©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Evidence in Support of a Role for Human T-cell Leukemia Virus Type I Tax in Activating NF-B via Stimulation of Signaling Pathways(*)

Tomohiko Kanno, Keith Brown, and Ulrich Siebenlist (§)

From the (1) Laboratory of Immunoregulation, NIAID, National Institutes of Health, Bethesda, Maryland 20892-1876

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The human T-cell leukemia virus type I Tax protein activates NF-B transcription factors from preformed cytoplasmic pools, including those pools that are retained by the IB- inhibitory protein. Degradation of IB- is enhanced by Tax, resulting in the liberation of some NF-B, which then translocates into the nucleus. Here we have investigated the mechanism by which Tax causes degradation of IB-. Two IB- mutants defective in extracellular signal-induced degradation of IB- also blocked Tax-mediated B-dependent transactivation when cotransfected into Jurkat T cells. Cotransfected wild-type IB- or an irrelevant mutant did not significantly effect transactivation induced by Tax. The signal-defective IB- proteins are mutated at either of two closely spaced serines in the N terminus of the protein (Ser and Ser). In wild-type IB-, one or both of these serines are inducibly phosphorylated with extracellular stimuli, and such phosphorylation appears necessary for subsequent degradation and thus activation of NF-B. These results suggest that Tax triggers IB- degradation and thus NF-B activation by a mechanism that converges with that induced by extracellular stimulation such as phorbol 12-myristate 13-acetate/ionomycin or tumor necrosis factor . A role for Tax in activating signal transduction pathways upstream of IB- is implied.


INTRODUCTION

The human T-cell leukemia virus type I Tax protein is a potent inducer of NF-B activity (1) . NF-B dimers, typically heterodimers of p50 (NF-B1) and p65 (RelA), are normally kept in the cytoplasm by association with their inhibitors, primarily with IB- but also including p100 (NF-B2), p105 (NF-B1) (2) , and IB- (3) . Tax induces translocation of NF-B from the cytoplasm into the nucleus, apparently by liberating NF-B from several distinct cytoplasmic complexes (4, 5, 6, 7) . In particular, degradation of IB- is enhanced in the presence of Tax, suggesting that some NF-B can be released by rapid turnover of this inhibitor (4) . IB- is also rapidly degraded during physiologic activation of NF-B by extracellular stimuli (2) . Such activation proceeds via signal-induced phosphorylation, which marks IB- for proteolytic degradation, presumably by proteasomes (8, 9) . The question arises how Tax induces degradation of IB-. Previous reports suggested at least two divergent explanations. Based on observed binding of Tax to various IB and NF-B proteins (6, 7, 10) , Tax may effect dissociation of the inhibitor, which in turn could lead to rapid turnover of the now uncomplexed and thus more unstable inhibitor; alternatively, Tax may have an indirect effect, possibly by affecting physiologic signal transduction pathways, which then lead to activation of NF-B. Indirect support for the latter model comes from the observation that Tax-expressing cells have detectable amounts of phosphorylated IB- (4, 5) ; in addition, the antioxidant pyrrolidinedithiocarbamate has been noted to inhibit both normal signal-induced as well as Tax-induced activation of NF-B, although the mechanism by which this occurs is unknown (11) .

Recently we have identified two serines (Ser and Ser) in the N terminus of IB- that are critical to activation of NF-B in response to signals such as phorbol 12-myristate 13-acetate (PMA)() /ionomycin or tumor necrosis factor (12) . IB- proteins in which either of these closely spaced serines is altered cannot be inducibly phosphorylated at these sites nor are the mutant proteins subject to signal-induced degradation, presumably as a consequence to the block in phosphorylation. As expected, these IB- mutants also prevent signal-induced activation of bound NF-B. Wild-type IB- is tagged by induced phosphorylation at these serines to undergo rapid proteolysis. Phosphorylation by itself is insufficient to dissociate the complex, suggesting that degradation of IB- is initiated while NF-B is still attached to the inhibitor. In addition to phosphorylation of the two N-terminal serine(s), signal-induced degradation of IB- also requires additional sequences rich in Pro, Glu/Asp, Ser, and Thr residues (PEST sequences) located in the C-terminal PEST region of the inhibitor (12) .

In the present study we have utilized the two signal-defective IB- mutants in cotransfection experiments together with Tax. We have determined that Tax-mediated B-dependent transactivation is potently inhibited in the presence of these mutant proteins, while wild-type IB- or an irrelevant mutant is a much less effective inhibitor. The results support a model in which Tax affects signaling proteins functioning at or upstream of IB- to effect phosphorylation and thus degradation of the inhibitor.


