|
Originally published In Press as doi:10.1074/jbc.M107322200 on November 28, 2001
J. Biol. Chem., Vol. 277, Issue 8, 6137-6142, February 22, 2002
HIV Nef Increases T Cell ERK MAP Kinase Activity*
Jeffrey A.
Schrager ,
Violette
Der Minassian, and
Jon W.
Marsh§
From the Laboratory of Molecular Biology, National Institute of
Mental Health, Bethesda, Maryland 20892-4034
Received for publication, August 1, 2001, and in revised form, November 1, 2001
 |
ABSTRACT |
The human immunodeficiency regulatory
protein Nef enhances viral replication and is central to viral
pathogenesis. Although Nef has displayed a capacity to associate with a
diverse assortment of cellular molecules and to increase T cell
activity, the biochemical activity of Nef in T cells remains poorly
defined. In this report we examine the bioactivity of Nef in primary
CD4 T cells and, in particular, focus on the biochemical pathways known
to be central to T cell activity. The extracellular signal-regulated
kinase (ERK) mitogen-activated protein (MAP) kinase pathway was
dramatically affected by Nef expression with increases in ERK, MEK, and
Elk induction. The capacity of Nef to increase the MAP kinase pathway activity was dependent on T cell receptor stimulation. By increasing ERK MAP kinase activity, Nef is functionally associated with a kinase
known to affect T cell activity, viral replication, and viral infectivity.
 |
INTRODUCTION |
Most of the viral gene products of
HIV1 have established
structural or biochemical functions. Nef, which is the predominant early transcript (1, 2), is largely defined by cellular and viral
phenotypes and by in vivo effects. Nef expression modulates cell surface receptors (3, 4), enhances virion infectivity (5-7), and
enhances in vivo viral replication and pathogenesis (8). The
study of CD4 and the major histocompatibility antigen I down-modulation
has been the most productive and has resulted in identification of
numerous cellular moieties, largely restricted to endocytotic
machinery, that can associate with Nef (for reviews, see Refs. 9 and
10). From this work, it has been suggested that Nef serves as an
adapter for coupling endocytotic molecules to the targeted membrane receptors.
The study of Nef-mediated effects on activation pathways has been less
conclusive. Studies addressing Nef function in T cells define
capacities that both inhibit and enhance T cell activity (for review,
see Refs. 10 and 11). From work with a CD8-Nef fusion protein, it was
proposed that cellular location defined whether Nef expression resulted
in negative or positive effects on T cell activity (12). Recent efforts
from our laboratory have demonstrated that the opposing effects of Nef
on T cell activation are also mediated by different Nef concentrations
(13). Nef can increase T cell interleukin-2 synthesis in both T cell
lines and human primary CD4 T cells (14-17). Furthermore, Nef has been shown to increase both T cell nuclear factor of activated T cells (17,
18) and NF- B (17) reporter activities. Thus, there is evolving
support for Nef as a positive T cell factor.
Attempts to define the biochemical capacity of Nef are numerous. Nef
can bind to a number of cellular signaling moieties. For example, Nef
binds to and activates the tyrosine kinase Hck (19, 20), and the
co-expression of Nef and Hck in Rat-2 fibroblasts resulted in cellular
transformation, although Nef expression alone had no effect (19). In a
macrophage cell line, Nef, through Hck and MAP kinase, induces the
transcription factor activating protein-1 (21). Hck is not expressed in
T cells, but with regard to T cell kinases, Nef has been shown to bind
and inhibit Lck and MAP kinase activity (22). Nef expression has also
been demonstrated to alter calcium signaling. When expressed in NIH3T3
cells, Nef inhibited inositol trisphosphate-mediated calcium flux (23), an effect similar to that seen in a Jurkat cell expressing a Nef-CD8 fusion protein (12). However, in Nef-expressing transgenic murine thymocytes, T cell stimulation resulted in elevated calcium responses (24), a finding more consistent with T cell activation enhancement. Nef
also binds to an activated serine kinase, p21-activated kinase or Pak
(25), and this association occurs in HIV-infected primary T cells (26).
In a recent exploration of Nef activity, it was demonstrated that the
co-expression of Nef with Vav, through Pak, increased c-Jun N-terminal
kinase (JNK) activity in NIH3T3 cells (27). However, it is unclear how
relevant these studies are to the activity of Nef in peripheral CD4 T
cells, the main target of HIV infection.
The biochemistry of T cell activation is highly complex, but many of
the molecular pathways leading to IL-2 expression have been
characterized (for reviews, see Refs. 28 and 29). As used here, IL-2 is
both a reporter for the metabolic activity of a T cell and the
end-product of a highly characterized and dissectible biochemical
process of the CD4 T cell. Per se, an increase in IL-2
levels does not significantly contribute to HIV replication (30).
However, with a recent demonstration that Nef, as expressed from HIV
infection, increased T cell activity (as defined by IL-2 secretion) and
viral production (31), an understanding of the biochemical activity of
Nef in these contexts appears relevant.
Events mediated by engagement of the T cell receptor and CD28
co-receptor result in activation of the MAP kinases extracellular signal-regulated kinase (ERK), JNK, and p38, in addition to
phosphorylation of I B, elevation of cytosolic calcium, and
activation of the kinase Akt. The pathways leading to these cytosolic
signaling moieties are briefly outlined in Fig.
1. The choice of kinases and signaling
molecules is based on the assumption that Nef, as a cytosolic protein,
would affect pathways in this cellular compartment. The choice was also
to look at late cytosolic events, yielding the greatest opportunity to
"capture" Nef effects. We make use of a system that expresses HIV
Nef at concentrations similar to those seen in HIV infection (13) in
hopes of identifying an indigenous pathway in primary CD4 T cells that
would permit relevant molecular dissection. In this report, we
demonstrate that Nef expression in primary CD4 T cells specifically
increases activity of the ERK MAP kinase cascade.

View larger version (11K):
[in this window]
[in a new window]
|
Fig. 1.
Summary of T cell activation pathways
examined in this report. The MAP kinases ERK, JNK, and p38 are
phosphorylated by specific MAP kinase kinases (MEK1/2, SEK1(MKK4)/MKK7,
and MKK3/6, respectively) (41, 43-45). Phosphatidylinositol 3-kinase
(PI3K) activity is required for induction of the ERK cascade
pathway (76, 77) as well as for activation of
3-phosphoinositide-dependent kinase (PDK), which
phosphorylates protein kinase B/Akt (46, 47, 78, 79). Protein kinase
C- activates the IKK/I B/NF- B cascade through phosphorylation
of IKK (80-82), which phosphorylates I B and promotes NF- B
activity (49, 50, 83, 84). IKK can also be phosphorylated by MEKK1 (85,
86) and by COT-activated NIK (87-89). Cytosolic calcium is released
from intracellular stores by an inositol trisphosphate-sensitive
receptor (90).
|
|
 |
EXPERIMENTAL PROCEDURES |
T Cell Cultures--
The peripheral lymphocyte fraction from
healthy donors was obtained by leukapheresis and countercurrent
centrifugal elutriation from the Department of Transfusion Medicine at
the National Institutes of Health (32) and purified as previously
described (16). Cells were grown in complete growth medium (RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum, 25 mM Hepes, 2 g/liter sodium bicarbonate, 1 mM
nonessential amino acids, 10 mM sodium pyruvate, 4 µl/liter -mercaptoethanol, and 50 µg/ml gentamicin, adjusted to
pH 7.4). Proliferation of purified CD4 T cells was achieved by the
addition of CD3 plus CD28 antibody immobilized on magnetic beads
(16).
