From the Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261 and the ¶ Program in Molecular Medicine, University of Massachusetts Medical Center, Worcester, Massachusetts 01605
Received for publication, April 11, 2001, and in revised form, April 24, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Human immunodeficiency virus Nef is a small
myristylated protein that plays a critical role in AIDS
progression. Nef binds with high affinity to the SH3 domain of the
myeloid-restricted tyrosine kinase Hck in vitro,
identifying this Src-related kinase as a possible cellular target for
Nef in macrophages. Here we show that Nef activates endogenous Hck in
the granulocyte-macrophage colony-stimulating
factor-dependent myeloid cell line, TF-1. Unexpectedly, Nef
induced cytokine-independent TF-1 cell outgrowth and constitutive activation of the Stat3 transcription factor. Induction of survival required the Nef SH3 binding and membrane-targeting motifs and was
blocked by dominant-negative Stat3 mutants. Nef also stimulated Stat3
activation in primary human macrophages, providing evidence for Stat3
as a Nef effector in a target cell for human immunodeficiency virus.
nef is a highly conserved gene unique to the primate
lentiviruses HIV-1,1 HIV-2,
and SIV (1, 2). Previous studies show that Nef is required for high
titer replication of HIV and SIV and greatly accelerates the
development of AIDS-like disease in Rhesus monkeys (3). HIV strains
with defective nef alleles have been isolated from patients
with long term, non-progressive HIV infection, implicating nef as a progression factor in AIDS (4, 5). Although
nef is not required for the growth of HIV in most cultured
cell lines, it has been shown to enhance HIV replication in resting
peripheral blood mononuclear cells (6, 7). Furthermore, selective
expression of nef in T-cells and monocytes/macrophages of
transgenic mice is sufficient to induce a severe AIDS-like syndrome
(8). However, the molecular mechanisms by which nef
contributes to the replication and pathogenicity of HIV and SIV are not clear.
The nef gene encodes a 25-30-kDa myristylated protein with
no identified catalytic activity and is believed to influence cellular signaling pathways by physically interacting with host cell proteins and modifying their functions (1, 2). In particular, non-receptor tyrosine kinases of the Src family have emerged as potential
intracellular targets for Nef. Like Nef, Src family kinases are also
myristylated and localize to cellular membranes (9). The SH3 domain of
the Src-related kinase Hck binds to Nef with the highest affinity known
for an SH3-mediated interaction (10, 11). Mutagenesis has established
that the Nef motif responsible for SH3 binding consists of the repeated
proline-rich sequence PXXPXXPXXP,
where X = any amino acid (10). X-ray crystallographic
and NMR structural studies of high affinity SH3-Nef complexes show that
these residues form the polyproline type II helix responsible for most
SH3-mediated interactions (12, 13). The Nef PXXP motif is
highly conserved among known HIV isolates (14) and is important for SIV
replication in macaques (3). These findings support the notion that the SH3 binding function of Nef and its interaction with Hck or other Src
family members are essential to its functions in HIV replication and
AIDS progression.
Recently, we reported that SH3-dependent interaction with
Nef functionally activates full-length Hck in Rat-2 fibroblasts, leading to cellular transformation (15-17). In addition, we
demonstrated that this transformation event correlates with increased
cellular phosphotyrosine content and Nef·Hck complex
formation, suggesting that Nef directly activates Hck in
vivo. In the present study, we extend these findings by showing
that Nef activates endogenous Hck in the cytokine-dependent
monocyte/macrophage precursor cell line, TF-1 (18). Surprisingly, Nef
produced a cytokine-independent phenotype in these cells that requires
the Nef SH3 binding motif and myristylation signal sequence. Nef also
induced constitutive activation of the Stat3 transcription factor in
the cytokine-independent TF-1 cells, providing further evidence for the
activation of endogenous Hck or a closely related tyrosine kinase by
Nef. Experiments with dominant-negative Stat3 mutants indicate that
Nef-induced TF-1 survival is dependent upon Stat3 activation. Nef also
induced Stat3 activation in primary human macrophages. These results
show for the first time that Nef can activate endogenous Src family kinases and downstream Stat factors in a cell lineage relevant to HIV infection.
Cell Culture--
The human myeloid leukemia cell line TF-1 (18)
was obtained from the American Type Culture Collection (ATCC) and grown
in RPMI 1640 medium supplemented with 10% fetal bovine serum, 50 µg/ml gentamycin, and 3 × 105 units/ml recombinant
human GM-CSF (a generous gift of the Immunex Corp.). The human cell
line 293T (19) was also obtained from the ATCC and grown in Dulbecco's
modified Eagle's medium containing 5% fetal bovine serum and 50 µg/ml gentamycin.
Hck and Nef Expression Constructs--
The nef gene
used in these studies is from the HIV-1 isolate SF2. The Nef mutants
used include Nef-PA, in which Pro residues 72 and 75 were converted to
Ala, and Nef-GA, in which the Gly residue at position 2 was converted
to Ala. Both mutants were produced using standard polymerase chain
reaction-based methods and subcloned along with wild-type Nef into the
retroviral vector pSR
Construction of activated (Y501F) and kinase-dead (K269E) Hck mutants
is described elsewhere (15, 22). These mutants together with wild-type
Hck were subcloned into the mammalian expression vector pCDNA3
(InVitrogen) for transient expression in 293T cells. The
pCDNA3-based expression vector for Stat3 has been described elsewhere (23).
The Stat3 dominant-negative mutant Stat3-EVA was supplied by Dr.
Richard Jove, Moffitt Cancer Center. A point mutation converting Tyr-705 of murine Stat3 to Phe (Stat3-YF) was introduced into pcDNA3-Stat3 using the GeneEditor in vitro site-directed
mutagenesis system (Promega). The dominant-negative Stat3 mutants were
subcloned into a variant of the retroviral vector pSR Soft Agar Colony Assay--
TF-1 cells were infected with
recombinant Nef retroviruses using a centrifugal enhancement procedure
described elsewhere (24). Briefly, TF-1 cells (5 × 104) were resuspended in retroviral supernatants and
centrifuged at 1,000 × g for 4 h in the presence
of 4 µg/ml Polybrene. After selection with G418 in the presence of
GM-CSF, 1 × 104 drug-resistant cells were plated in
triplicate 35-mm dishes in RPMI 1640 medium containing 0.3% agar and
20% fetal bovine serum in the presence and absence of GM-CSF. Colony
formation was assessed 2 weeks later.