MATERIALS AND METHODS

IB- Mutants

IB- mutants used in this study have been described (12) . Ser was changed to Gly; Ser was changed to Ala; and Ser was changed to Ala. The mutants are referred to as m32, m36, and m63, respectively.

Expression Vectors

pMT2T-p65, pMT2T-Tax, pMT2T-IB-, pMT2T-IB/m32, pMT2T-IB-/m36, and pMT2T-IB-/m63 have been described (4, 8, 12) . The luciferase reporter construct, Ig-B-Luc, which contains three repeats of the immunoglobulin -light chain enhancer B site (13) , was kindly provided by T. Fujita.

Cells, Transfection, and Luciferase Assay

Jurkat human T-lymphocytes and EL-4 mouse T-lymphocytes stably expressing either the wild type or the m32 mutant of human IB- (12) were grown in RPMI 1640 medium supplemented with 10% fetal calf serum, glutamine, and antibiotics. Electroporation (using Gene Pulser (Bio-Rad) at 960 microfarads, 250 V) was performed with 10 cells in 300 µl of the culture medium at room temperature. The luciferase assay was performedas described (14) using a luminometer (Analytical Luminescence Laboratory).

RESULTS

Jurkat T cells were transfected with a B-dependent reporter construct and expression vectors for p65, Tax, and/or IB- (Fig. 1). The amount of IB- used for cotransfection was able to significantly inhibit the p65-mediated transactivation of the reporter (lanes1 and 2). However, the same amount of IB- was unable to inhibit the Tax-mediated activation of endogenous NF-B complexes when assayed with the same reporter (lanes3 and 4). The results suggest that Tax neutralized the inhibitory effects of these levels of transfected IB-. Tax may have induced degradation not only of endogenous but also of exogenously introduced IB- proteins.


Figure 1: Tax can neutralize inhibitory effects of cotransfected wild-type IB-. Jurkat cells (10 cells) were transfected with Ig-B-Luc (5 µg) and: lane1, pMT2T-p65 (3 µg)/pMT2T (12 µg); lane2, pMT2T-p65 (3 µg)/pMT2T-IB- (0.6 µg)/pMT2T (11.4 µg); lane3, pMT2T-Tax (2 µg)/pMT2T (13 µg); and lane4, pMT2T-Tax (2 µg)/pMT2T-IB- (0.6 µg)/pMT2T (12.4 µg). The cells were harvested after 24 h, and luciferase activity was assayed. Results are expressed as -fold induction relative to the luciferase activity from cells transfected with Ig-B-Luc (5 µg)/pMT2T (15 µg). Each value represents the mean of three experiments.



We then evaluated the potential inhibitory effects of signal-defective IB- mutants on Tax-mediated activation. Mutations at either Ser or Ser in IB- have recently been shown to prevent phosphorylation that normally occurs at these sites; the two mutant proteins (m32 and m36) are also not degraded in response to signals because of the loss in phosphorylation (12) . In the same series of experiments reported previously, wild-type IB- and a mutant in which Ser (m63) was altered were fully signal-responsive. As shown in Fig. 2, wild-type and all mutant IB-s were equally effective in inhibiting p65-mediated transactivation of the B-dependent reporter construct, displaying quantitatively similar dose-dependent effects. By contrast, differential inhibitory effects were evident when the same IB-s were tested for their ability to inhibit PMA plus ionomycin-mediated transactivation of the B-dependent reporter in Jurkat T cells (Fig. 3). As expected, the signal-defective mutants were much more potent in their inhibitory effects than the wild-type form or the irrelevant mutant; this is true over a wide range of concentrations (0.6 and 6 µg are shown). At the higher concentration even the wild-type IB- can partially overcome the signal-induced activation, presumably because the cellular capacity to degrade IB- in response to signals is limited.


Figure 2: Inhibitory effects of wild type (wt) and mutant (m32, m36, and m63) IB- on transactivation by p65. Jurkat cells were transfected with Ig-B-Luc (5 µg) and pMT2T-p65 (3 µg) with or without the indicated amounts of pMT2T-driven IB- expression vectors (wild type or mutants). Total amount of DNA was kept constant (20 µg) by adding appropriate amounts of the empty expression vector pMT2T. Cells were harvested after 24 h and analyzed as described in Fig. 1.




Figure 3: Effects of wild type (wt) and mutant IB- on B-dependent transactivation by PMA plus ionomycin. Jurkat cells were transfected with Ig-B-Luc (5 µg) with or without the indicated amounts of pMT2T-driven IB- expression vectors (wild type, m32, m36, or m63). The total amount of DNA (20 µg) was kept constant by adding appropriate amounts of the empty pMT2T expression vector. After 16 h, cells were stimulated with PMA (20 ng/ml) and ionomycin (2 µM) for 8 h and harvested. Results are presented as described in Fig. 1.