Transduction and Detection of Nef--
Primary CD4 T cells were
transduced with the PA-317 retroviral LXSN system (3) expressing either
the NL4-3 Nef or the nonmyristylated NL4-3 Nef mutant, which was
generated by a glycine to alanine switch at residue position 2 (G2A)
(33). Following selection in G418, Nef was detected by Western analysis
(16). For T cell functions, the G2A cells were found to be similar to
the nontransduced cells, as previously noted (16). All transductions
were tested and found positive for Nef expression.
Kinase Assays--
For cell activation studies, magnetic beads
were removed from proliferating cell cultures after gentle pipetting of
the cells, followed by re-exposure to a magnetic field. Cells no longer
bound to beads were removed. The bead-containing fraction was cycled through this process two or three times. The cells were resuspended at
2 million cells/ml in fresh RPMI with fetal calf serum and then rested
overnight. The next day 4 million cells were resuspended in 1 ml of
serum-free RPMI 25 mM Hepes, pH 7.4. At time 0, either 10 µg of anti-CD3 (clone HIT3A) or anti-CD3 plus anti-CD28 (clone CD28.2) beads (five beads per cell) was added at 37 °C with
mixing. At defined times, cells were centrifuged at 4 °C for 30 s, and the cell pellet was resuspended in 200 µl of lithium dodecyl
sulfate sample buffer (Novex), heated for 10 min at 70 °C,
and sonicated for 20 s to shear DNA, and similar aliquots were
applied to a 10% NuPage gel (Novex). The electrophoresis gel
run was then transferred to nitrocellulose and probed with kinase-,
substrate-, or phosphospecific antibodies (Cell Signaling Technology or
Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Binding of antibodies
was assayed by secondary peroxidase conjugate antibody (Kirkegaard and
Perry) and developed with West Dura (Pierce) substrate. Measurements of
generated light were achieved on an Alpha Innotech ChemiImager with a
cooled CCD camera. For reprobing, blots were treated with Restore
stripping solution (Pierce).
For analysis of ERK phosphorylation of recombinant Elk-1, 4 million
cells were suspended in 1 ml of serum-free RPMI, 25 mM Hepes, pH 7.4. At time 0, cells were activated by the addition of
antibody as described above. At defined times, cells were centrifuged at 4 °C for 30 s, and the cell pellet was resuspended into 1 ml of lysis buffer (20 mM Tris (pH 7.5), 150 mM
NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100,
2.5 mM sodium pyrophosphate, 1 mM -glycerolphosphate, 1 mM Na3VO4,
1 µg/ml leupeptin, 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride). Lysates were sonicated and centrifuged at 14 K for 10 min, and the supernatant was aliquoted for kinase assays or frozen at
70 °C. Total cellular protein in the lysate was determined by
micro-BCA protein assay, and then all samples to be compared were made
equivalent in protein concentration. Immobilized phospho-ERK kinase
antibody (Thr202/Tyr204) was used to purify
active ERK kinase from the cell lysate, followed by an in
vitro kinase assay utilizing recombinant Elk-1 protein (as
purchased in assay kit form from New England Biolabs). A Western analysis for phospho-Ser383 Elk-1 was then performed as
above. For this assay, we followed the protocols of the manufacturer.
Cytosolic Calcium--
Free cytosolic calcium was determined by
the procedure outlined for fluo-3 acetoxymethyl ester (AM) (34), but
using the fluo-4 AM plus Pluronic F-127 reagent from Molecular Probes,
Inc. (Eugene, OR) with minor modifications. Cell loading of 4 µM fluo-4 AM was achieved in Hanks' balanced salt buffer
with Hepes 25 mM, pH 7.4, 37 °C, 1 h. Loaded and
washed cells were aliquoted in a 96-well plate (105 cells).
Cells were stimulated through the addition of 2 µg of anti-CD3
antibody, and calcium levels were determined by measurement of emitted
fluorescence at 37 °C on a CytoFluor 4000 (Perseptive Biosystems)
fluorimeter fitted with a 485-nm excitation filter and a 530-nm
emission filter. Calculation of intracellular calcium was achieved
following the sequential addition of ionomycin and EGTA, as previously
described (35, 36), but a value of Kd = 345 nM was used for fluo-4. For each experiment, cell number and volume were determined on a Coulter Z2 particle analyzer.
 |
RESULTS |
We transduced purified primary CD4 T cells with either wild type
NL4-3 Nef or the nonmyristylated G2A mutant of NL4-3 Nef retroviral
expression vectors. The G2A mutant has previously been demonstrated to
lack T cell enhancement capacity (16, 17) but serves as a control for
the cellular manipulations of transduction and selection. Under the
conditions used in this report, the G2A Nef-transduced cell performed
identically to a nontransduced cell. Both native and G2A Nef-transduced
cells specifically expressed Nef protein (Refs. 13 and 16; data not shown).
Activation of the ERK MAP kinase pathway is essential for IL-2
synthesis (37, 38). ERK is also central to the induction of cellular
transcription and translation factors and activation of proliferation
machinery (39). In T cells, stimulation of the T cell receptor (CD-28
co-stimulus is not necessary) leads to ERK activity (40). T cell
activation involves two ERK species (ERK1 and ERK2) of different
molecular weights (~44,000 and 42,000, respectively). Activation of
this kinase is achieved by MEK1/2 phosphorylation of a pair of
threonine and tyrosine residues of ERK (41). This active dual
phosphorylated species was quantitated by phosphospecific antibody
binding on Western blots developed from electrophoresis gels of cell
lysates. Control and Nef-expressing primary CD4 T cells were maintained
by stimulation with CD3-CD28 antibodies immobilized on magnetic beads
(see "Experimental Procedures"). Prior to activation, the cells
were removed from beads and rested overnight. As shown in Fig.
2A, active phosphorylated
forms of both ERK1 (p44) and ERK2 (p42) are generated following
stimulation with soluble CD3 antibody. The level of activated ERK was
dramatically increased in Nef-expressing cells, but not in the
nonmyristylated G2A Nef mutant transduced cell. Probing this blot for
total ERK protein (activated plus nonactivated) displayed no
differences, demonstrating that the Nef-mediated increase in ERK1 and
-2 activity was not due to changes in total ERK protein. For
comparative purposes, we have plotted the induction of ERK activity
with time (Fig. 2B). The inability of the nonmyristylated
G2A mutant Nef to mediate the Nef increase in ERK activity is
consistent with its lack of effect on T cell activation enhancement
(16, 17). The ERK activity was increased by Nef in cells from three
different donors and, in addition, was evident in cells stimulated by
CD3-CD28 beads (data not shown).

