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From the
Received for publication, December 5, 1999, and in revised form, January 5, 2000
Nef is a lentiviral protein involved in
pathogenesis of AIDS, but its molecular mechanisms of action remain
incompletely understood. Here we report a novel effect of Nef on
lymphocyte signaling, which is mediated via a T cell receptor
(TCR)-independent contribution of Nef to induction of nuclear factor of
activated T cells (NFAT), a transcription factor that plays a central
role in coordinating T cell activation. Expression of Nef did not
significantly alter the basal level of NFAT activity in Jurkat cells
nor the increased activity following T cell receptor stimulation by
anti-CD3 or anti-CD3 + anti-CD28. We also mimicked NFAT induction by
TCR triggering by simultaneous activation of the Ras and calcium
signaling pathways with phorbol 12-myristate 13-acetate and ionomycin,
respectively. Strikingly, whereas activation of either of these
pathways individually did not induce NFAT activity in control cells, in
Nef-expressing cells phorbol 12-myristate 13-acetate treatment alone
resulted in a 100-fold increase in NFAT-directed gene expression.
Experiments with different dominant negative mutant signaling proteins,
inhibitory chemicals, and Lck-deficient Jurkat cells revealed that this
effect was mediated via activation of calcineurin by Nef-induced
changes in calcium metabolism, but was independent of TCR-associated
signaling events. This ability of Nef to substitute for triggering of
the calcium pathway in induction of NFAT could promote activation of
human immunodeficiency virus (HIV)-infected T cells in response to
stimuli mediated via TCR or other cell surface receptors under conditions when activation of Ras rather than calcium signaling would
otherwise predominate.
Nef is a 25-34-kDa myristoylated viral protein that has an
important role in the development of AIDS in
HIV1-infected persons and in
SIV-infected monkeys. Studies in cell culture have revealed a number of
functions for Nef, including enhancement of HIV replication kinetics
and particle infectivity, down-regulation of cell surface expression of
CD4 and major histocompatability complex I, and modulation of
intracellular signaling. In agreement with a role in signal
transduction, Nef has been reported to bind to a large number of
proteins involved in cellular signaling cascades (for a review, see
Refs. 1-3).
An extreme example of the potential of Nef to alter cellular signaling
is provided by the malignant transformation of fibroblasts by the
mutant Nef allele of the SIVpbj strain (4). Also, co-expression in rat
fibroblasts of native Nef and the Src family tyrosine kinase Hck can
lead to malignant transformation of these cells via an increased
enzymatic activity of Hck (5). Remarkably, Stevenson and colleagues (6)
recently demonstrated that in infected macrophages Nef could cause
secretion of chemotactic and mitogenic cytokines, which in turn could
render resting T cell susceptible to HIV infection. In addition to
acting via such paracrine mechanisms, Nef has also been shown to alter
signal transduction of T cells directly. However, most such studies
have indicated that the role of Nef is to inhibit T cell receptor
(TCR)-induced signaling events, such as induction of the transcription
factors NF- Nevertheless, because T cell activation positively correlates with HIV
replication, it would seem logical that the effects of Nef on T cell
activation pathways would be positive, and it could be speculated that
the opposite results might have somehow arisen via a paradoxical
manifestation of the regulatory potential of Nef. Although this
hypothesis is attractive, there has been only limited evidence that Nef
could under any conditions contribute to T cell activation.
Hyperactivation of T lymphocytes has been observed in Nef transgenic
mice (15). The SIVpbj Nef allele has been shown to enable this virus to
productively infect resting peripheral blood mononuclear cell cultures
and activate T cells in these cultures (4). This unusual property of
SIVpbj Nef is because of an immunoreceptor tyrosine-based activation
motif created by mutations in its amino terminus (16, 17). Forced expression on the cell surface of a Nef chimera containing the extra-
and transmembrane parts of CD8 can result in activation of several
signaling cascades in Jurkat T-lymphoid cells (18). However, as in the
case of SIVpbj Nef, it is not clear how well the effects caused by the
CD8-Nef fusion protein reflect the normal function of Nef. Desrosiers
and colleagues (19) have demonstrated that wild-type Nef can allow SIV
to replicate in an IL-2-dependent T cell clone even upon
IL-2 withdrawal, apparently because of Nef-induced secretion of IL-2.
Perhaps related to this observation, two groups have recently shown
that under certain experimental conditions HIV-1 Nef can clearly
synergize (rather than interfere) with TCR stimulation as measured by
TCR-induced IL-2 secretion or other indicators of T cell activation
(20, 21).