A similar set of experiments was performed to test the inhibitory effects of the various IB-s on cells that were activated for NF-B by cotransfection of Tax rather than by stimulation with PMA/ionomycin (Fig. 4). The Tax-mediated B-dependent transactivation was inhibited in ways indistinguishable from that seen with extracellular stimulation. The signal-defective mutants were significantly more potent in inhibiting Tax activation than the wild-type or the irrelevant mutant (cf. lanes2 and 5 with lanes3 and 4). As seen before with PMA/ionomycin stimulation, high concentrations of wild-type IB- (or the irrelevant mutant) partially inhibited transactivation (lanes6 and 9); however, even at this high concentration the signal-defective mutants were significantly more effective (lanes7 and 8). The data indicate that alterations at Ser or Ser prevent Tax-mediated activation of NF-B.


Figure 4: Effects of wild type (wt) and mutant IB- on Tax-mediated B-dependent transactivation. Jurkat cells were transfected with Ig-B-Luc (5 µg) and pMT2T-Tax (2 µg) with or without the indicated amounts of pMT2T-driven IB- expression vectors (wild type, m32, m36, or m63). Total amount of DNA (20 µg) was kept constant by adding appropriate amounts of the empty pMT2T expression vector. Cells were harvested after 24 h and analyzed as described in Fig. 1.



We also examined the effects of wild-type or m32 IB- on Tax-mediated activation in mouse EL-4 cells that stably expressed these human IB-s (see Ref. 12). The exogenously expressed human IB-s are present at a level comparable with that of the endogenous mouse IB-. Thus approximately equal amounts of endogenous NF-B are inhibited and bound by the endogenous mouse and by the exogenous human IB-. The B-dependent transactivation of the transiently transfected reporter was significantly more inhibited in cells harboring the signal-defective mutant than in cells with the wild-type form (Fig. 5). Again this is true with both PMA/ionomycin-induced activation of NF-B as well as with activation via cotransfected Tax. We had shown previously that PMA/ionomycin activation does not allow activation of NF-B complexes bound by the signal-defective form (12) . It is thus significant that the relative inhibition due to the presence of the m32 mutant was the same regardless of whether NF-B was activated by Tax or by extracellular signals.


Figure 5: B-dependent transactivation stimulated by PMA plus ionomycin (A) or by Tax (B) in EL-4 cells stably expressing wild type (wt) or m32 mutant human IB-. EL4 cells (10 cells) stably expressing human IB- (either wild type or m32 mutant) were transfected with: A, Ig-B-Luc (20 µg); or B, Ig-B-Luc (10 µg) plus pMT2T-Tax (10 µg). For A, half of the cells were not stimulated, and the other half was stimulated with PMA (20 ng/ml) plus ionomycin (2 µM) 16 h after electroporation; cells were harvested 8 h later. Results are expressed as -fold induction of stimulated over unstimulated cells. For B, cells were harvested 24 h after electroporation, and results are expressed as -fold induction of cells expressing Tax over those that do not (these cells were transfected with 10 µg of the empty pMT2T expression vector instead). Representative data of two independent experiments are shown.



The level of activation that remains with the m32 mutant cells is due to release from the endogenous, signal-responsive NF-BIB- complexes; these complexes are about equal in amount to the complexes inhibited by the signal-defective mutant. Unlike the transiently transfected cells that can express extremely high levels of IB-, resulting in substantial levels of free, unbound IB-,() the permanently transfected cells contain lower levels of the exogenously introduced form. Therefore the reduced level of induced transactivation seen in the m32 cells is consistent with a block to activation of signal-defective mutant-bound complexes regardless of whether NF-B was activated by Tax or with PMA/ionomycin.

DISCUSSION

IB- proteins that contain a mutation at either Ser or Ser effectively inhibit Tax-mediated B-dependent transactivation; wild-type IB- or an irrelevant mutant does not. We demonstrated this with transient transfection experiments into Jurkat T cells as well as with EL-4 T cell lines stably transfected with mutant and wild-type IB-. Ser and Ser are critical also to activation of NF-B via extracellular signals; they are phosphorylated in wild-type IB- in response to extracellular signals, and alterations at either site stop signal-induced phosphorylation and degradation of the respective mutants (12) . Since we have demonstrated previously that Tax is capable of inducing degradation of IB- as well (4) , our data indicate that Tax-mediated activation of NF-B is convergent with activation through extracellular signals. This provides evidence for a model in which Tax induces phosphorylation of IB- at the serine sites whose phosphorylation is required also for signal-induced degradation of the inhibitor.