View larger version (51K):
[in this window]
[in a new window]
|
Fig. 2.
Nef expression increases ERK MAP kinase
induction in primary T cells. Nontransduced (control) cells or
cells transduced with either wild type NL4-3 Nef (Nef) or the NL4-3
G2A Nef mutant (G2A) were rested overnight, stimulated with soluble
anti-T cell receptor antibody (CD3) for the indicated times (0-20
min), lysed, and then applied to an electrophoresis gel. A,
the Western blot was probed with an anti-phospho-p44/p42 ERK
(Thr202/Tyr204) antibody (Active
ERK) and developed with a horseradish peroxidase-conjugated
secondary antibody. The chemiluminescence was captured by a CCD camera.
The blot was then stripped and reprobed with an antibody specific for
total ERK1/2 protein (Total ERK). Time course is
shown for nontransduced cells (lanes 1-4), Nef
(lanes 5-8), and G2A Nef (lanes
9-12). B, relative units of light emission for
the p42 and p44 bands (NL4-3 Nef (closed
circles), G2A Nef (diamonds), and control
(triangles)) are plotted. The enhancement of ERK activity
was reproducible in cells from three donors.
|
|
T cells also possess two other inducible MAP kinases, JNK and p38. JNK
is induced in T cells by CD3 plus CD28 co-stimulation (42). JNK
activation in T cells is achieved by SEK1 (also known as MKK4)-mediated
phosphorylation of a proximal threonine and tyrosine pair in JNK (43).
Rested primary human CD4 T cells were stimulated with beads containing
immobilized anti-CD3 and CD28 antibodies. As shown in Fig.
3A, JNK activation was
dependent on stimulation. Unlike the activity of MAP kinase ERK, there
is no measurable effect on JNK activity in primary CD4 T cells by HIV
Nef. Comparison of total JNK protein is also displayed in Fig.
3A.

View larger version (48K):
[in this window]
[in a new window]
|
Fig. 3.
Lack of Nef effect on multiple signaling
pathways. A, Nef does not increase JNK induction in T
cells. Cells were rested overnight and then activated by the addition
of beads bound with anti-CD3 plus anti-CD28 antibody. The blot was
developed as in Fig. 2 with the following adaptations. The blot was
probed for activated kinase (active JNK) with antibody specific for
phospho-JNK (Thr183/Tyr185) or for total JNK
protein. Time course (0-40 min) is shown for nontransduced
(Control; lanes 1-4) and NL4-3 Nef
cells (lanes 5-8). The lack of enhancement in JNK induction
was seen in cells from three donors. B, induction of Akt and
p38 kinase activity is unaffected by Nef. NL4-3 Nef and control cells
were activated as described above and simultaneously probed with either
antibody detecting (Active Species) phospho-p38
(Thr180/Tyr182) or phospho-Akt
(Ser473). Heavy chain of IgG (HC) is present on
this blot as well as the blot for total Akt and p38 protein
(Total Kinase). C, Nef does not alter
induction of I B phosphorylation. Cells were activated and
analyzed as above. Detection of I B phosphorylation was achieved
with a phospho-I B (Ser32)-specific antibody
(phospho-I B). The Western analysis for total I B protein is
also shown. D, measurement of intracellular calcium
following T cell receptor stimulation. Cells were rested overnight,
loaded with fluo-4 AM, and then stimulated by anti-CD3 soluble
antibody. Fluorescence (485-nm excitation/530-nm emission) was measured
in quadruplicate samples every minute. Plotted data represent the
mean ± S.D. Data for control cells are depicted as
squares; NL4-3 Nef cells are circles.
Open symbols are activated populations, whereas
nonstimulated populations are represented by the filled
symbols.
|
|
A third MAP kinase, p38, is also activated by threonine-tyrosine
phosphorylation (44, 45). Relative to ERK, the phosphorylation of p38
was less intense and required higher exposure. As shown in Fig.
3B, activation of p38 in the absence or presence of Nef was similar.
Akt kinase (also known as protein kinase B) is downstream of
phosphoinositide 3-kinase activity and is phosphorylated on
Thr308 by a phosphatidylinositol
trisphosphate-dependent Akt/protein kinase B kinase (46)
and on Ser473 by autophosphorylation (47). Engagement of
the surface receptors resulted in the anticipated phosphorylation of
Akt, as shown in Fig. 3B, and Nef expression appeared to
have no effect.
The NF- B transcription factor is inactive when bound to the
inhibitory factor I B (48). The phosphorylation of
Ser32/Ser36 (49, 50) leads to release and
degradation of I B , which correlates with NF- B nuclear
localization following T cell activation (51, 52). To examine the
effect of Nef on I B phosphorylation, Nef-expressing and control
cells were stimulated with CD3-CD28 beads. Cells were lysed and applied
to gel electrophoresis as above, blotted onto cellulose nitrate, and
probed by anti-phospho-I B antibody (see Fig. 3C).
Stimulation of cells led to rapid phosphorylation of I B , and
expression of Nef appeared to play no role in enhancing this process.
Stimulation of the T cell receptor also results in a rapid
elevation of intracellular calcium, a downstream effect of
phospholipase C 1 activity. Cytosolic calcium was determined by
fluorimetric measurement of free calcium binding by fluo-4. Response of
control and Nef cells to T cell receptor stimulation resulted in
similar cellular calcium elevations as shown in Fig. 3D.
Nef increased the induction of ERK MAP kinase but did not
appear to affect other pathways. To further define pathway specificity, we then compared the phosphosphorylation state of MEK1/2 and SEK1 (MKK4), the specific upstream T cell kinases for ERK and JNK, respectively. As with the previous studies, T cells were rested overnight, then activated by antibody engagement of the T cell receptor. Like ERK, MEK1/2 activation-associated phosphorylation was
found to be transient, with the highest levels around 5 min (Fig.
4A). Compared with the
nontransduced controls and the G2A mutant Nef cells, the Nef-expressing
cells displayed an increase in the induction of MEK1/2 activity, as
measured by antibody recognition of the dual serine
(Ser217/Ser221) phosphorylation by Raf. As with
the ERK induction, we did not see Nef-mediated activation in the
absence of T cell receptor stimulation. An examination of the induction
of the corresponding kinase in the JNK pathway, SEK1 (MEKK4),
demonstrated a similar dependence on surface receptor stimulation (Fig.
4B). Consistent with the lack of Nef on JNK activation, SEK1
activity was unchanged by Nef expression.

View larger version (72K):
[in this window]
[in a new window]
|
Fig. 4.