In this study we have examined the ability of HIV-1 Nef to influence
basal and stimulated levels of transcription directed by NFAT, a
pivotal transcription factor that coordinates the effects of two
important signaling pathways upon activation of T cell gene expression
(22-24). We did not find evidence of positive or negative modulation
by Nef of TCR-stimulated NFAT activation. Instead, we report here that
Nef can activate calcium signaling in T lymphocytes independently of
TCR, and thereby potently synergize with inducers of the Ras pathway to
cause a dramatic (~100-fold) increase in NFAT-dependent transcription.
Plasmids, Antibodies, and Reagents--
The pEF-BOS-Nef plasmid
was created by subcloning the HIV-1 NL4-3R71 Nef allele (25) into the
pEF-BOS expression vector (26). The NFAT-luciferase construct
containing three tandem short binding sites for NFAT promoter (-286 to
-257 of human IL-2 enhancer) in front of minimal IL-2 promoter provided
by Dr. Y. Choi (Rockefeller University, New York). pLacZ reporter
plasmid was created by inserting Cells, DNA Transfections, and Luciferase Assays--
J.CaM-1
(kindly provided by Tomas Mustelin, The Burnham Institute, La Jolla,
CA), Jurkat E-6 (JE-6; from ATCC), A3.01 (a CEM, human T cell line,
derivative obtained from National Institutes of Health AIDS Research
and Reference Reagent Program), and MT-4 (kindly provided by Anssi
Lagerstedt from our Institute) cell lines were maintained in RPMI 1640 (BioWhittaker) medium supplemented with 2 mM glutamine
(HyClone) and 10% fetal bovine serum without antibiotics. Cultures
were diluted one day prior to transfection into 5-6 × 105 cells/ml. Two to three million cells were transfected
using Fugene (Roche Molecular Biochemicals) according to the
manufacturer's instructions. Twenty hours later cells were either
directly stimulated with 50 ng/ml anti-CD3 antibody and/or 1 µg/ml
anti-CD28 antibody for 6 h or with PMA and/or A23187 for 4 h.
When harvested the cells were washed once with 0.5 ml of standard
phosphate-buffered saline and lysed in 200 µl of 1× cell culture
lysis buffer (Promega, Madison, WI) luciferase activity with Promega
luciferase reagents and a Bio-Orbit (Turku, Finland) luminometer. For
measurement of Triggering of the TCR results in induction of
NFAT-dependent transcription via simultaneous activation of
calcium/calcineurin and Ras/MAPK pathways (22, 24, 27). To investigate
if expression of HIV-1 Nef could substitute for TCR stimulation in
activating these pathways, we transfected Jurkat T lymphoid cells with
a reporter plasmid expressing luciferase under the control of three tandem short binding sites for NFAT (-286 to -257 of human IL-2 enhancer; Ref. 28) with or without a Nef expression vector. Twenty
hours after transfection NFAT-dependent luciferase activity in these cells was analyzed either without further treatment or following a 4-h stimulation with a calcium/calcineurin
pathway-activating ionophore (A23187; 2 µM), a Ras/MAPK
pathway-activating phorbol ester (PMA, 100 nM), or a
combination of these (Fig. 1).
As expected, stimulation of either pathway alone did not result in
significant NFAT activation, but co-stimulation with PMA plus ionophore
caused a robust increase (~150-fold) in luciferase activity. A small
(~2-fold) but reproducible increase in NFAT-dependent transcription was observed in Nef-transfected cells that were left
unstimulated. However, when Nef-transfected cells were treated with PMA
a 100-fold increase in NFAT-driven transcription was observed. The
addition of ionophore to Nef-transfected and PMA-stimulated cells
caused only a small (~2-fold) additional increase in NFAT activity.
These results suggested that Nef could activate the calcium arm but not
the Ras arm of TCR signaling.
We also examined the effect of Nef expression on NFAT activity after
anti-CD3-mediated TCR triggering. Nef expression did not enhance or
inhibit NFAT activation in cells stimulated by anti-CD3 or anti-CD3 + anti-CD28 co-ligation (Fig. 1). Similar negative results were observed
regardless of whether suboptimal or saturating concentrations of
anti-CD3 were used (data not shown). Thus, despite the ability of Nef
to synergize with PMA stimulation in inducing NFAT, no potentiation
with TCR stimulation was observed, suggesting that the Ras pathway
rather than the calcium pathway is limiting in TCR-mediated NFAT induction.
