(Received for publication, July 5, 1995; and in revised form, November 8, 1995)
From the
The nef gene is unique to the primate lentiviruses and
encodes a cytoplasmic membrane-associated protein that affects T-cell
signaling and is essential for both maintenance of a high virus load in vivo and for disease progression. Here we investigated the
perturbation of cell signaling by Nef in T-cells and found
that Nef interacts with the T-cell restricted Lck tyrosine kinase both in vitro and in vivo. The molecular basis for this
interaction was analyzed. We show that cell-derived Nef is precipitated
in a synergistic manner by the recombinant Src homology 2 (SH2) and SH3
domains from Lck. A functional proline-rich motif and the tyrosine
phosphorylation of Nef were evidenced as likely participants in this
interaction. The precipitation of Nef by the Lck recombinant proteins
was specific, since neither Fyn, Csk, p85 phosphatidylinositol 3-kinase
nor phospholipase C SH2 domains coprecipitated Nef from T-cells.
Finally, depressed Lck kinase activity resulted from the presence of
Nef, both in vitro and in intact cells, and nef expression resulted in impairment of both proximal and distal
Lck-mediated signaling events. These results provide a molecular basis
for the Nef-induced T-cell signaling defect and its role in AIDS
pathogenesis.
HIV infection is associated with a severe CD4+ T-cell
depletion. This quantitative defect is preceded by immune qualitative
dysfunctions resulting in a profound disturbance of the complex network
of cytokines that maintains immune homeostasis, in particular within
the Th1-produced cytokines (IL-2, ()interferon-
). Three
HIV-1-encoded proteins exhibit immunosuppressive effects that may, at
least partly, account for these T-cell dysfunctions. First, soluble
gp120 envelope gene product added to T-cells affects their
activation(1) . Second, immunosuppressive effects of Tat have
also been reported (2) but may not play an important role in vivo, since these in vitro immunosuppressive
functions of Tat are not observed in the presence of accessory cells
and Tat did not affect recall antigen-mediated T-cell
proliferation(3) . Finally, the altered T-cell activation and
development associated with nef transgene expression in mice (4, 5, 6) make this viral gene a candidate
for the severe immunodeficiency induced by HIV-1 infection.
nef is an early and abundantly (7) transcribed viral gene
conserved in human (HIV-1 and HIV-2) and in simian (SIV)
immunodeficiency viruses. nef encodes a 25-32-kDa
myristoylated and membrane-associated protein in infected
cells(8) . Although its function remains to be defined, in
vivo experiments indicate that nef is required for both
viral replication and full development of the pathogenesis associated
with SIV infection of rhesus monkeys (9) or HIV-1 infection in
SCID-Hu mouse (10) . In contrast to these in vivo data, nef was initially described as a negative regulator
of the viral replication (11, 12, 13, 14, 15) , but
this observation remains controversial as other reports demonstrated a
lack of effect (16, 17, 18) or a moderate
positive effect (19, 20) on rates of viral
transcription or replication. More recently, nef was shown to
contribute to the induction of viral replication in primary quiescent
T-cells, its positive role being readily discernible in the primary
cell setting of virus induction through T-cell
activation(21, 22) . A known consequence of nef expression is CD4 membrane
down-regulation(23, 24, 25, 26) ,
which has been proposed to prevent cell superinfection(27) .
This down-regulation occurs at the post-translational level (23, 25) and might involve either a critical dileucine
motif (28) or the Src family protein-tyrosine kinase Lck
binding site in CD4(29) . However, the precise mechanism by
which Nef down-modulates CD4 remains unclear. Recent studies provide
further evidence that HIV-1 nef gene function is closely
related to T-cell signaling pathways as demonstrated by its ability to
affect T-cell activation in nef-transgenic
mice(4, 5, 6) , to inhibit growth of
CD4+ T lymphocytes(30) , and to down-regulate both IL-2
induction (31) ()and activation of NF-
B and
AP-1 (32, 33, 34) transcription factors in
human T-cells. Nef appears to exert different effects on T-cell
signaling, depending on its intracellular localization(29) ,
and has been reported to associate with various cellular proteins from
T-cell lysates(35) , notably serine/threonine protein kinases (36) and
-COP(37) , an essential component of the
molecular machinery of the membrane trafficking. These molecular
interactions of Nef with different families of cellular proteins
indicate that Nef contains at least one domain that can mediate
protein-protein interactions. Indeed, a proline-rich motif has been
identified within Nef (38) that allows binding to the SH3
domain from the Src family protein-tyrosine kinases Hck and Lyn and is
required for enhanced growth of nef+ viruses in
monocytes(39) . However, as the expression of these proteins is
mainly restricted to monocytes, the Nef-Hck interaction does not
account for the Nef-induced T-cell signaling defects.