Nef affects MEK1/2 but not SEK1
activity. A, Nef expression increases T cell
receptor-induced phosphorylation of MEK1/2. The assay was performed as
in Fig. 2, except the blot was probed with antibodies recognizing the
dual serine (Ser217/Ser221) phosphorylation
(Active MEK1/2) or total MEK1/2 protein.
B, Nef expression does not alter SEK1 activation. Cells were
activated as in Fig. 3A and probed with phospho-SEK1
(Thr261) (Active Sek1) or
antisera that recognize total SEK1.
|
|
The increased phosphorylation of ERK
Thr202/Tyr204 by MEK (Fig. 2) and of MEK
Ser217/Ser221 by Raf (Fig. 4) defines the
pathway specifically enhanced by Nef expression. These phosphorylation
measurements define activities upstream to ERK MAP kinase. Once active,
ERK MAP kinase induces numerous downstream transcription factors,
including the phosphorylation (Ser383) of Elk-1 (53). We
performed Western analysis on the lysate of activated control and
Nef-expressing primary CD4 T cells. Increased specific phosphorylation
of the Elk-1 Ser383 was evident in the Nef-expressing
cells, compared with the control G2A Nef mutant cell (Fig.
5A) or nontransduced cells
(data not shown). The increased phosphorylation of Elk was not due to
an increase in Elk protein levels in the Nef-positive cell. In addition to MAP kinase ERK, Elk-1 can be phosphorylated by the MAP kinases JNK
and p38 (54-56), although activation of JNK or p38 in these studies
does not appear to be affected by Nef. We then measured, in an in
vitro kinase assay, Elk-1 phosphorylation by ERK kinase activity
that was purified (separated from JNK and p38) from lysates of
activated cells. As shown in Fig. 5B, there was an increased induction of ERK MAP kinase activity in Nef-expressing T cells following T cell stimulation, consistent with increased Elk-1 phosphorylation in Nef-expressing cells (Fig. 5A).

View larger version (34K):
[in this window]
[in a new window]
|
Fig. 5.
Nef increases Elk-1 phosphorylation
(Ser383). A, Western analysis of cell
lysate of activated control and Nef-expressing primary CD4 T cells,
performed as in Fig. 2, comparing the control G2A Nef mutant cell to
wild type Nef. G2A control and nontransduced cells were equivalent. The
Western for total Elk-1 is also shown. B, Western analysis
for Elk-1 phosphorylated by immunoprecipitated active ERK1/2 in an
in vitro kinase assay.
|
|
 |
DISCUSSION |
We conclude that the ERK MAP kinase activation cascade is enhanced
in Nef-expressing primary CD4 T cells. The lack of effect on other
pathways emanating from the T cell surface receptor suggests that this
activity is responsible for the enhancement in T cell activity. In
addition, the mediated increase in ERK MAP kinase activity also
connects Nef to previously characterized HIV infection processes. ERK
activity has been demonstrated to increase HIV retroviral long terminal
repeat expression (57) and HIV replication (58). Furthermore, viral
infectivity has been found to be adversely affected by ERK
pathway-specific inhibitors and enhanced by constitutive activation of
this pathway (59, 60). Vif (viral infectivity factor) increases infectivity of HIV (61, 62), and its
phosphorylation by ERK remarkably increases the activity of this viral
regulatory protein (63). Thus, the capacity of Nef to increase ERK
activity suggests that a role of this viral protein in enhancing virion infectivity may, in part, be upstream to Vif activity.
Our work with CD4 T cells suggests that Nef expression by itself does
not activate the MAP kinase pathway. In our system, there is a
requirement for stimulation of the surface T cell receptor. This is
consistent with previous demonstrations that the Nef-mediated IL-2
induction in primary human CD4 T cells requires activation (16, 17) and
that the HIV infection-induced hypersensitization of T cell IL-2
secretion is also dependent on T cell receptor stimulation (31,
64).
Our finding appears to differ from a recent demonstration that
co-expression of Nef with Vav, through Pak, increases JNK activity in
NIH3T3 cells (27). We do not find an increase in JNK activity with Nef
in the absence of or following T cell receptor stimulation. Two
systematic differences may be responsible. First, the injection of
plasmids into NIH3T3 cells would probably result in higher expression
levels of Nef and Vav than those seen in the cells examined here.
Second, while Pak can induce JNK in numerous cells (65-67), Pak
activity in T cells, following receptor stimulation, has been shown to
be essential for ERK activity but not JNK (68). The role of Pak in ERK
activity is unresolved, and experimental approaches involving Nef are
complicated by the potential for Nef to physically associate with
different Pak isoforms and different Pak-associated nucleotide exchange
factors (26, 69-71).
The ability of Nef to affect ERK but not the JNK or p38 MAP kinase
pathways suggests biochemical specificity for Nef molecular targeting,
but our present understanding of the biochemical nature of T cell
activation suggests broad possibilities. Differentiation of ERK from
JNK and p38 MAP kinase activation can occur as early as the CD3-
chain (72) and, obviously, as late in the pathway as the kinase species
studied here. In fact, the effect of Nef at a distal biochemical site
that is responsible for the enhanced ERK response could affect cellular
or viral function not associated with the ERK activity. Previous
efforts have demonstrated that recombinant Nef can associate with
purified ERK MAP kinase (22); however, this interaction inhibited MAP
kinase activity. Nef has also been reported to bind Raf (73), but the
effect of this association on kinase activity is unknown.
The positive effect of Nef on T cell IL-2 secretion following
stimulation has been documented in murine, simian, and human T cell
lines, as well as in primary human T cells (14-17). The use of this
system to resolve the biochemical activity of Nef in T cells is
validated by the recent demonstration that Nef from HIV infection also
increases this T cell response (31). Given that Nef increases both
murine and human T cell activity, we believe the conclusion that Nef
expression results in increased ERK activity in human T cells is
corroborated by the transgenic study of Hanna et al. (74).
These authors found that Nef-expressing, CD3-stimulated murine
thymocytes display enhanced phosphorylation of several proteins,
including the Thr202/Tyr204 phosphorylation of
ERK, characteristic of its activation by MEK1 (41, 75). Thus, the
findings for Nef bioactivity in primary CD4 T cells presented here
appear to be reproducible in other cellular systems. Although the
actual pathogenesis may indeed be different, this Nef activity is
correlated with a state in these mice that is remarkably similar to the
disease seen in humans; thus, further studies of the molecular activity
of Nef that affects the MAP kinase pathway is warranted. In conclusion,
the specificity for ERK MAP kinase activity over other T cell
activation pathways offers an opportunity for biochemical resolution of
Nef molecular activity.
 |
ACKNOWLEDGEMENTS |
We thank Thomas Trischmann of the Blood
Services Section, Department of Transfusion Medicine, National
Institutes of Health, for providing elutriated lymphocytes; Dr.
Sundararajan Venkatesan for the Nef expression vectors; and Drs.