Cell Lysis and Immune Complex Kinase Assay--
TF-1 cells
(106) were incubated in the absence of GM-CSF for 48 h
and then lysed in 1.0 ml of modified radioimmune precipitation buffer
(50 mM Tris-HCl, pH 7.4, 50 mM NaCl, 1 mM EDTA, 10 mM MgCl2, 1% Triton
X-100, 1% sodium deoxycholate, 0.1% SDS). TF-1 cell lysates were
clarified by centrifugation at 100,000 × g for 30 min
at 4 °C. Clarified cell lysates were incubated with rabbit antibodies to Hck or Fyn (Santa Cruz Biotechnology) and protein G-Sepharose (20 µl of a 50% slurry) for 2 h at 4 °C. The
kinase immunoprecipitates were collected by centrifugation and washed twice with 1.0 ml of radioimmune precipitation buffer followed by two
washes with kinase buffer (50 mM HEPES, pH 7.4, 10 mM MgCl2). Kinase buffer (20 µl) containing 1 µg of the p50 substrate (50-kDa glutathione
S-transferase-Sam 68 fusion protein; Santa Cruz
Biotechnology) and 5 µCi [ Electrophoretic Mobility Shift Assay--
TF-1 or 293T cells
were washed once with phosphate-buffered saline containing 1 mM Na3VO4 and 5 mM NaF.
Cells were resuspended with 0.5 ml of hypotonic buffer (40 mM HEPES, pH 7.9, 2 mM EDTA, 2 mM
EGTA, 20 mM NaF, 1 mM
Na3VO4, 1 mM
Na4P2O7, 1 mM
dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 50 µg/ml leupeptin, 25 µg/ml aprotinin, and 150 mM
Na2MoO4), sonicated for 10 s, and then
centrifuged for 30 s at 14,000 rpm and 4 °C. Nuclei were
resuspended in high salt buffer (40 mM HEPES, pH 7.9, 840 mM NaCl, 2 mM EDTA, 2 mM EGTA, 40%
glycerol, 20 mM NaF, 1 mM
Na3VO4, 1 mM
Na4P2O7, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 50 µg/ml leupeptin, 25 µg/ml aprotinin, and 150 mM
Na2MoO4) and incubated for 30 min on a rotator
at 4 °C. Nuclear extracts were clarified by microcentrifugation for
5 min at 14,000 rpm and 4 °C. The protein concentrations of the
nuclear extracts were determined using the Coomassie Plus protein assay
reagent (Pierce) and bovine serum albumin as standard. For the
gel-shift assay, 10 µg of TF-1 and 5 µg of 293T nuclear extracts
were used. The probe used for the Stat3 gel-shift assay is based on the
sis-inducible element (SIE) (25). Oligonucleotides (20 pmol)
were annealed in 10 µl of TE buffer (10 mM Tris-HCl, pH
8.0, 1 mM EDTA) by heating to 70 °C and slowly cooling
to room temperature. The probes were labeled by combining 4 µl of
annealed oligonucleotide with 2 µl of Labeling Mix-dATP (Amersham
Pharmacia Biotech), 2 µl of [ Expression of Hck and STAT3 in 293T Cells--
Human 293T cells
were transfected with Hck and Stat3 expression plasmids using a calcium
phosphate-mediated transfection procedure described elsewhere (26).
Cells were lysed in modified radioimmune precipitation buffer, and
Stat3 expression and tyrosine phosphorylation were detected by
immunoblotting with antibodies to phosphotyrosine or Stat3 (Santa Cruz
Biotechnology). Control blots were performed with anti-Hck antibodies
(Santa Cruz) to verify Hck expression.
Primary Macrophage Culture, Adenovirus Infection, and Gel Shift
Assay--
Monocytes were isolated by counter-current centrifugal
elutriation of mononuclear leukocyte-rich cell preparations obtained from normal HIV-1 and hepatitis B seronegative donors after
leukapheresis (27). Monocytes were cultured as adherent monolayers for
2 days in the presence of M-CSF (R & D Systems) and for a
further 5 days in the absence of cytokine before plating.
Monocyte-derived macrophages were infected 7 days after plating.
The parental adenovirus vector is a derivative of pJM17 (28) in
which the entire E1- and E3-coding sequences have been deleted. The adenovirus shuttle vector (452/JL1) was constructed from pXC/JL1 (28). The HIV-1 Nef and green fluorescent protein (GFP)-coding regions
were inserted in the shuttle vector under control of the cytomegalovirus immediate early promoter. The recombinant GFP and Nef
adenovirus vectors were generated by homologous recombination of the
parent adenovirus vector with the corresponding shuttle vector after
calcium phosphate transfection of early passage 293 cells. Recombinants
were propagated on 293 cells and purified through two rounds of plaque
purification and concentrated by cesium chloride banding. Titers of
recombinant viruses were determined by plaque formation on 293 cells
(29).
To prepare nuclear extracts from macrophages, cells were washed twice
with phosphate-buffered saline containing 1 mM
Na3VO4 and 20 mM NaF and then lysed
in buffer A (50 mM NaCl, 10 mM HEPES, pH 8.0, 500 mM sucrose, 1 mM EDTA, 0.5 mM
spermidine, 0.15 mM spermine, 1 mM
dithiothreitol, 0.2% Triton X-100). Nuclei were collected by
centrifugation at 6500 × g for 3 min at 4 °C,
washed in 600 µl of buffer B (buffer A without Triton X-100), and
collected again. Nuclear proteins were extracted at 4 °C on a
rotating wheel for 30 min in buffer C (350 mM NaCl, 10 mM HEPES, pH 8.0, 25% glycerol, 0.1 mM EDTA,
0.5 mM spermidine, 0.15 mM spermine). Protease and phosphatase inhibitors were added to all buffers (20 mM
NaF, 1 mM Na3VO4, 1 mM
Na4P2O7, 1 mM
phenylmethylsulfonyl fluoride, 100 µM leupeptin, 0.3 µM aprotinin, 10 µM tosylphenylalanyl
chloromethyl ketone (TPCK), 800 µM
1-chloro-3-tosylamido-7-amino-2-heptanone (TLCK), 10 µM
bestatin, 1 µM pepstatin, 10 µM MG132).
Extracts were clarified by centrifugation at 6500 × g
for 5 min, and protein concentrations were determined by the Bradford
assay according to the manufacturer's instructions (Bio-Rad). For the
gel shift assay, 100 ng of wild-type or mutant Stat3 double-stranded
oligonucleotide probes (Santa Cruz) were end-labeled with
polynucleotide kinase in the presence of 20 µCi of
[ Nef Induces Cytokine-independent Growth in the Human Myeloid
Leukemia Cell Line, TF-1--
Previous work from our laboratory has
shown that co-expression of Nef and Hck in Rat-2 fibroblasts induces
cellular transformation (15). Hck is strongly expressed in macrophages
(30-34), an important target cell for primate lentivirus infection
(35, 36). To investigate whether endogenous Hck is activated by Nef in
cells of the monocyte/macrophage lineage, we utilized the human myeloid leukemia cell line, TF-1 (18). This cell line is dependent on GM-CSF,
IL-3, or erythropoietin for growth; removal of cytokine induces growth
arrest and programmed cell death. Upon treatment with phorbol esters,
TF-1 cells differentiate into macrophages.
Recombinant retroviruses were used to establish populations of TF-1
cells that stably express Nef (15, 16). After infection with Nef
retroviruses, cells were placed under G418 selection in the presence or
absence of GM-CSF. As shown in Fig.
1A, Nef expression was readily
detected in the G418-resistant cells. To our surprise, TF-1 cells
expressing Nef no longer required GM-CSF for growth (Fig.