The Src family of protein-tyrosine kinases comprises nine identified members (Src, Lck, Fyn, Yes, Blk, Fgr, Lyn, Hck, and Yrk), defined by the presence of the catalytic domain, Src homology domain 1 (SH1), a specific phosphotyrosine residue binding domain (SH2), a proline-rich binding region (SH3), and a myristoylated membrane-targeting domain (SH4)(40, 41, 42) . Importantly, the T-cell-specific Lck product binds to the CD4 cytoplasmic tail(43, 44) , hence regulating its cell surface expression by a post-translational mechanism(45) , and is also involved in T-cell activation(46, 47, 48) , IL-2 induction(49) , thymic development (50) , and HIV-1 expression(51) .
Here, we describe the physical and functional interaction of HIV-1 Nef with Lck in human T-cells and show that this interaction is associated with impaired Lck kinase activity as well as with defects in both proximal and distal signaling events mediated by Lck. Our results provide a molecular basis by which Nef affects T-cell functions and also possibly CD4 cell surface expression and viral growth.
GST-Lck and -Csk fusion proteins were a
kind gift from P. Jullien and C. Bougéret (ICGM,
Paris), and p50NF-B
was kindly provided by P. Lecine
(U119 INSERM, Marseille). The GST-Nef and Nef
production
was described previously (35, 57) as well as that of
the GST-Lck SH2, GST-Lck SH3, GST-Lck SH2+SH3, GST-Fyn SH2,
GST-phospholipase C
SH2
, and
phosphatidylinositol 3-OH-kinase
SH2
(73, 74, 75) .
Nef peptides produced by Neosystem S.A. (Strasbourg, France) and distributed by ANRS were coupled to activated agarose beads (Steragene) at a ratio of 1 mg of peptide/ml of activated beads according to instructions provided by the manufacturer. PMA and Ionomycin were purchased from Sigma. Peptides Glu-Pro-Gln-Tyr(P)-Glu-Glu-Ile-Pro-Ile and Glu-Pro-Gln-Tyr-Glu-Glu-Ile-Pro-Ile were kindly provided by O. Acuto (Institut Pasteur, Paris, France).
For immunoblotting, fractionated proteins were transferred to polyvinylidene difluoride membranes (Millipore), and filters were blocked for 2 h at room temperature in 5% bovine serum albumin (Sigma) in phosphate-buffered saline containing 0.01% Tween 20 detergent. Filters were successively incubated for 1 h at room temperature with appropriate primary and secondary antibodies. Each incubation period was followed by three washes in phosphate-buffered saline with 0.01% Tween 20. Proteins were finally detected by enhanced chemiluminescence following instructions of the manufacturer (Amersham Corp.).
For
kinase assays, precipitates were further washed in kinase buffer (50
mM Tris-Cl, pH 7.4; 10 mM MnCl) and
resuspended in 25 µl of this buffer. For autophosphorylation
analysis, the reaction was processed in the presence of 1 µCi of
[
-
P]ATP (5000 Ci/mmol, ICN) for 15 min at
room temperature and stopped by the addition of SDS-PAGE reducing
sample buffer. To analyze for the phosphorylation of exogenous
substrates, experiments were identically performed except that 5 µg
of acid-denatured enolase and 3 µM of unlabeled ATP were
added. Phosphorylated proteins were then analyzed by SDS-PAGE and
autoradiography. Integrated signal intensity of phosphorylated proteins
was determined by use of the BioImage system (Millipore Corp.).
For determination of CAT activity, cells were harvested, washed in phosphate-buffered saline, and resuspended in lysis buffer (0, 25 M Tris-Cl, pH 7.8), and proteins were extracted by successive freezing/thawing cycles. After centrifugation of the lysates, supernatants were collected, and the protein concentration was determined by the Bradford method (Bio-Rad). CAT enzyme activity present in the sample was then determined as described previously(78) , using an equivalent amount of total protein. Reaction products were analyzed by thin-layer chromatography followed by autoradiography and determination of integrated signal intensity by use of the BioImage system (Millipore). CAT activity was expressed as the ratio of radioactivity present in the acetylated forms of chloramphenicol to the sum of both acetylated and unacetylated forms.
Figure 1:
Specific precipitation of Nef by
immobilized GST-Lck-SH2+SH3, GST-Lck-SH3, and GST-Lck-SH2
recombinant proteins. A, Jurkat and JBru.2 T-cell lysates
(10 cell equivalent in 500 µl) were precipitated with
immobilized GST-Lck SH2+SH3 fusion protein (10 µg of
recombinant protein recoupled to glutathione-agarose beads).
Precipitates were fractionated on a 12% SDS-PAGE, followed by
immunoblotting with the Nef monoclonal antibody and enhanced
chemiluminescence. Cells were left uninduced or induced by PMA (15
ng/ml) and ionomycin (0.5 µg/ml) to increase nef expression, and whole cell lysates from Jurkat (lane 1)
and JBru.2 (lane 2) induced cells were included as control. Lane 3, induced Jurkat cells; Lanes 4 and 5,
respectively, induced and uninduced JBru.2 cells. B and C, as A with the exception that lysates from induced
cells were precipitated with the indicated GST-Lck (B) or
GST-Lck, GST-Fyn, GST-Csk, GST-p85, or GST-phospholipase C
(C) recombinant proteins. WCL, whole cell lysate; AU, arbitrary units.