Kuan-Teh Jeang, Xiaolan Qian, and Yuntao Wu for critical review of the manuscript.
 |
FOOTNOTES |
*
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.
Present address: Laboratory of Pathology, NCI, Bldg. 10, Rm.
2N212, Bethesda, MD 20892.
§
To whom correspondence should be addressed: LMB, NIMH, Bldg. 36, Rm. 1B08, 36 Convent Dr., MSC 4034, Bethesda, MD 20892-4034. Tel.:
301-402-3655; Fax: 301-402-0245; E-mail: jon@codon.nih.gov.
Published, JBC Papers in Press, November 28, 2001, DOI 10.1074/jbc.M107322200
 |
ABBREVIATIONS |
The abbreviations used are:
HIV-1, human
immunodeficiency virus type 1;
ERK, extracellular signal-regulated
kinase;
MAP, mitogen-activated protein;
MEK, extracellular
signal-regulated kinase kinase 1/2;
MKK, MAP kinase kinase;
JNK, c-Jun
N-terminal kinase;
SEK1, stress-activated kinase kinase 1;
IL-2, interleukin-2;
CCD, charge-coupled device;
AM, acetoxymethyl
ester.
 |
REFERENCES |
| 1.
|
Robert-Guroff, M.,
Popovic, M.,
Gartner, S.,
Markham, P.,
Gallo, R. C.,
and Reitz, M. S.
(1990)
J. Virol.
64,
3391-3398[Abstract/Free Full Text]
|
| 2.
|
Guatelli, J. C.,
Gingeras, T. R.,
and Richman, D. D.
(1990)
J. Virol.
64,
4093-4098[Abstract/Free Full Text]
|
| 3.
|
Garcia, J. V.,
and Miller, A. D.
(1991)
Nature
350,
508-511[CrossRef][Medline]
[Order article via Infotrieve]
|
| 4.
|
Schwartz, O.,
Marechal, V., Le,
Gall, S.,
Lemonnier, F.,
and Heard, J. M.
(1996)
Nat. Med.
2,
338-342[CrossRef][Medline]
[Order article via Infotrieve]
|
| 5.
|
de Ronde, A.,
Klaver, B.,
Keulen, W.,
Smit, L.,
and Goudsmit, J.
(1992)
Virology
188,
391-395[CrossRef][Medline]
[Order article via Infotrieve]
|
| 6.
|
Spina, C. A.,
Kwoh, T. J.,
Chowers, M. Y.,
Guatelli, J. C.,
and Richman, D. D.
(1994)
J. Exp. Med.
179,
115-123[Abstract/Free Full Text]
|
| 7.
|
Miller, M. D.,
Warmerdam, M. T.,
Gaston, I.,
Greene, W. C.,
and Feinberg, M. B.
(1994)
J. Exp. Med.
179,
101-113[Abstract/Free Full Text]
|
| 8.
|
Kestler, H. W.,
Ringler, D. J.,
Mori, K.,
Panicali, D. L.,
Sehgal, P. K.,
Daniel, M. D.,
and Desrosiers, R. C.
(1991)
Cell
65,
651-662[CrossRef][Medline]
[Order article via Infotrieve]
|
| 9.
|
Piguet, V.,
Schwartz, O., Le,
Gall, S.,
and Trono, D.
(1999)
Immunol. Rev.
168,
51-63[CrossRef][Medline]
[Order article via Infotrieve]
|
| 10.
|
Skowronski, J.,
Greenberg, M. E.,
Lock, M.,
Mariani, R.,
Salghetti, S.,
Swigut, T.,
and Iafrate, A. J.
(1999)
Cold Spring Harbor Symp. Quant. Biol.
64,
453-463[CrossRef][Medline]
[Order article via Infotrieve]
|
| 11.
|
Marsh, J. W.
(1999)
Arch. Biochem. Biophys.
365,
192-198[CrossRef][Medline]
[Order article via Infotrieve]
|
| 12.
|
Baur, A. S.,
Sawai, E. T.,
Dazin, P.,
Fantl, W. J.,
Cheng-Mayer, C.,
and Peterlin, B. M.
(1994)
Immunity
1,
373-384[CrossRef][Medline]
[Order article via Infotrieve]
|
| 13.
|
Liu, X.,
Schrager, J. A.,
Lange, G. D.,
and Marsh, J. W.
(2001)
J. Biol. Chem.
276,
32763-32770[Abstract/Free Full Text]
|
| 14.
|
Rhee, S. S.,
and Marsh, J. W.
(1994)
J. Immunol.
152,
5128-5134[Abstract]
|
| 15.
|
Alexander, L., Du, Z.,
Rosenzweig, M.,
Jung, J. U.,
and Desrosiers, R. C.
(1997)
J. Virol.
71,
6094-6099[Abstract]
|
| 16.
|
Schrager, J. A.,
and Marsh, J. W.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
8167-8172[Abstract/Free Full Text]
|
| 17.
|
Wang, J. K.,
Kiyokawa, E.,
Verdin, E.,
and Trono, D.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
394-399[Abstract/Free Full Text]
|
| 18.
|
Manninen, A.,
Renkema, G. H.,
and Saksela, K.
(2000)
J. Biol. Chem.
275,
16513-16517[Abstract/Free Full Text]
|
| 19.
|
Briggs, S. D.,
Sharkey, M.,
Stevenson, M.,
and Smithgall, T. E.
(1997)
J. Biol. Chem.
272,
17899-17902[Abstract/Free Full Text]
|
| 20.
|
Moarefi, I.,
LaFevre-Bernt, M.,
Sicheri, F.,
Huse, M.,
Lee, C. H.,
Kuriyan, J.,
and Miller, W. T.
(1997)
Nature
385,
650-653[CrossRef][Medline]
[Order article via Infotrieve]
|
| 21.
|
Biggs, T. E.,
Cooke, S. J.,
Barton, C. H.,
Harris, M. P.,
Saksela, K.,
and Mann, D. A.
(1999)
J. Mol. Biol.
290,
21-35[CrossRef][Medline]
[Order article via Infotrieve]
|
| 22.
|
Greenway, A.,
Azad, A.,
Mills, J.,
and McPhee, D.
(1996)
J. Virol.
70,
6701-6708[Abstract/Free Full Text]
|
| 23.
|
De, S. K.,
and Marsh, J. W.
(1994)
J. Biol. Chem.
269,
6656-6660[Abstract/Free Full Text]
|
| 24.
|
Skowronski, J.,
Parks, D.,
and Mariani, R.
(1993)
EMBO J.
12,
703-713[Medline]
[Order article via Infotrieve]
|
| 25.
|
Nunn, M. F.,
and Marsh, J. W.
(1996)
J. Virol.
70,
6157-6161[Abstract]
|
| 26.
|
Brown, A.,
Wang, X.,
Sawai, E.,
and Cheng-Mayer, C.