1B). Whereas cells infected with a control retrovirus did
not survive in the absence of GM-CSF, the TF-1 cells expressing Nef
readily emerged from G418 selection in the absence of cytokine.
To confirm that Nef was producing a survival signal in TF-1 cells, we
also performed soft-agar colony assays. TF-1 cells were infected with
recombinant Nef retroviruses, selected with G418, and plated in soft
agar in the presence or absence of GM-CSF. As shown in Fig.
2A, both the parental and
Nef-expressing TF-1 cells readily formed colonies in the presence of
GM-CSF. However, TF-1 cells expressing Nef also formed soft agar
colonies in the absence of cytokine, whereas the parental cells did
not. Immunoblot analysis of protein extracts from parallel liquid
cultures shows that Nef protein levels were unchanged 24 h after
GM-CSF removal, indicating that Nef expression is independent of GM-CSF
treatment (Fig. 2B). GM-CSF-independent outgrowth of TF-1
cells expressing Nef results from suppression of apoptosis as
determined by flow cytometry of propidium iodide-stained cells as well
as DNA fragmentation assays (data not shown). These results indicate
that Nef is sufficient to initiate a signal transduction event in TF-1
cells permissive for survival in the absence of cytokine.
Induction of Cytokine-independent Growth in TF-1 Cells Requires the
Nef SH3 Binding Motif and Myristylation Signal Sequence--
We next
investigated the structural features of Nef that are required for the
induction of cytokine independence by Nef in TF-1 cells. For these
experiments, we employed a Nef mutant in which the N-terminal Gly
residue essential for myristylation is replaced with Ala (Nef-GA
mutant). This mutation blocks Nef myristylation, which is essential for
membrane targeting. We also tested a Nef mutant lacking the highly
conserved PXXP repeat essential for interaction with the Hck
SH3 domain (Nef-PA mutant). TF-1 cells expressing these Nef mutants
were tested for outgrowth in liquid culture as well as colony-forming
activity in soft agar in the presence or absence of GM-CSF. As shown in
Figs. 1 and 2, neither of these Nef mutants was able to induce GM-CSF
independence despite strong expression of the mutant proteins. These
results indicate that both myristylation and the SH3 binding motif are
required for cytokine independence. Thus, the TF-1 cellular target
responsible for the Nef survival signal is likely to be a
membrane-localized protein with an SH3 domain.
Activation of Endogenous Hck by Nef in TF-1 Cells--
Previous
studies have shown that Hck and other members of the Src tyrosine
kinase family are activated in response to GM-CSF, IL-3, and/or
erythropoietin (37-41), the three cytokines known to promote survival
of TF-1 cells (18). In addition, the SH3 domain of Hck binds to Nef
with high affinity, leading to Hck kinase activation both in
vitro and in vivo (10, 11, 15, 16, 42). These findings
suggest that Nef may activate endogenous Hck in TF-1 cells as part of a
signaling pathway leading to cytokine-independent growth. To determine
whether Nef activates endogenous Hck, control and Nef-expressing TF-1
cells were washed free of GM-CSF and lysed, and Hck was
immunoprecipitated and assayed in vitro with
[
We also investigated the activity of Hck in TF-1 cells expressing the
Nef-PA and Nef-GA mutants. As shown in Fig. 3, Nef-PA was unable to
induce endogenous Hck activation in TF-1 cells, indicating that the Nef
SH3 binding motif is essential for kinase activation. This result is
consistent with our previous work in Rat-2 fibroblasts co-expressing
Nef and Hck (15, 16). On the other hand, mutagenesis of the
myristylation signal sequence did not markedly impair the ability of
Nef to induce Hck activation despite the myristylation requirement for
the induction of cytokine independence by Nef. Because the Nef-GA
mutant retains a functional SH3 binding motif, it is possible that this
mutant interacts with the cytoplasmic pool of Hck (45, 46), resulting
in kinase activation. However, activation of Hck at the plasma membrane may be required for initiation of the signaling response required for
cytokine independence.
Nef Induces Activation of Endogenous Stat3 in GM-CSF-independent
TF-1 Cells--
As an alternative test for the activation of
endogenous tyrosine kinase signaling in TF-1 cells by Nef, we
investigated activation of the transcription factor, Stat3. Stat3 is
one member of a family of transcription factors with SH2 domains that
require tyrosine phosphorylation for activation (47, 48). Tyrosine
phosphorylation induces Stat dimerization by reciprocal
SH2-phosphotyrosine interaction, leading to nuclear translocation, DNA
binding, and gene activation. Thus, Stat activation is a good indicator
of endogenous tyrosine kinase activity.
Stat3 activation was investigated in TF-1 cells using an
electrophoretic mobility shift (gel-shift) assay with an
oligonucleotide probe based on the SIE, which is potently bound by
activated Stat3 (23, 25). As shown in Fig.
4, nuclear extracts prepared from the
GM-CSF-independent TF-1/Nef cells produced a strong shift in SIE
mobility. The gel-shifted complex was significantly reduced in control
cells or in cells expressing either the SH3 binding or
myristylation-defective mutants of Nef, which do not produce cytokine
independence. SIE-protein complex formation was completely blocked in
the presence of a 100-fold excess of unlabeled probe, indicative of
specific DNA binding. No complexes were observed with a mutant SIE
probe lacking nucleotides critical for Stat3 binding, and addition of
anti-Stat3 antibodies to the reaction mixture completely inhibited
complex formation (data not shown). Treatment of control TF-1 cells
with GM-CSF produced a gel-shifted complex with electrophoretic
mobility identical to that observed with the factor-independent
TF-1/Nef cells, consistent with other data showing that GM-CSF induces
Stat3 activation in this cell line (49). Taken together, these results
demonstrate that Nef expression leads to the activation of endogenous
Stat3 in TF-1 cells.
Direct Activation of Stat3 by Hck in a Heterologous Expression
System--
Data presented so far have demonstrated a strong
correlation between Nef-induced cytokine independence and endogenous
Hck and Stat3 activation. These results led us to speculate that Hck may be the kinase responsible for Stat activation downstream of Nef. To
test this idea more directly, we expressed Hck and Stat3 either alone
or together in 293T cells and assayed Stat3 activation using the
gel-shift assay. As shown in Fig.
5A, co-expression of Hck and
Stat3 in 293T cells produced a dramatic shift in probe mobility,
indicative of potent Stat3 activation. In contrast, expression of
either protein alone was without effect, consistent with the direct
activation of Stat3 by Hck in this system. As an additional negative
control, we co-expressed Stat3 with a kinase-inactive mutant of Hck in
this system. No gel-shift response was detected, demonstrating the
requirement for tyrosine phosphorylation in the activation mechanism.
Finally, we also tested a transforming variant of Hck in which the
negative regulatory tyrosine residue in the C-terminal tail (Tyr-501)
has been converted to Phe (15). This Hck mutant activated Stat3 to the
same extent as wild type, suggesting that 293T cells lack sufficient
Csk for effective negative regulation of wild-type Hck. Csk is the
kinase responsible for the negative regulation of Src family kinases
and works by phosphorylating the conserved tyrosine residue in Src
family kinase C-terminal tails. As a result, the phosphorylated tail
engages the SH2 domain in an intramolecular fashion, contributing to
kinase down-regulation.