To confirm that our observations are not unique to the NL4-3/R71
allele of Nef, which is derived from a laboratory-adapted isolate of
HIV-1 (25), we also subcloned into the same potent expression vector
(pEF-BOS) the Nef gene of the HIV-1 SF2 isolate (29), which has not
been passaged extensively before its molecular cloning and whose Nef
gene is more similar to sequences obtained directly from patients (30).
We found that SF2 Nef was equally if not more potent than NL4-3/R71
Nef in activating NFAT in PMA-treated Jurkat cells and also did not
positively or negatively modulate TCR-induced NFAT activity (data not
shown). Thus, the ability to contribute to NFAT activation via the
calcium signaling appears to be a general property of different HIV-1
Nef alleles.
Although Jurkat cells are a widely used model for studies on T
lymphocyte activation, we also examined the effect of Nef in other T
lymphocytic human cell lines. Like in Jurkat cells, PMA treatment of
A3.01 cells did not induce the expression of the NFAT-luciferase
reporter when transfected with the control vector (empty pEF-BOS) but
did result in a marked NFAT activation (~10-fold) when co-transfected
with Nef (data not shown). Whereas the magnitude of this effect was not
as great as that of Nef + PMA in Jurkat cells, it was in accordance
with a comparably smaller activation of NFAT-mediated transcription
(30-40-fold) seen in A3.01 cells also in response to PMA + ionophore
treatment (data not shown). Likewise, we were also able to consistently
observe a small (2-3-fold) induction of NFAT by Nef + PMA in MT-4
cells, even if PMA + ionophore stimulation of these cells
resulted in only a modest 6-fold increase in NFAT-dependent
luciferase expression (data not shown). Thus, whereas the contribution
of Nef to NFAT activation was most pronounced in Jurkat cells, this
effect could also be observed in other T cell lines, suggesting that
it is mediated by a mechanism that has general biological significance.
To study the molecular mechanisms underlying the synergistic NFAT
activation by Nef and PMA, we co-transfected Jurkat cells with dominant
negative (DN) mutants of signaling molecules known to act at distinct
positions in TCR-initiated signaling cascades (Fig.
2). Expression of either DN-Ras or
DN-MEKK1, two potent blockers of Ras signaling leading to the c-Jun
NH2-terminal kinase and extracellular signal-regulated
kinase family of mitogen-activated protein kinases (31) efficiently
blocked NFAT activation induced by Nef plus PMA. This result confirmed
that the synergistic effect of PMA stimulation with Nef was indeed
mediated via the Ras pathway. As expected, by interfering with the Ras
arm of TCR-signaling, DN-Ras and DN-MEKK1 also inhibited
anti-CD3-stimulated NFAT induction.
Synergistic Activation of NFAT by HIV-1 Nef and the Ras/MAPK
Pathway*
,§, and¶
Institute of Medical Technology, University
of Tampere, P. O. Box 607, FIN-33101 and the ¶ Department of
Clinical Chemistry, Tampere University Hospital, P. O. Box 2000,
FIN-33521, Tampere, Finland
ABSTRACT
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B and activating protein-1 (AP-1) and cell surface
expression of CD69 (7-12). On the other hand, some reports have
concluded that Nef has neither positive nor negative effects on T cell
signaling (13, 14).
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-galactosidase into pEF-BOS.
Dominant negative vector for p21-activated kinase-1 (PAK1)
(pEF-BOS-PAK1-K299R) was a gift from Dr. B. Mayer (Harvard University,
Boston). Dominant negative constructs for Ras (pSR
3-RASN17) and
mitogen-activated protein kinase/extracellular signal-regulated kinase
kinase kinase 1 (MEKK1) were provided by Dr. T. Kallunki (Danish Cancer
Society, Copenhagen). Polyclonal anti-CD3 (HIT3a) antibody, targeted
against the CD3-T-cell antigen receptor complex, was purchased from
Pharmingen (San Diego, CA), the anti-CD28 monoclonal antibody was from
CLB (Amsterdam, Netherlands), cyclosporin A and wortmannin were from Sigma, and PP1 was from Calbiochem.