(1999)
J. Virol.
73,
9899-9907[Abstract/Free Full Text]
|
| 27.
|
Fackler, O. T.,
Luo, W.,
Geyer, M.,
Alberts, A. S.,
and Peterlin, B. M.
(1999)
Mol. Cell
3,
729-739[CrossRef][Medline]
[Order article via Infotrieve]
|
| 28.
|
van Leeuwen, J. E. M.,
and Samelson, L. E.
(1999)
Curr. Opin. Immunol.
11,
242-248[CrossRef][Medline]
[Order article via Infotrieve]
|
| 29.
|
Kane, L. P.,
Lin, J.,
and Weiss, A.
(2000)
Curr. Opin. Immunol.
12,
242-249[CrossRef][Medline]
[Order article via Infotrieve]
|
| 30.
|
Kovacs, J. A.,
Vogel, S.,
Albert, J. M.,
Falloon, J.,
Davey, R. T., Jr.,
Walker, R. E.,
Polis, M. A.,
Spooner, K.,
Metcalf, J. A.,
Baseler, M.,
Fyfe, G.,
and Lane, H. C.
(1996)
N. Engl. J. Med.
335,
1350-1356[Abstract/Free Full Text]
|
| 31.
|
Wu, Y.,
and Marsh, J. W.
(2001)
Science
293,
1503-1506[Abstract/Free Full Text]
|
| 32.
|
Czerniecki, B. J.,
Carter, C.,
Rivoltini, L.,
Koski, G. K.,
Kim, H. I.,
Weng, D. E.,
Roros, J. G.,
Hijazi, Y. M., Xu, S. W.,
Rosenberg, S. A.,
and Cohen, P. A.
(1997)
J. Immunol.
159,
3823-3837[Abstract]
|
| 33.
|
Guy, B.,
Riviere, Y.,
Dott, K.,
Regnault, A.,
and Kieny, M. P.
(1990)
Virology
176,
413-425[CrossRef][Medline]
[Order article via Infotrieve]
|
| 34.
|
Kao, J. P.,
Harootunian, A. T.,
and Tsien, R. Y.
(1989)
J. Biol. Chem.
264,
8179-8184[Abstract/Free Full Text]
|
| 35.
|
Sharp, B. M.,
Shahabi, N. A.,
Heagy, W.,
McAllen, K.,
Bell, M.,
Huntoon, C.,
and McKean, D. J.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
8294-8299[Abstract/Free Full Text]
|
| 36.
|
Tsien, R. Y.,
Pozzan, T.,
and Rink, T. J.
(1982)
Nature
295,
68-71[CrossRef][Medline]
[Order article via Infotrieve]
|
| 37.
|
Owaki, H.,
Varma, R.,
Gillis, B.,
Bruder, J. T.,
Rapp, U. R.,
Davis, L. S.,
and Geppert, T. D.
(1993)
EMBO J.
12,
4367-4373[Medline]
[Order article via Infotrieve]
|
| 38.
|
Izquierdo, M.,
Bowden, S.,
and Cantrell, D.
(1994)
J. Exp. Med.
180,
401-406[Abstract/Free Full Text]
|
| 39.
|
Whitmarsh, A. J.,
and Davis, R. J.
(2000)
Nature
403,
255-256[CrossRef][Medline]
[Order article via Infotrieve]
|
| 40.
|
Izquierdo, M.,
Leevers, S. J.,
Marshall, C. J.,
and Cantrell, D.
(1993)
J. Exp. Med.
178,
1199-1208[Abstract/Free Full Text]
|
| 41.
|
Payne, D. M.,
Rossomando, A. J.,
Martino, P.,
Erickson, A. K.,
Her, J. H.,
Shabanowitz, J.,
Hunt, D. F.,
Weber, M. J.,
and Sturgill, T. W.
(1991)
EMBO J.
10,
885-892[Medline]
[Order article via Infotrieve]
|
| 42.
|
Su, B.,
Jacinto, E.,
Hibi, M.,
Kallunki, T.,
Karin, M.,
and Ben-Neriah, Y.
(1994)
Cell
77,
727-736[CrossRef][Medline]
[Order article via Infotrieve]
|
| 43.
|
Derijard, B.,
Raingeaud, J.,
Barrett, T., Wu, I. H.,
Han, J. H.,
Ulevitch, R. J.,
and Davis, R. J.
(1995)
Science
267,
682-685[Abstract/Free Full Text]
|
| 44.
|
Han, J.,
Lee, J. D.,
Bibbs, L.,
and Ulevitch, R. J.
(1994)
Science
265,
808-811[Abstract/Free Full Text]
|
| 45.
|
Raingeaud, J.,
Gupta, S.,
Rogers, J. S.,
Dickens, M.,
Han, J. H.,
Ulevitch, R. J.,
and Davis, R. J.
(1995)
J. Biol. Chem.
270,
7420-7426[Abstract/Free Full Text]
|
| 46.
|
Stephens, L.,
Anderson, K.,
Stokoe, D.,
Erdjument-Bromage, H.,
Painter, G. F.,
Holmes, A. B.,
Gaffney, P. R. J.,
Reese, C. B.,
McCormick, F.,
Tempst, P.,
Coadwell, J.,
and Hawkins, P. T.
(1998)
Science
279,
710-714[Abstract/Free Full Text]
|
| 47.
|
Toker, A.,
and Newton, A. C.
(2000)
J. Biol. Chem.
275,
8271-8274[Abstract/Free Full Text]
|
| 48.
|
Baeuerle, P. A.,
and Baltimore, D.
(1988)
Science
242,
540-546[Abstract/Free Full Text]
|
| 49.
|
Traenckner, E. B. M.,
Pahl, H. L.,
Henkel, T.,
Schmidt, K. N.,
Wilk, S.,
and Baeuerle, P. A.
(1995)
EMBO J.
14,
2876-2883[Medline]
[Order article via Infotrieve]
|
| 50.
|
Brown, K.,
Gerstberger, S.,
Carlson, L.,
Franzoso, G.,
and Siebenlist, U.
(1995)
Science
267,
1485-1488[Abstract/Free Full Text]
|
| 51.
|
Kalli, K.,
Huntoon, C.,
Bell, M.,
and McKean, D. J.
(1998)
Mol. Cell. Biol.
18,
3140-3148[Abstract/Free Full Text]
|
| 52.
|
Khoshnan, A.,
Kempiak, S. J.,
Bennett, B. L.,
Bae, D., Xu, W.,
Manning, A. M.,
June, C. H.,
and Nel, A. E.
(1999)
J. Immunol.
163,
5444-5452[Abstract/Free Full Text]
|
| 53.
|
Gille, H.,
Kortenjann, M.,
Thomae, O.,
Moomaw, C.,
Slaughter, C.,
Cobb, M. H.,
and Shaw, P. E.
(1995)
EMBO J.
14,
951-962[Medline]
[Order article via Infotrieve]
|
| 54.
|
Janknecht, R.,
and Hunter, T.