To establish that Stat3 DNA binding activity induced by Hck correlated
with Stat3 tyrosine phosphorylation, lysates from the transfected 293T
cells were immunoblotted with anti-phosphotyrosine antibodies. Fig.
5B shows that Stat3 is strongly phosphorylated on tyrosine
when co-expressed with either of the active forms of Hck, consistent
with the gel-shift result. In contrast, lysates from cells expressing
Stat3 alone or in the presence of the kinase-inactive form of Hck
showed no evidence of Stat3 tyrosine phosphorylation. Control
immunoblots show that approximately equivalent levels of Stat3 and Hck
proteins were expressed. These results provide strong evidence that Hck
can induce direct activation of Stat3 under defined conditions and
suggest that Nef may trigger Stat3 activation via Hck in TF-1 cells.
Activation of Stat3 Is Required for Nef-induced Survival of TF-1
Cells--
To determine whether activation of Stat3 is necessary for
Nef-induced cell survival, we employed two dominant-negative mutants of
Stat3. The first mutant (YF) lacks the site of tyrosine phosphorylation (Tyr-705), whereas the second (EVA) has mutations in Glu residues required for DNA binding. These and related Stat3 mutants have been
used to block Stat3-mediated biological responses in other systems
(50-52) including transformation of fibroblasts by v-Src (53, 54).
TF-1 cells were co-infected with recombinant retroviruses carrying Nef
and the dominant-negative Stat3 mutants. Nef and the Stat3 mutants were
linked to different drug selection markers (neo and hygromycin,
respectively) to ensure that cells were infected with both retroviruses
after double selection. Drug-resistant cells were then plated in soft
agar and scored for colony outgrowth in the absence of GM-CSF. Nef
readily induced colony formation in cells that were also infected with
a control retrovirus carrying only the hygromycin resistance marker
(Fig. 6). However, when cells were doubly
infected with Nef and either of the Stat3 dominant-negative mutants, no
colony outgrowth was observed. These data suggest that activation of
Stat3 is an essential part of the survival response induced by Nef in
TF-1 cells.
Nef Activates Stat3 in Primary Human Macrophages--
A final
series of experiments investigated whether Nef can induce Stat3
activation in primary human monocyte-derived macrophages, a target cell
for HIV infection. For these experiments, recombinant adenovirus
vectors were used to introduce Nef into primary cultures of macrophages
isolated from normal donors. Parallel cultures were infected with a
control adenovirus carrying GFP. Nuclear protein extracts were prepared
from Nef-infected and control macrophages and analyzed for the presence
of activated Stat3 using the SIE probe. As shown in Fig.
7A, extracts from macrophages
infected with the Nef adenovirus produced a marked shift in SIE
mobility, whereas extracts from control cells infected with the GFP
adenovirus or mock-infected cells had no effect. Duplicate gel shift
assays using an SIE probe with mutations in the sequence required for Stat3 binding produced no detectable protein-DNA complexes. Similarly, the addition of an excess of unlabeled SIE probe to the reaction completely inhibited formation of the gel-shifted complex, whereas the
mutant SIE did not (Fig. 7B). These results are consistent with the observations made in TF-1 cells after expression of Nef (Fig.
4) and provide direct evidence that Nef expression is sufficient to
activate both tyrosine kinase activity and Stat3 in primary macrophages.
Previous work from our laboratory established that SH3-mediated
interaction of Nef with Hck is sufficient to induce oncogenic transformation when these proteins are co-expressed in Rat-2
fibroblasts (15-17). This result suggested that Nef may constitutively
induce Hck activation in target cells for HIV that express this
Src-related kinase, such as macrophages. In this study, we provide
evidence that Nef can induce endogenous Hck activation in the
monocyte/macrophage precursor cell line, TF-1. Expression of Nef in
TF-1 cells also revealed a new biological property of Nef: its ability
to induce cytokine-independent myeloid cell growth. This finding
suggests that Nef is able to stimulate some of the signal transduction pathways normally activated by GM-CSF that lead to survival and proliferation in this cell line. Several observations suggest that
activation of Hck may contribute to the effect of Nef on TF-1 cell
survival. First, Nef-induced activation of Hck (Fig. 3) but not Fyn
(data not shown) in the factor-independent cells correlates with the
binding affinities of Nef for the SH3 domains of these Src family
kinases (11, 44). Second, the Nef mutant lacking the polyproline motif
responsible for SH3 binding does not produce the cytokine-independent
phenotype in TF-1 cells (Figs. 1 and 2) and is unable to activate
endogenous Hck in these cells (Fig. 3). In previous work, we and others
show that the Nef SH3 binding motif is essential for interaction with
Hck and subsequent kinase activation (10, 15). Finally, Hck is normally
activated downstream of cytokine receptors, including those for GM-CSF
and IL-3 (38-40). Both of these cytokines have been shown to promote growth and survival of TF-1 cells (18).
Previous work has established that Nef may mimic the effect of IL-2 on
T-lymphocyte activation (55). This study employed a monkey T-lymphoid
cell line, known as 221, which requires IL-2 to proliferate. SIV was
found to replicate efficiently in IL-2-stimulated 221 cells
independently of the presence of Nef. However, nef+ viruses
replicated as much as 100-fold more efficiently than
nef-defective viruses in the absence of IL-2. This
difference was attributed to the ability of Nef to induce IL-2
production in the host cell line. These results suggest that our
observation of Nef-induced survival in myeloid cells may also involve
an autocrine mechanism, possibly through the induction of GM-CSF, IL-3,
or other cytokines known to promote TF-1 cell survival. However, we
found that culture medium conditioned by cytokine-independent TF-1/Nef
cells was unable to support naive TF-1 cell growth (data not shown).
Thus, Nef appears to promote TF-1 cell survival via constitutive
stimulation of a host survival pathway rather than through the
induction of anti-apoptotic cytokines.
The observation that Nef-induced activation of Hck correlates with
cytokine-independent survival of TF-1 cells suggests that activation of
Hck alone may be sufficient to induce the survival response. We have
tested this possibility by expressing two different constitutively
activated forms of Hck in TF-1 cells using recombinant retroviruses.