-galactosidase activity in the same samples, 100 µl
of lysate was mixed with 10 µl of 10xLacZ buffer (500 mM
NaCl, 100 mM MgCl2, 100 mM
-mercaptoethanol) followed by the addition of 100 µl of 10 mM 0-nitrophenyl
-D-galactopyranoside (Sigma). Reactions were incubated
overnight at 37 °C, after which their absorbances were measured at
420 nm. The absorbance values of each sample were divided by the mean
value of all samples from that transfection experiment. The
corresponding luminometer readings were then divided by this ratio to
normalize for transfection efficiency.
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View larger version (31K):
[in a new window]
Fig. 1.
HIV-1 Nef induces NFAT in synergy with
activation of the Ras/MAPK pathway. Jurkat cells were
co-transfected with a NFAT-driven luciferase reporter plasmid together
with a Nef expression vector (dark bars) or an empty control
vector (light bars). Twenty hours after transfection cells
were either left untreated, or treated with ionophore
(IONO), PMA, or both of these, or in some cases stimulated
with anti-CD3 or anti-CD28 or a combination of these, as indicated
under the graph, followed by analysis of luciferase activity 4 h
later. The mean values for the relative increase in luciferase activity
in six or more independent transfections as compared with the
unstimulated control cells in the same experiment are shown in a
logarithmic scale on the y axis and indicated numerically
under each bar. Variation in the fold increase values between different
experiments is indicated by standard error bars.
View larger version (24K):
[in a new window]
Fig. 2.
Inhibition of NFAT activation by Nef plus PMA
or anti-CD3 stimulation by dominant negative signaling proteins.
Jurkat cells were transfected with a NFAT reporter together with Nef
(dark bars) or a control vector (light bars) as
in Fig. 1, except that expression vectors for the DN version of Ras,
MEKK1, or PAK1 were also included in some transfections, as indicated.
Nef-transfected cells were stimulated with PMA, and the control cells
were stimulated with anti-CD3. Shown are mean values of luciferase
activity from duplicate transfections from one of several similar
experiments expressed as a percentage of the values as compared with
corresponding cells that received no DN plasmids. The latter were
normalized to 100% from their original values of 5559 (anti-CD3-stimulated control cells) and 16590 (PMA-stimulated
Nef-transfected cells).
Weiss and colleagues (32) have recently shown that PAK1 plays a
critical role in TCR signaling by acting at a proximal step downstream
of Lck, Cdc42, and Vav but before the calcium and Ras pathways
bifurcate. Although we could confirm the ability of DN-PAK1 to block
anti-CD3-stimulated NFAT activity, it had only a very minor effect on
Nef-induced NFAT activation (Fig. 2). As expected, DN-PAK1 did not
inhibit NFAT-directed gene transcription in response to PMA + ionophore
treatment (data not shown). This observation suggested that Nef did not
mediate its co-stimulatory function by interacting with TCR or
associated factors. To substantiate this conclusion, we tested the
ability of Nef to activate NFAT in the Jurkat-derived J.CaM1 cells,
which are deficient in Lck (Fig. 3) (33).
Lck is the major Src kinase of T cells, plays a critical role as the
initiation of intracellular TCR signaling (34), and has also been
suggested as one of the cellular partners of Nef (10, 35). As expected,
no NFAT activation was seen in anti-CD3-stimulated J.CaM1 cells,
whereas a response similar to that seen in Jurkat cell was observed
following PMA plus ionophore treatment. Notably, the ability of Nef
expression to induce NFAT in these Lck-negative cells was not
compromised, providing further proof that this effect is independent of
TCR signaling.
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To further characterize Nef-induced NFAT activation, we tested a panel
of chemicals for their ability to block this effect. To confirm that
these agents did not decrease cell viability or the ability to respond
to the PMA co-stimulation, we compared in parallel the effect of the
same compounds on PMA-induced activation of
NF-B-dependent transcription. As shown in Fig.
4 preincubation with cyclosporin A (200 nM), a potent inhibitor of calcineurin (36) efficiently
blocked NFAT activation induced by Nef plus PMA but caused only a
modest reduction in PMA-stimulated NF-B-driven luciferase expression
(35% decrease from 1,918,224 to 1,250,188 relative units; data not
shown).
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Together with the observation that ionophore did not significantly
potentiate the effect of Nef (Fig. 1), this finding suggested that Nef
acts in the calcium pathway upstream of calcineurin and probably by
regulating intracellular calcium concentration. To test if prevention
of calcium influx from extracellular sources could block the effect of
Nef on NFAT, we supplemented the medium of the transfected Jurkat cells
with 500 µM calcium chelating agent EGTA. As seen in Fig.
