminus of Ikappa Bbeta plays a role in regulation of its inducible degradation, although a C-terminal truncation mutant of Ikappa Bbeta lacking these phosphorylation sites becomes unresponsive to the degradation signals (Fig. 3B). Studies are in progress to determine possible functions of the Ikappa Bbeta C-terminal PEST domain and to map specific amino acids in the C terminus required for inducible degradation.


FOOTNOTES

*   This study was supported by United States Public Health Service Grant 1 R01 CA68471-01, an AmFAR grant made in memory of Richard Lee Schiffman, and an American Society of Hematology Junior Faculty Scholar Award (to S.-C. S.). 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.
Dagger    To whom correspondence should be addressed: Dept. of Microbiology and Immunology, Pennsylvania State University College of Medicine, Hershey Medical Center, P. O. Box 850, Hershey, PA. Tel.: 717-531-4164; Fax: 717-531-6522.
1    The abbreviations used are: TNF-alpha , tumor necrosis factor-alpha ; HTLV-I, type I human T cell leukemia virus; EMSA, electrophoresis mobility shift assay; PAGE, polyacrylamide gel electrophoresis; HA, hemagglutinin.

Acknowledgment

We gratefully acknowledge Dr. S. Ghosh for the Ikappa Bbeta cDNA expression vector.


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