The first of these was activated by Phe substitution of the conserved
Tyr residue in the C-terminal tail, whereas the second has mutations of
two proline residues in the SH2-kinase linker region that interact
intramolecularly with the SH3 domain in the inactive form of the kinase
(15, 16). Although both of these mutants are strongly transforming in
Rat-2 fibroblasts, neither was able to produce cytokine-independent
outgrowth of TF-1 cells. This result suggests that activation of Hck
alone may not be sufficient to produce the survival signal. Nef may activate other members of the Src kinase family, which could in turn
contribute to Stat activation and the observed biological effect. One
possibility is Lyn, which has been shown to mediate the GM-CSF signal
for survival in neutrophils (56). The Lyn SH3 domain has been shown to
interact with Nef in vitro (10), suggesting that Nef may
induce Lyn activation in a manner analogous to Hck. However, we have
observed that co-expression of Lyn with Nef in fibroblasts does not
induce Lyn activation under conditions where Hck is potently activated
by Nef (17). This result argues against a contribution of Lyn to the
observed survival signal produced by Nef in TF-1 cells. Alternatively,
Nef-mediated activation of serine/threonine kinases may be required for
full transcriptional activation of Stat3 (57). Nef has been recently
shown to activate the c-Jun N-terminal kinase (Jnk) downstream of Nak
(58), a Pak-related kinase that interacts directly with Nef (59, 60). Interestingly, Jnk and the related p38 kinase have recently been identified as the activities responsible for serine phosphorylation and
transcriptional activation of Stat3 in v-Src-transformed fibroblasts (61). Thus it is possible that Nef may activate multiple kinase signals
that are convergent on Stat3.
Data presented here show for the first time that expression of HIV Nef
is sufficient to activate a Stat signal transduction pathway. Data in
Fig. 4 demonstrate that Nef activates Stat3 DNA binding activity to the
same extent as GM-CSF treatment in TF-1 cells. Stat3 activation is
required for the generation of survival signals by Nef, as two
different dominant-negative Stat3 mutants were able to interfere with
cytokine-independent colony formation (Fig. 6). This result agrees with
the work of Fukada et al. (50), who demonstrate that Stat3
dominant-negative mutants were able to interfere with survival signals
generated by gp130, the signal-transducing receptor subunit shared by
the IL-6 family of cytokines. Hck has been implicated in leukemia
inhibitory factor signal transduction, which is linked to gp130 (62,
63). Data presented in Fig. 5 show that Hck can directly induce Stat3
DNA binding activity and tyrosine phosphorylation in a defined
expression system (293T cells). These results are consistent with the
possibility that activation of Hck by Nef may be sufficient to induce
Stat3 tyrosine phosphorylation in TF-1 cells. In addition, a recent
study has shown that Src kinases, and not Jaks, are responsible for
Stat activation in response to IL-3 treatment in some cases (64). More
recent work has shown that constitutive activation of Stat3 suppresses
apoptosis in human multiple myeloma cells (65). This study also
demonstrated the Stat3-dependent induction of the Bcl-2 family member Bcl-XL as a possible mechanism for the
observed anti-apoptotic effects of Stat3. Whether a similar mechanism
accounts for the Nef-induced survival of TF-1 cells will require
further investigation.
In addition to the TF-1 cell line, we observed that Nef expression is
sufficient to activate Stat3 in primary human macrophages, raising the
possibility that Nef may mimic cytokine signaling in this target cell
for HIV. Although the biological significance of this effect is
currently unknown, it is reasonable to speculate that Nef may
contribute to macrophage survival by a mechanism similar to that
observed with TF-1 cells. Interestingly, HIV-1 infection of primary
human macrophages promotes their survival in
culture.2 Because Hck is a
myeloid-restricted tyrosine kinase, constitutive activation of a
Hck-Stat3 pathway by Nef may promote virus replication and
dissemination. Recently, it was demonstrated that monocytotropism promotes amplification and spread of SIV in macaques (36). This finding
raises the possibility that Nef modifies the physiology of macrophages
in a way that promotes their role as persistent viral reservoirs. Our
observation that Nef may serve as a survival factor is consistent with
this role. The identification of cellular pathways through which Nef
mediates survival and other effects may shed further light on the role
of this accessory gene product in virus replication and pathogenicity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
MSVtkneo (20). The resulting constructs were
used to generate high titer stocks of recombinant retroviruses by
co-transfection of 293T cells with an amphotropic packaging vector as
described elsewhere (15-17, 21).
MSVtkneo in
which the neo-antibiotic resistance marker was replaced by a DNA
fragment coding for the hygromycin-resistance gene. The resulting
plasmids were used to make recombinant retroviruses in 293T cells as
described above.
-32P]ATP (3,000 Ci/mmol;
PerkinElmer Life Sciences) was added to the washed
immunoprecipitates and incubated for 15 min at 30 °C. Reactions were
quenched by adding SDS-polyacrylamide gel electrophoresis sample buffer
and heating to 95 °C for 5 min. Proteins were resolved by
SDS-polyacrylamide gel electrophoresis and transferred to
polyvinylidene difluoride, and levels of the immunoprecipitated kinases
were visualized by immunoblotting with monoclonal antibodies to Hck or
Fyn (Transduction Laboratories). Radiolabeled p50 was visualized and
quantitated by storage phosphor technology (Molecular Dynamics PhosphorImager).
-32P]dATP (10mCi/ml;
Amersham Pharmacia Biotech), and 2 µl of the Klenow fragment of DNA
polymerase (Life Technologies, Inc., 3.7 units/µl). The mixture was
incubated at room temperature for 30 min, and the unincorporated
nucleotides were removed using a G-25 Sephadex spin column (Millipore).
DNA binding reactions contained 40,000 cpm of labeled probe in a final
volume of 20 µl. Reactions were incubated at 30 °C for 30 min and
run on 5% polyacrylamide gels in 0.25× Tris borate-EDTA buffer. Gels
were fixed with 10% acetic acid, 10% methanol, rinsed with water,
dried, and exposed to a storage phosphor screen. Images were visualized
using a Molecular Dynamics PhosphorImager.
-32P]ATP (6000Ci/mmol, PerkinElmer Life Sciences).
After removal of free ATP, 1 ng of labeled probe was incubated with 2 µg of nuclear proteins and 2 µg of poly(dI·dC) in binding buffer
(50 mM KCl, 4% glycerol, 20 mM HEPES, pH 7.9, 1 mM dithiothreitol, 5 mM MgCl2, 1 mM spermidine, 0.85 mM EDTA, and 0.17% Nonidet
P-40) for 15 min. Bound proteins were separated on a 5% polyacrylamide gel in 0.5× Tris-borate EDTA and visualized by autoradiography.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (17K):
[in a new window]
Fig. 1.
Expression of HIV-1 Nef promotes
cytokine-independent outgrowth of the human
GM-CSF-dependent myeloid leukemia cell line, TF-1.
A, stable expression of Nef and Nef mutants in TF-1 cells.
TF-1 cells were infected with recombinant retroviruses carrying
wild-type Nef (WT), Nef mutants with alanine substitutions
for prolines 72 and 75 in the SH3-binding motif (PA), or
with an alanine substitution for glycine 2 in the myristylation signal
sequence (GA). Control cells (Con) were infected
with an empty retrovirus carrying only the neo selection
marker. Cells were selected with G418 in the presence of GM-CSF, and
Nef protein expression was verified by immunoblotting. Nef expression
was also verified in TF-1 cells that emerged after infection with the
wild-type Nef retrovirus and withdrawal of GM-CSF (far right
lane). B, Nef promotes GM-CSF-independent outgrowth of
TF-1 cells. Triplicate cultures of TF-1 cells expressing wild-type Nef
( ), Nef-PA (
), Nef-GA (
), or control cells (
) were washed
and replated at 1.25 × 105 cells/ml in the absence of
GM-CSF. Viable cells were counted with a hemocytometer every other day
for 10 days. Results shown are the mean cell counts ± S.E.