4., this efficiently blocked NFAT activation in Nef-transfected plus
PMA-stimulated cells but did not significantly affect PMA-induced
NF-B-activated luciferase expression (17% decrease; data not shown).
Phosphatidylinositol 3-kinase has been implicated as an effector molecule in TCR signaling (37) and in regulation of calcium metabolism (38, 39). The addition of 200 µM wortmannin, an irreversible inhibitor of the enzymatic activity of phosphatidylinositol 3-kinase, resulted in significant but not complete blocking of anti-CD3-stimulated NFAT activity (Fig. 4). By contrast, wortmannin failed to decrease the effect of Nef on NFAT, indicating that the catalytic activity of phosphatidylinositol 3-kinase was not involved in this process.
Finally, we tested PP1, a tyrosine kinase inhibitor with relative
specificity toward the Src family kinases (40). We found PP1 (5 nM) to be an efficient inhibitor of anti-CD3-induced NFAT activation, whereas it only had a minimal effect on NFAT activity induced by Nef plus PMA (Fig. 4). This observation was in good agreement with our results using the Lck-negative J.CaM1 cells and
indicated that activation of the calcium/calcineurin pathway by Nef was
also independent of other Src kinases, such as Fyn, which has been
implicated in calcium regulation via the TCR (41, 42).
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The present study describes a strong positive effect by wild-type HIV-1 Nef on T cell signaling. This effect was found to be mediated via activation of the calcium/calcineurin pathway and thus was strongly synergistic with the Ras pathway in inducing NFAT-dependent gene expression. Notably, this contribution of Nef to NFAT activation did not require TCR stimulation and was not dependent on the activity of the TCR-associated signaling protein complex.
Although some previous studies have also reported positive effects by Nef on T cell activation, in these cases the action of Nef has been mediated through modulation of TCR signaling. Expression of certain mutant Nef alleles (Nef-pbj (4, 17, 43) and CD8-Nef (18, 44)) has been reported to be sufficient to activate T cells, apparently by mimicking extracellular TCR ligation via action of Nef on the intracellular components of the TCR signaling complex. Although a similar dominant function for wild-type Nef has not been shown, it has recently been clearly demonstrated that native HIV-1 Nef has the potential to cooperate with TCR stimulation in promoting T cell activation (20, 21). The mechanism of this enhancement is still unknown but was suggested to involve a lowered threshold for T cell activation caused by Nef-mediated priming of the TCR signaling complex (20, 21). It remains unclear, however, why other studies have concluded that the effect of Nef on TCR signaling is negative (such as Ref. 12) or that Nef does not modify the outcome of TCR stimulation at all (such as Ref. 14). Our present data concur best with the latter conclusion, as we saw little or no effect by Nef on NFAT activity induced by anti-CD3 or anti-CD3 + anti-CD28 stimulation. The variables that account for these differential effects by Nef on TCR signaling will need to be clarified by future research and could provide important insights into Nef function.
Whatever the role of Nef in modulating TCR signaling will prove to be, our current data indicate that Nef can also promote T cell activation by a different mechanism that involves a more direct effect on calcium/calcineurin-regulated cellular processes. One reason why this novel function of Nef has not been revealed by earlier studies could be that these may have focused on transcriptional effects induced by Nef alone or addressed a possible co-stimulatory role of Nef only in the context of TCR activation, which in itself results in the activation of calcium signaling. Furthermore, the NFAT-inducing effect of Nef described here requires a relatively high level of Nef expression. We have noted that Nef expression in transiently transfected Jurkat cells was significantly lower if a cytomegalovirus promoter-driven vector (pcDNA3) was used instead of pEF-BOS and that this lower level of Nef expression was insufficient to cause any increase in NFAT activity in PMA-treated Jurkat cells.2 Thus, the expression levels achieved in earlier studies may not have been as high as the relatively strong expression of Nef used here, which might better recapitulate the abundance of Nef thought to be produced in acutely HIV-infected lymphocytes (45).