(1997)
EMBO J.
16,
1620-1627[CrossRef][Medline]
[Order article via Infotrieve]
|
| 55.
|
Price, M. A.,
Cruzalegui, F. H.,
and Treisman, R.
(1996)
EMBO J.
15,
6552-6563[Medline]
[Order article via Infotrieve]
|
| 56.
|
Whitmarsh, A. J.,
Yang, S. H., Su, M. S. S.,
Sharrocks, A. D.,
and Davis, R. J.
(1997)
Mol. Cell. Biol.
17,
2360-2371[Abstract]
|
| 57.
|
Yang, X.,
Chen, Y.,
and Gabuzda, D.
(1999)
J. Biol. Chem.
274,
27981-27988[Abstract/Free Full Text]
|
| 58.
|
Flory, E.,
Weber, C. K.,
Chen, P.,
Hoffmeyer, A.,
Jassoy, C.,
and Rapp, U. R.
(1998)
J. Virol.
72,
2788-2794[Abstract/Free Full Text]
|
| 59.
|
Jacque, J. M.,
Mann, A.,
Enslen, H.,
Sharova, N.,
Brichacek, B.,
Davis, R. J.,
and Stevenson, M.
(1998)
EMBO J.
17,
2607-2618[CrossRef][Medline]
[Order article via Infotrieve]
|
| 60.
|
Yang, X.,
and Gabuzda, D.
(1999)
J. Virol.
73,
3460-3466[Abstract/Free Full Text]
|
| 61.
|
Fisher, A. G.,
Ensoli, B.,
Ivanoff, L.,
Chamberlain, M.,
Petteway, S.,
Ratner, L.,
Gallo, R. C.,
and Wong-Staal, F.
(1987)
Science
237,
888-893[Abstract/Free Full Text]
|
| 62.
|
Strebel, K.,
Daugherty, D.,
Clouse, K.,
Cohen, D.,
Folks, T.,
and Martin, M. A.
(1987)
Nature
328,
728-730[CrossRef][Medline]
[Order article via Infotrieve]
|
| 63.
|
Yang, X.,
and Gabuzda, D.
(1998)
J. Biol. Chem.
273,
29879-29887[Abstract/Free Full Text]
|
| 64.
|
Ott, M.,
Emiliani, S.,
VanLint, C.,
Herbein, G.,
Lovett, J.,
Chirmule, N.,
McCloskey, T.,
Pahwa, S.,
and Verdin, E.
(1997)
Science
275,
1481-1485[Abstract/Free Full Text]
|
| 65.
|
Polverino, A.,
Frost, J.,
Yang, P.,
Hutchison, M.,
Neiman, A. M.,
Cobb, M. H.,
and Marcus, S.
(1995)
J. Biol. Chem.
270,
26067-26070[Abstract/Free Full Text]
|
| 66.
|
Frost, J. A., Xu, S.,
Hutchison, M. R.,
Marcus, S.,
and Cobb, M. H.
(1996)
Mol. Cell. Biol.
16,
3707-3713[Abstract]
|
| 67.
|
Brown, J. L.,
Stowers, L.,
Baer, M.,
Trejo, J.,
Coughlin, S.,
and Chant, J.
(1996)
Curr. Biol.
6,
598-605[CrossRef][Medline]
[Order article via Infotrieve]
|
| 68.
|
Yablonski, D.,
Kane, L. P.,
Qian, D.,
and Weiss, A.
(1998)
EMBO J.
17,
5647-5657[CrossRef][Medline]
[Order article via Infotrieve]
|
| 69.
|
Arora, V.,
Molina, R.,
Foster, J.,
Blakemore, J.,
Chernoff, J.,
Fredericksen, B.,
and Garcia, J.
(2000)
J. Virol.
74,
11081-11087[Abstract/Free Full Text]
|
| 70.
|
Fackler, O. T., Lu, X. B.,
Frost, J. A.,
Geyer, M.,
Jiang, B.,
Luo, W.,
Abo, A.,
Alberts, A. S.,
and Peterlin, B. M.
(2000)
Mol. Cell. Biol.
20,
2619-2627[Abstract/Free Full Text]
|
| 71.
|
Renkema, G. H.,
Manninen, A.,
Mann, D. A.,
Harris, M.,
and Saksela, K.
(1999)
Curr. Biol.
9,
1407-1410[CrossRef][Medline]
[Order article via Infotrieve]
|
| 72.
|
Delgado, P.,
Fernandez, E.,
Dave, V.,
Kappes, D.,
and Alarcon, B.
(2000)
Nature
406,
426-430[CrossRef][Medline]
[Order article via Infotrieve]
|
| 73.
|
Hodge, D. R.,
Dunn, K. J.,
Pei, G. K.,
Chakrabarty, M. K.,
Heidecker, G.,
Lautenberger, J. A.,
and Samuel, K. P.
(1998)
J. Biol. Chem.
273,
15727-15733[Abstract/Free Full Text]
|
| 74.
|
Hanna, Z.,
Kay, D. G.,
Rebai, N.,
Guimond, A.,
Jothy, S.,
and Jolicoeur, P.
(1998)
Cell
95,
163-175[CrossRef][Medline]
[Order article via Infotrieve]
|
| 75.
|
Seger, R.,
Ahn, N. G.,
Posada, J.,
Munar, E. S.,
Jensen, A. M.,
Cooper, J. A.,
Cobb, M. H.,
and Krebs, E. G.
(1992)
J. Biol. Chem.
267,
14373-14381[Abstract/Free Full Text]
|
| 76.
|
King, W. G.,
Mattaliano, M. D.,
Chan, T. O.,
Tsichlis, P. N.,
and Brugge, J. S.
(1997)
Mol. Cell. Biol.
17,
4406-4418[Abstract]
|
| 77.
|
Eder, A. M.,
Dominguez, L.,
Franke, T. F.,
and Ashwell, J. D.
(1998)
J. Biol. Chem.
273,
28025-28031[Abstract/Free Full Text]
|
| 78.
|
Alessi, D. R.,
James, S. R.,
Downes, C. P.,
Holmes, A. B.,
Gaffney, P. R. J.,
Reese, C. B.,
and Cohen, P.
(1997)
Curr. Biol.
7,
261-269[CrossRef][Medline]
[Order article via Infotrieve]
|
| 79.
|
Stokoe, D.,
Stephens, L. R.,
Copeland, T.,
Gaffney, P. R. J.,
Reese, C. B.,
Painter, G. F.,
Holmes, A. B.,
McCormick, F.,
and Hawkins, P. T.
(1997)
Science
277,
567-570[Abstract/Free Full Text]
|
| 80.
|
Lin, X.,
O'Mahony, A., Mu, Y. J.,
Geleziunas, R.,
and Greene, W. C.
(2000)
Mol. Cell. Biol.