View larger version (51K):
[in a new window]
Fig. 2.
HIV-1 Nef induces cytokine-independent colony
formation in TF-1 cells. A, human TF-1 cells were
infected with recombinant retroviruses carrying wild-type Nef
(WT) or the Nef SH3 binding (PA) or
myristylation-defective (GA) mutants as described in the
legend to Fig. 1. TF-1 cells were also infected with the parent
retrovirus carrying only the neo selection marker as a
negative control (Con). Cells were selected with G418 in the
presence of GM-CSF and plated in soft agar in the presence (+) or
absence ( ) of GM-CSF. After incubation for 14 days, colonies were
photographed under the microscope. This experiment was repeated in
triplicate and revealed that nearly the same numbers of colonies were
obtained with TF-1 cells expressing Nef in the presence or absence of
GM-CSF (data not shown). B, immunoblot analysis of Nef
expression of the TF-1 cultures used in A. Whole cell
protein extracts were prepared from TF-1 cultures maintained in the
presence of GM-CSF (+ GM, right) or 24 h
after GM-CSF withdrawal (
GM, left). The
arrow indicates the position of Nef on the blot.
-32P]ATP and a glutathione
S-transferase-Sam 68 fusion protein (p50) as substrate. As
shown in Fig. 3, TF-1 cells expressing
Nef showed a greater than 3-fold increase in endogenous Hck kinase
activity relative to control cells. These results suggest that
activation of endogenous Hck may contribute to the survival effects of
Nef in TF-1 cells. Similar levels of Hck activation have been observed after stimulation of other myeloid leukemia cell lines with IL-3 (39)
or cross-linking of cell-surface Fc receptors (43). In contrast to Hck,
Fyn tyrosine kinase activity was not increased by the presence of Nef
in TF-1 cells (data not shown). This result is consistent with the
observation that the Fyn SH3 domain binds to Nef in vitro
with low affinity (11, 44).
View larger version (40K):
[in a new window]
Fig. 3.
Nef activates endogenous Hck in TF-1
cells. Endogenous Hck proteins were immunoprecipitated from
protein extracts of TF-1 cells stably expressing wild-type Nef
(WT), mutant forms of Nef lacking the SH3 binding function
(PA), or the myristylation signal sequence (GA)
and from vector control cells (Con) and incubated in
vitro with [ -32P]ATP and a glutathione
S-transferase-Sam 68 fusion protein of 50 kDa (p50) as
substrate. After incubation, phosphorylated p50 was resolved by
SDS-polyacrylamide gel electrophoresis and visualized by quantitative
storage phosphorimaging (upper panel). The relative extent
of p50 phosphorylation is shown in the bar graph
(middle). The levels of the p59 and p61 forms of Hck present
in each immunoprecipitate were determined by immunoblotting
(bottom). Phosphoamino acid analysis verified the
phosphorylation of p50 on tyrosine residues (data not shown).
View larger version (40K):
[in a new window]
Fig. 4.
Activation of endogenous Stat3 DNA binding
activity by Nef in TF-1 cells. Nuclear extracts were prepared from
TF-1 cells expressing wild-type Nef (WT), mutant forms of
Nef lacking the SH3 binding function (PA), or the
myristylation signal sequence (GA) or from vector control
cells (Con) as described under "Experimental Procedures..
Extracts were tested for the presence of activated Stat3 by gel-shift
analysis with an SIE probe. To control for the specificity of DNA
binding, assays were performed in the presence (+) or absence ( ) of a
100-fold molar excess of unlabeled SIE oligonucleotide. As a positive
control, gel-shift assays were conducted on extracts from uninfected
TF-1 cells treated with (+) or without (
) GM-CSF. The positions of
the shifted Stat3·SIE complex and the free probe (FP) are
indicated by the arrows. Control immunoblots revealed
equivalent levels of Stat3 in each of the cellular extracts (data not
shown).
View larger version (28K):
[in a new window]
Fig. 5.
Activation of Stat3 by Hck in 293T
cells. Human 293T cells were transfected with Stat3, wild-type Hck
(WT), kinase-inactive Hck (KE), or a Hck mutant
with a Phe substitution for the conserved tail Tyr residue
(YF) either alone or in the combinations shown.
A, gel-shift assay. Cytosolic extracts were prepared and
tested for the presence of active Stat3 using a gel-shift assay and an
SIE probe. Extracts from cells transfected with the empty vector were
included as an additional negative control (Con). To control
for the specificity of DNA binding, assays were performed in the
presence (+) or absence ( ) of a 100-fold molar excess of unlabeled
SIE probe. The positions of the shifted Stat3·SIE complex and the
free probe (FP) are indicated by the arrows.
B, Stat3 tyrosine phosphorylation. Transfected 293T cell
lysates were immunoblotted with antibodies to phosphotyrosine
(P-Tyr, top), Stat3 (middle), or Hck
(bottom). The arrows indicate the positions of
Stat3 and Hck.
View larger version (17K):
[in a new window]
Fig. 6.
Suppression of Nef-induced TF-1 cell colony
formation by dominant-negative mutants of Stat3. TF-1 cells were
infected with recombinant retroviruses carrying Stat3 mutants lacking
either the tyrosine phosphorylation site (Stat3 YF) or
glutamic acid residues essential for DNA binding (Stat3
EVA). As a control, cells were infected with a virus carrying the
hygromycin selection marker alone (Vector). Twenty-four
hours later, the cells were superinfected with the Nef retrovirus
(linked to the neo selection marker) and selected with
hygromycin and G418 in the presence of GM-CSF. Drug-resistant cells
were plated in soft agar in the presence of hygromycin and G418 and in
the absence of GM-CSF. Macroscopic colonies were stained and counted
after 14 days. The experiment was plated in triplicate, and the average
number of colonies is shown ±S.E.
View larger version (38K):
[in a new window]
Fig. 7.
Nef activates endogenous Stat3 in human
macrophages. Primary cultures of macrophages were infected with
recombinant adenovirus vectors carrying Nef (Ad-Nef) or
green fluorescent protein (Ad-GFP) cDNAs. Mock-infected
cells were also included as an additional negative control.
A, nuclear extracts were prepared and tested for the
presence of activated Stat3 by gel-shift analysis with wild-type or
mutant (m) SIE probes. B, to control for
specificity in the gel-shifted complexes, duplicate assays were
conducted in the presence (+) of an excess of unlabeled wild-type or
mutant SIE probes. The arrows indicate the presence of the
gel-shifted complexes.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Michael Widmer, Immunex Corp., for the generous gift of recombinant human GM-CSF, Dr. James A. Hoxie, University of Pennsylvania, for the anti-Nef antibody, and Drs. Richard Jove and Tammy Bowman at the Moffitt Cancer Center, University of South Florida for the Stat3/EVA dominant-negative mutant.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants CA81398 (to T. E. S.) and RR11589 (to M. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Dept. of Biochemistry and Molecular Genetics,
University of Virginia Health Sciences Center, Rm. 6222 Jordan Hall,
1300 Jefferson Park Ave., Charlottesville, VA 22908.
§ Present address: Eppley Institute for Research in Cancer, 986805 Nebraska Medical Center, Omaha, NE 68198-6805.