The detailed mechanism that connects Nef to cellular calcium metabolism remains to be characterized. Interestingly, two recent studies (46, 47) have shown that chronic Nef expression in certain stably transfected cells resulted in the enlargement of their intracellular calcium stores, although no changes in cellular signaling or steady state intracellular free calcium concentrations were observed. It is possible, however, that such covert changes in calcium homeostasis could have been caused by an adaptive response to constitutive Nef expression. Our experiments on direct measurement of calcium levels in transiently Nef-transfected Jurkat cells have so far been complicated by the low numbers of positively transfected cells but do support the idea that acute Nef expression would result in an increased concentration of intracellular calcium.3
The ability of Nef to cooperate with the Ras pathway in activation of NFAT-dependent gene expression independently of TCR signaling raises the intriguing possibility that HIV-infected T lymphocytes expressing Nef might become abnormally activated in response to extracellular stimuli that would induce Ras but not the calcium pathway. We have observed a modest up-regulation of NFAT in Nef-transfected cells treated with IL-1, as compared with Nef or IL-1 alone (~2-fold),2 but are looking for soluble or cell-associated molecules that would show more potent synergy with Nef in activating NFAT. On the other hand, it is also possible that lymphocytes under some conditions might react to TCR stimulation with a signaling response that would be dominated by activation of the Ras pathway (unlike the anti-TCR-activated Jurkat cells studied here) and that the independent effect of Nef on calcium signaling could then contribute to complete T cell activation.
Besides merely promoting T cell activation, Nef-mediated up-regulation of NFAT signaling could also contribute to HIV replication and AIDS pathogenesis by other mechanisms. A recent study by Nolan and colleagues (48) reported that ectopic expression of NFATc in resting CD4+ T lymphocytes induced a permissive state, which despite the lack of evidence of T cell-activating effects caused by NFATc overexpression, supported HIV replication in these cells in the absence of further stimulation. The target genes of NFATc that were responsible for this permissive state were not identified but presumably were distinct from those that depend on NFAT complexes formed upon co-stimulation of the calcium/calcineurin and Ras/MAPK pathways during T cell activation.
Besides IL-2, a known member of the latter category of NFAT targets is
the gene encoding FasL, the ligand for the proapoptotic receptor
CD95/Fas, which has been suggested to be involved in HIV-induced immune
destruction and in interfering with elimination of HIV-infected cells
by cytotoxic T cells (reviewed in Ref. 49). Interestingly,
up-regulation of cell surface FasL expression has also been correlated
with Nef expression both in SIV and HIV infection as well as in
transfection studies (44, 50-52). Although the regulation of FasL is
complex, it is conceivable that Nef-induced NFAT activity may
contribute to this phenomenon. Finally, in addition to cellular genes
involved in T cell activation, NFAT has also been shown to activate
HIV-1 long terminal repeat-directed transcription by interacting with
an unusual binding site that overlaps with the NF-B-responsive
element (53).
Identification of the relevant target genes regulated by Nef via NFAT
that are involved in Nef-induced enhanced HIV replication and
pathogenicity in vivo, as well as a better understanding of the molecular mechanisms by which Nef induces the calcium/calcineurin pathway provide important challenges for future studies. Elucidation of
these outstanding issues should help to clarify the enigmatic role of
Nef in T cell physiology and could provide a novel means for
interfering with the pathogenic function of Nef in AIDS.
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ACKNOWLEDGEMENTS |
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We thank Tomas Mustelin, Andreas Baur, Tuula Kallunki, Yongwon Choi, Anssi Lagerstedt, and Hannu Kankaanranta for reagents, help, and discussions.
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FOOTNOTES |
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* This work was supported by Grant SA152304 (to K. S.) from the Academy of Finland and the Medical Research Fund of Tampere University Hospital.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.
§ A European Union Biomed Marie Curie program Fellow.
To whom correspondence should be addressed: Inst. of Medical
Technology, University of Tampere, Finn-Medi II Bldg., Rm. 4-137; Lenkkeilijankatu 8, FIN-33520, Tampere, Finland. Tel.: 358-3-215-7029; Fax: 358-3-215-8597; E-mail: kalle.saksela@uta.fi.
Published, JBC Papers in Press, March 16, 2000, DOI 10.1074/jbc.M910032199
2 A. Manninen, unpublished results.
3 A. Manninen, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are:
HIV, human
immunodeficiency virus;
SIV, Simian immunodeficiency virus;
TCR, T cell
receptor;
NF-B, nuclear factor B;
IL, interleukin;
PAK1, p21-activated kinase-1;
PMA, phorbol 12-myristate 13-acetate;
NFAT, nuclear factor of activated T cells;
MAPK, mitogen-activated protein
kinase;
MEKK1, mitogen-activated protein kinase/extracellular
signal-regulated kinase kinase kinase 1;
PP1, 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-D]pyrimidine;
DN, dominant negative.
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