20,
2933-2940[Abstract/Free Full Text]
|
| 81.
|
Coudronniere, N.,
Villalba, M.,
Englund, N.,
and Altman, A.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
3394-3399[Abstract/Free Full Text]
|
| 82.
|
Sun, Z. M.,
Arendt, C. W.,
Ellmeier, W.,
Schaeffer, E. M.,
Sunshine, M. J.,
Gandhi, L.,
Annes, J.,
Petrzilka, D.,
Kupfer, A.,
Schwartzberg, P. L.,
and Littman, D. R.
(2000)
Nature
404,
402-407[CrossRef][Medline]
[Order article via Infotrieve]
|
| 83.
|
Regnier, C. H.,
Song, H. Y.,
Gao, X.,
Goeddel, D. V.,
Cao, Z. D.,
and Rothe, M.
(1997)
Cell
90,
373-383[CrossRef][Medline]
[Order article via Infotrieve]
|
| 84.
|
DiDonato, J. A.,
Hayakawa, M.,
Rothwarf, D. M.,
Zandi, E.,
and Karin, M.
(1997)
Nature
388,
548-554[CrossRef][Medline]
[Order article via Infotrieve]
|
| 85.
|
Kempiak, S. J.,
Hiura, T. S.,
and Nel, A. E.
(1999)
J. Immunol.
162,
3176-3187[Abstract/Free Full Text]
|
| 86.
|
Lee, F. S.,
Hagler, J.,
Chen, Z. J. J.,
and Maniatis, T.
(1997)
Cell
88,
213-222[CrossRef][Medline]
[Order article via Infotrieve]
|
| 87.
|
Lin, X.,
Cunningham, E. T., Mu, Y. J.,
Geleziunas, R.,
and Greene, W. C.
(1999)
Immunity
10,
271-280[CrossRef][Medline]
[Order article via Infotrieve]
|
| 88.
|
Woronicz, J. D.,
Gao, X.,
Cao, Z.,
Rothe, M.,
and Goeddel, D. V.
(1997)
Science
278,
866-869[Abstract/Free Full Text]
|
| 89.
|
Malinin, N. L.,
Boldin, M. P.,
Kovalenko, A. V.,
and Wallach, D.
(1997)
Nature
385,
540-544[CrossRef][Medline]
[Order article via Infotrieve]
|
| 90.
|
Berridge, M. J.
(1993)
Nature
361,
315-325[CrossRef][Medline]
[Order article via Infotrieve]
|
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

CiteULike Complore Connotea Del.icio.us Digg Reddit Technorati What's this?
This article has been cited by other articles:

|
 |

|
 |
 
N. Laguette, S. Benichou, and S. Basmaciogullari
Human Immunodeficiency Virus Type 1 Nef Incorporation into Virions Does Not Increase Infectivity
J. Virol.,
January 15, 2009;
83(2):
1093 - 1104.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
V. Witte, B. Laffert, P. Gintschel, E. Krautkramer, K. Blume, O. T. Fackler, and A. S. Baur
Induction of HIV Transcription by Nef Involves Lck Activation and Protein Kinase C{theta} Raft Recruitment Leading to Activation of ERK1/2 but Not NF{kappa}B
J. Immunol.,
December 15, 2008;
181(12):
8425 - 8432.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
A. W. Lee, E. R. Sharp, A. O'Mahony, M. G. Rosenberg, D. M. Israelski, G. P. Nolan, and D. F. Nixon
Single-Cell, Phosphoepitope-Specific Analysis Demonstrates Cell Type- and Pathway-Specific Dysregulation of Jak/STAT and MAPK Signaling Associated with In Vivo Human Immunodeficiency Virus Type 1 Infection
J. Virol.,
April 1, 2008;
82(7):
3702 - 3712.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
G. Mangino, Z. A. Percario, G. Fiorucci, G. Vaccari, S. Manrique, G. Romeo, M. Federico, M. Geyer, and E. Affabris
In Vitro Treatment of Human Monocytes/Macrophages with Myristoylated Recombinant Nef of Human Immunodeficiency Virus Type 1 Leads to the Activation of Mitogen-Activated Protein Kinases, I{kappa}B Kinases, and Interferon Regulatory Factor 3 and to the Release of Beta Interferon
J. Virol.,
March 15, 2007;
81(6):
2777 - 2791.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
J. Choi, J. Walker, K. Talbert-Slagle, P. Wright, J. S. Pober, and L. Alexander
Endothelial Cells Promote Human Immunodeficiency Virus Replication in Nondividing Memory T Cells via Nef-, Vpr-, and T-Cell Receptor-Dependent Activation of NFAT
J. Virol.,
September 1, 2005;
79(17):
11194 - 11204.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
S. B. Lee, J. Park, J. U. Jung, and J. Chung
Nef induces apoptosis by activating JNK signaling pathway and inhibits NF-{kappa}B-dependent immune responses in Drosophila
J. Cell Sci.,
May 1, 2005;
118(9):
1851 - 1859.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
E. A. Acheampong, Z. Parveen, L. W. Muthoga, M. Kalayeh, M. Mukhtar, and R. J. Pomerantz
Human Immunodeficiency Virus Type 1 Nef Potently Induces Apoptosis in Primary Human Brain Microvascular Endothelial Cells via the Activation of Caspases
J. Virol.,
April 1, 2005;
79(7):
4257 - 4269.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
R. R. Valiathan and M. D. Resh
Expression of Human Immunodeficiency Virus Type 1 Gag Modulates Ligand-Induced Downregulation of EGF Receptor
J. Virol.,
November 15, 2004;
78(22):
12386 - 12394.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
M. Maccarrone, M. Di Rienzo, A. Finazzi-Agro, and A. Rossi
Leptin Activates the Anandamide Hydrolase Promoter in Human T Lymphocytes through STAT3
J. Biol. Chem.,
April 4, 2003;
278(15):
13318 - 13324.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
E. Y. Choe, E. S. Schoenberger, J. E. Groopman, and I.-W. Park
HIV Nef Inhibits T Cell Migration
J. Biol. Chem.,
November 22, 2002;
277(48):
46079 - 46084.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
D. Messmer, J.-M. Jacque, C. Santisteban, C. Bristow, S.-Y. Han, L. Villamide-Herrera, E. Mehlhop, P. A. Marx, R. M. Steinman, A. Gettie, et al.
Endogenously Expressed nef Uncouples Cytokine and Chemokine Production from Membrane Phenotypic Maturation in Dendritic Cells
J. Immunol.,
October 15, 2002;
169(8):
4172 - 4182.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
S. Kinet, F. Bernard, C. Mongellaz, M. Perreau, F. D. Goldman, and N. Taylor
gp120-mediated induction of the MAPK cascade is dependent on the activation state of CD4+ lymphocytes
Blood,
September 18, 2002;
100(7):
2546 - 2553.
[Abstract]
[Full Text]
[PDF]
|
 |
|
Copyright © 2002 by the American Society for Biochemistry and Molecular Biology.
|
Advertisement
Advertisement
|