To whom correspondence should be addressed: Dept. of Molecular
Genetics and Biochemistry, University of Pittsburgh School of Medicine,
E1240 Biomedical Science Tower, Pittsburgh, PA 15261. Tel.:
412-648-9495; Fax: 412-624-1401; tsmithga{at}pitt.edu.
Published, JBC Papers in Press, April 27, 2001, DOI 10.1074/jbc.M103244200
2 B. Brichacek and M. Stevenson, unpublished observation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: HIV, human immunodeficiency virus; GM-CSF, granulocyte-macrophage colony-stimulating factor; SIV, simian immunodeficiency virus; GFP, green fluorescent protein; IL, interleukin; SIE, sis-inducible element.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Herna, R. G., and Saksela, K. (2000) Front Biosci. 5, 268-283 |
2. | Stevenson, M. (1996) Nat. Struct. Biol. 3, 303-306[Medline] [Order article via Infotrieve] |
3. | Kestler, H., Ringler, D. J., Mori, K., Panicali, D. L., Sehgal, P. K., Daniel, M. D., and Desrosiers, R. C. (1991) Cell 65, 651-662[Medline] [Order article via Infotrieve] |
4. | Deacon, N. J., Tsykin, A., Solomon, A., Smith, K., Ludford-Menting, M., Hooker, D. J., McPhee, D. A., Greenway, A. L., Ellett, A., Chatfield, C., Lawson, V. A., Crowe, S., Maerz, A., Sonza, S., Learmont, J., Sullivan, J. S., Cunningham, A., Dwyer, D., Dowton, D., and Mills, J. (1995) Science 270, 988-991[Abstract] |
5. |
Kirchhoff, F.,
Greenough, T. C.,
Brettler, D. B.,
Sullivan, J. L.,
and Desrosiers, R. C.
(1995)
N. Engl. J. Med.
332,
228-232 |
6. | Miller, M. D., Warmerdam, M. T., Gaston, I., Greene, W. C., and Feinberg, M. B. (1994) J. Exp. Med. 179, 101-113[Abstract] |
7. | Spina, C., Kwoh, T. J., Chowers, M. Y., Guatelli, J. C., and Richman, D. D. (1994) J. Exp. Med. 179, 115-123[Abstract] |
8. | Hanna, Z., Kay, D. G., Rebai, N., Guimond, A., Jothy, S., and Jolicoeur, P. (1998) Cell 95, 163-175[Medline] [Order article via Infotrieve] |
9. | Resh, M. D. (1994) Cell 76, 411-413[Medline] [Order article via Infotrieve] |
10. | Saksela, K., Cheng, G., and Baltimore, D. (1995) EMBO J. 14, 484-491[Abstract] |
11. | Lee, C.-H., Leung, B., Lemmon, M. A., Zheng, J., Cowburn, D., Kuriyan, J., and Saksela, K. (1995) EMBO J. 14, 5006-5015[Abstract] |
12. | Grzesiek, S., Bax, A., Clore, G. M., Gronenborn, A. M., Hu, J.-S., Kaufman, J., Palmer, I., Stahl, S. J., and Wingfield, P. T. (1996) Nat. Struct. Biol. 3, 340-345[Medline] [Order article via Infotrieve] |
13. | Lee, C.-H., Saksela, K., Mirza, U. A., Chait, B. T., and Kuriyan, J. (1996) Cell 85, 931-942[Medline] [Order article via Infotrieve] |
14. | Shugars, D. C., Smith, M. S., Glueck, D. H., Nantermet, P. V., Seillier-Moiseiwitsch, F., and Swanstrom, R. (1993) J. Virol. 67, 4639-4650[Abstract] |
15. |
Briggs, S. D.,
Sharkey, M.,
Stevenson, M.,
and Smithgall, T. E.
(1997)
J. Biol. Chem.
272,
17899-17902 |
16. |
Briggs, S. D.,
and Smithgall, T. E.
(1999)
J. Biol. Chem.
274,
26579-26583 |
17. | Briggs, S. D., Lerner, E. C., and Smithgall, T. E. (2000) Biochemistry 39, 489-495[CrossRef][Medline] [Order article via Infotrieve] |
18. | Kitamura, T., Tange, T., Terasawa, T., Chiba, S., Kuwaki, T., Miyagawa, K., Piao, Y.-F., Miyazono, K., Urabe, A., and Takaku, F. (1989) J. Cell. Physiol. 140, 323-334[Medline] [Order article via Infotrieve] |
19. |
Pear, W. S.,
Nolan, G. P.,
Scott, M. L.,
and Baltimore, D.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
8392-8396 |
20. | Muller, A. J., Young, J. C., Pendergast, A. M., Pondel, M., Landau, R. N., Littman, D. R., and Witte, O. N. (1991) Mol. Cell. Biol. 11, 1785-1792[Medline] [Order article via Infotrieve] |
21. |
Li, J.,
and Smithgall, T. E.
(1998)
J. Biol. Chem.
273,
13828-13834 |
22. |
Briggs, S. D.,
Bryant, S. S.,
Jove, R.,
Sanderson, S. D.,
and Smithgall, T. E.
(1995)
J. Biol. Chem.
270,
14718-14724 |
23. |
Nelson, K.,
Rogers, J. A.,
Bowman, T. L.,
Jove, R.,
and Smithgall, T. E.
(1998)
J. Biol. Chem.
273,
7072-7077 |
24. | Bahnson, A. B., Dunigan, J. T., Baysal, B. E., Mohney, T., Atchison, R. W., Nimgaonkar, M. T., Ball, E. D., and Barranger, J. A. (1995) J. Virol. Methods 54, 131-143[CrossRef][Medline] [Order article via Infotrieve] |
25. | Yu, C.-L., Meyer, D. J., Campbell, G. S., Larner, A. C., Carter-Su, C., Schwartz, J., and Jove, R. (1995) Science 269, 81-83[Medline] [Order article via Infotrieve] |
26. |
Rogers, J. A.,
Read, R. D.,
Li, J.,
Peters, K. L.,
and Smithgall, T. E.
(1996)
J. Biol. Chem.
271,
17519-17525 |
27. |
Kalter, D. C.,
Nakamura, M.,
Turpin, J. A.,
Baca, L. M.,
Hoover, D. L.,
Dieffenbach, C.,
Ralph, P.,
Gendelman, H. E.,
and Meltzer, M. S.