Our evidence in support of the involvement of normal signaling paths in activation of NF-B by Tax is consistent with prior more indirect evidence cited in the Introduction. On the other hand, these results are less easily reconciled with a recent report by Munoz et al.(15) , which suggests that Tax-induced turnover of IB- is not critical to Tax-mediated activation of NF-B. It is possible, however, that induced turnover is necessary but not sufficient to observe optimal Tax-induced NF-B activity in cells. It has also been shown that Tax can activate NF-B from cytoplasmic complexes that are inhibited by the IB-like p105 and p100 precursor proteins, instead of IB- (6, 7, 15) ; the mechanism by which Tax activates via the precursor complexes is not known.

Our results rule out a model wherein Tax might have activated NF-B via dissociation of IB--inhibited complexes merely through physical association with these complexes; for example, Tax might have bound to proteins competitively, preventing proper association and thus inhibition by IB-, as hypothesized for IB- (10) . Contrary to such a view our results indicate that Tax must utilize normal physiologic signaling paths to cause degradation of the IB- inhibitor. It remains to be shown precisely how Tax accomplishes this. It is possible that Tax may induce phosphorylation/degradation through direct binding to the cytoplasmic complexes, for example by promoting or targeting the activity of an IB- kinase in this way. Alternatively, Tax may stimulate a signaling component acting further upstream in the cascade of signals that transduces an extracellular signal from the membrane to the cytoplasmic NF-B complexes.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Bldg. 10, Rm. 11B-16, NIH, Bethesda, MD 20892-1876. Tel.: 301-496-7662; Fax: 301-402-0070.

The abbreviation used is: PMA, phorbol 12-myristate 13-acetate.

T. Kanno, K. Brown, and U. Siebenlist, unpublished observation.


ACKNOWLEDGEMENTS

We are grateful to A. S. Fauci for support and review of the manuscript.


REFERENCES
  1. Smith, M. R., and Greene, W. C.(1991) J. Clin. Invest. 87, 761-766 [Medline] [Order article via Infotrieve]
  2. Siebenlist, U., Franzoso, G., and Brown, K.(1994) Annu. Rev. Cell Biol. 10, 405-455 [CrossRef]
  3. Thompson, J. E., Phillips, R. J., Erdjument-Bromage, H., Tempst, P., and Ghosh, S.(1995) Cell 80, 573-582 [Medline] [Order article via Infotrieve]
  4. Kanno, T., Brown, K., Franzoso, G., and Siebenlist, U.(1994) Mol. Cell. Biol. 14, 6443-6451 [Abstract]
  5. Sun, S.-C., Elwood, J., Beraud, C., and Greene, W. C.(1994) Mol. Cell. Biol. 14, 7377-7384 [Abstract]
  6. Kanno, T., Franzoso, G., and Siebenlist, U.(1994) Proc. Natl. Acad. Sci. U. S. A. 91, 12634-12638 [Abstract/Free Full Text]
  7. Watanabe, M., Muramatsu, M., Hirai, H., Suzuki, T., Fujisawa, J., Yoshida, M., Arai, K., and Arai, N.(1993) Oncogene 8, 2949-2958 [Medline] [Order article via Infotrieve]
  8. Brown, K., Park, S., Kanno, T., Franzoso, G., and Siebenlist, U.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2532-2536 [Abstract]
  9. Lin, Y.-C., Brown, K., and Siebenlist, U.(1995) Proc. Natl. Acad. Sci. U. S. A. 92, 552-556 [Abstract]
  10. Hirai, H., Suzuki, T., Fujisawa, J.-I., Inoue, J.-I., and Yoshida, M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 3584-3588 [Abstract]
  11. Schreck, R., Grassmann, R., Fleckenstein, B., and Baeuerle, P. A. (1992) J. Virol. 66, 6288-6293 [Abstract]
  12. Brown, K., Gerstberger, S., Carlson, L., Franzoso, G., and Siebenlist, U.(1995) Science 267, 1485-1488 [Medline] [Order article via Infotrieve]
  13. Fujita, T., Nolan, G. P., Liou, H.-C., Scott, M. L., and Baltimore, D. (1993) Genes & Dev. 7, 1354-1363
  14. DeWet, J. R., Wood, D. V., Deluca, M., Helinski, D. R., and Subramani, S.(1987) Mol. Cell. Biol. 7, 725-737 [Medline] [Order article via Infotrieve]
  15. Munoz, E., Courtois, G., Veschambre, P., Jalinot, P., and Israel, A. (1994) J. Virol. 68, 8035-8044 [Abstract]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.