(1991)
J. Immunol.
146,
298-306 |
28. | McGrory, W. J., Bautista, D. S., and Graham, F. L. (1988) Virology 163, 614-617[Medline] [Order article via Infotrieve] |
29. | Hitt, M., Bett, A. J., Addison, C. L., Prevec, L., and Graham, S. L. (1995) Methods Mol. Genet. 7, 13-30 |
30. | Ziegler, S. F., Marth, J. D., Lewis, D. B., and Perlmutter, R. M. (1987) Mol. Cell. Biol. 7, 2276-2285[Medline] [Order article via Infotrieve] |
31. | Quintrell, N., Lebo, R., Varmus, H., Bishop, J. M., Pettenati, M. J., Le Beau, M. M., Diaz, M. O., and Rowley, J. D. (1987) Mol. Cell. Biol. 7, 2267-2275[Medline] [Order article via Infotrieve] |
32. | English, B. K., Ihle, J. N., Myracle, A., and Yi, T. (1993) J. Exp. Med. 178, 1017-1022[Abstract] |
33. | Ziegler, S. F., Wilson, C. B., and Perlmutter, R. M. (1988) J. Exp. Med. 168, 1801-1810[Abstract] |
34. | Boulet, I., Ralph, S., Stanley, E., Lock, P., Dunn, A. R., Green, S. P., and Phillips, W. A. (1992) Oncogene 7, 703-710[Medline] [Order article via Infotrieve] |
35. |
Balter, M.
(1996)
Science
274,
1464-1465 |
36. | Hirsch, V. M., Sharkey, M. E., Brown, C. R., Brichacek, B., Goldstein, S., Wakefield, J., Byrum, R., Elkins, W. R., Hahn, B. H., Lifson, J. D., and Stevenson, M. (1998) Nat. Med. 4, 1401-1408[CrossRef][Medline] [Order article via Infotrieve] |
37. | Torigoe, T., O'Connor, R., Santoli, D., and Reed, J. C. (1992) Blood 80, 617-624[Abstract] |
38. |
Linnekin, D.,
Howard, O. M. Z.,
Park, L.,
Farrar, W.,
Ferris, D.,
and Longo, D. L.
(1994)
Blood
84,
94-103 |
39. | Anderson, S. M., and Jorgensen, B. (1995) J. Immunol. 155, 1660-1670[Abstract] |
40. |
Burton, E. A.,
Hunter, S.,
Wu, S. C.,
and Anderson, S. M.
(1997)
J. Biol. Chem.
272,
16189-16195 |
41. |
Tilbrook, P. A.,
Ingley, E.,
Williams, J. H.,
Hibbs, M. L.,
and Klinken, S. P.
(1997)
EMBO J.
16,
1610-1619 |
42. | 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] |
43. |
Durden, D. L.,
Kim, H. M.,
Calore, B.,
and Liu, Y.
(1995)
J. Immunol.
154,
4039-4047 |
44. | Arold, S., O'Brien, R., Franken, P., Strub, M. P., Hoh, F., Dumas, C., and Ladbury, J. E. (1998) Biochemistry 37, 14683-14691[CrossRef][Medline] [Order article via Infotrieve] |
45. | Lock, P., Ralph, S., Stanley, E., Boulet, I., Ramsay, R., and Dunn, A. R. (1991) Mol. Cell. Biol. 11, 4363-4370[Medline] [Order article via Infotrieve] |
46. | Robbins, S. M., Quintrell, N. A., and Bishop, J. M. (1995) Mol. Cell. Biol. 15, 3507-3515[Abstract] |
47. |
Darnell, J. E., Jr.
(1997)
Science
277,
1630-1635 |
48. | Ihle, J. N. (1996) Cell 84, 331-334[Medline] [Order article via Infotrieve] |
49. |
Rajotte, D.,
Sadowski, H. B.,
Haman, A.,
Gopalbhai, K.,
Meloche, S.,
Liu, L.,
Krystal, G.,
and Hoang, T.
(1996)
Blood
88,
2906-2916 |
50. | Fukada, T., Hibi, M., Yamanaka, Y., Takahashi-Tezuka, M., Fujitani, Y., Yamaguchi, T., Nakajima, K., and Hirano, T. (1996) Immunity 5, 449-460[Medline] [Order article via Infotrieve] |
51. | Nakajima, K., Yamanaka, Y., Nakae, K., Kojima, H., Ichiba, M., Kiuchi, N., Kitaoka, T., Fukada, T., Hibi, M., and Hirano, T. (1996) EMBO J. 15, 3651-3658[Abstract] |
52. |
Minami, M.,
Inoue, M.,
Wei, S.,
Takeda, K.,
Matsumoto, M.,
Kishimoto, T.,
and Akira, S.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
3963-3966 |
53. |
Turkson, J.,
Bowman, T. L.,
Garcia, R.,
Caldenhoven, E.,
de Groot, R. P.,
and Jove, R.
(1998)
Mol. Cell. Biol.
18,
2545-2552 |
54. |
Bromberg, J. F.,
Horvath, C. M.,
Besser, D.,
Lathem, W. W.,
and Darnell, J. E., Jr.
(1998)
Mol. Cell. Biol.
18,
2553-2558 |
55. | Alexander, L., Du, Z., Rosenzweig, M., Jung, J. U., and Desrosiers, R. C. (1997) J. Virol. 71, 6094-6099[Abstract] |
56. | Wei, S., Liu, J. H., Epling-Burnette, P. K., Gamero, A. M., Ussery, D., Pearson, E. W., Elkabani, M. E., Diaz, J. I., and Djeu, J. Y. (1996) J. Immunol. 157, 5155-5162[Abstract] |
57. | Wen, Z., Zhong, Z., and Darnell, J. E., Jr. (1995) Cell 82, 241-250[Medline] [Order article via Infotrieve] |
58. | 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] |
59. | Sawai, E. T., Baur, A., Struble, H., Peterlin, B. M., Levy, J. A., and Cheng-Mayer, C. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 1539-1543[Abstract] |
60. | Sawai, E. T., Khan, I. H., Montbriand, P. M., Peterlin, B. M., Cheng-Mayer, C., and Luciw, P. A. (1996) Curr. Biol. 6, 1519-1527[Medline] [Order article via Infotrieve] |
61. |
Turkson, J.,
Bowman, T.,
Adnane, J.,
Zhang, Y.,
Djeu, J. Y.,
Sekharam, M.,
Frank, D. A.,
Holzman, L. B.,
Wu, J.,
Sebti, S.,
and Jove, R.
(1999)
Mol. Cell. Biol.
19,
7519-7528 |
62. | Ernst, M., Gearing, D. P., and Dunn, A. R. (1994) EMBO J. 13, 1574-1584[Abstract] |
63. |
Ernst, M.,
Novak, U.,
Nicholson, S. E.,
Layton, J. E.,
and Dunn, A. R.
(1999)
J. Biol. Chem.
274,
9729-9737 |
64. | Chaturvedi, P., Reddy, M. V. R., and Reddy, E. P. (1998) Oncogene 16, 1749-1758[CrossRef][Medline] [Order article via Infotrieve] |
65. | Catlett-Falcone, R., Landowski, T. H., Oshiro, M. M., Turkson, J., Levitzki, A., Savino, R., Ciliberto, G., Moscinski, L., Fernández-Luna, J. L., Nuñez, G., Dalton, W. S., and Jove, R. (1999) Immunity 10, 105-115[Medline] [Order article via Infotrieve] |