Figure 2:
HIV-1 Nef is tyrosine-phosphorylated. A, JBru.2 cell lysates were precipitated by the GST-Lck SH2
recombinant protein as described in Fig. 1, except that 5
µM unphosphorylated (YEEI, lane 3) or
tyrosine-phosphorylated (pYEEI, lane 4) EPQYEEIPI peptide was
added during the precipitation step. Precipitates were subsequently
fractionated by SDS-PAGE and analyzed by Nef immunoblotting. Whole cell
lysates from Jurkat and JBru.2 cells were added as control (lanes 1 and 2, respectively). B, lysates from induced
Jurkat(-) and JBru.2 (+) cells were precipitated by use of
GST-Lck SH2+SH3 recombinant protein, separated by SDS-PAGE and
analyzed by Nef immunoblotting (left panel) followed by
phosphotyrosine immunoblotting (right panel). Whole cell
lysates were added as control where indicated. C, cells were
either left unstimulated(-) or stimulated (+) in the
presence of PMA (20 ng/ml) for 15 h to increase nef expression. Cell lysates (4 10
cells
equivalent) were immunoprecipitated with a Nef polyclonal sheep
antiserum, and analyzed by anti-phosphotyrosine immunoblotting (left panel). After stripping, the same membrane was also
probed with a Nef monoclonal antibody (right panel). Lane
1, stimulated Jurkat cells; lanes 2 and 3:
unstimulated and stimulated JBru.2 cells, respectively. PTYR,
anti-phosphotyrosine; Ig, light chain
immunoglobulins.
The binding of the GST-Lck SH3 fusion protein to Nef suggested that Nef might contain a proline-rich domain that interacted with the Lck SH3 domain. Fig. 3A identifies a proline-rich domain that matches the consensus proline-rich motif defined by Yu et al.(71) and present in the nefBru/Laï primary sequence (residues 68-78) but also in various HIV-1, HIV-2, and SIV nef isolates. Interestingly, this motif corresponds to the prototypic class II proline-rich motif with the consensus sequence Pro-X-q-Pro-X-Arg, where X represents any amino acid residue and q represents a hydrophobic residue. To determine whether this motif of Nef mediated interaction with Lck SH3 domain, a peptide encompassing HIV-1 NefBru residues 66-80 was directly coupled to activated agarose beads and used to precipitate GST fusion proteins (Fig. 3B). This proline-rich domain of Nef precipitated the GST-Lck SH2+SH3 fusion protein as efficiently as glutathione-agarose beads (Fig. 3B, compare lanes 2 and 5). Similarly, GST-Lck SH3 fusion protein could be efficiently precipitated (Fig. 3B, lanes 3 and 6), whereas GST-Lck SH2 was not (Fig. 3B, lanes 1 and 4). This peptide accounts for most of Nef binding to GST-Lck SH3 recombinant protein in vitro, since peptides encompassing residues 34-71 or 137-168 of Nef were poorly or less efficient, respectively, as compared with peptide encompassing residues 66-100 (Fig. 3C). Nef interacted specifically with SH3 domains, since the GST recombinant protein was not precipitated (Fig. 3D) and also because a gradual affinity for Src-like derived SH3 domains, in particular for Lck, was observed (Fig. 3D).
Figure 3:
Nef contains a functional Lck SH3 binding
domain. A, sequence alignment of HIV-1
Bru/Laï(70) , HIV-1
consensus(38) , HIV-2 consensus(38) , and SIV consensus (38) reveals the presence of a consensus (71) proline-rich domain in Nef that corresponds to a class II
motif (-Pro-X-
-Pro-X-Arg). Position
numbering is indicated on the left of each sequence. The
single letter amino acid code is used, X represents
nonconserved residues,
represents hydrophobic residues, and P
represents residues that are likely to be proline. B, a Nef
peptide encompassing amino acid residues 66-80 from HIV-1
Bru/Laï Nef was immobilized on agarose beads
(1 mg/ml). 10 µg of peptide equivalent were then incubated for 2 h
at 4 °C in 500 µl of lysis buffer with 2 µg of soluble
GST-Lck SH2, GST-Lck SH2+SH3, and GST-Lck SH3 fusion proteins.
Precipitates were extensively washed and subsequently fractionated by
10% SDS-PAGE. Precipitated proteins were visualized by Coomassie Blue
staining. The various GST-recombinant proteins were also precipitated
by use of glutathione-agarose beads (GA-beads) as a loading control. C, peptides encompassing the indicated amino acid residues
from HIV-1 NefBru were immobilized on agarose beads and used to
precipitate soluble GST-Lck SH3 recombinant protein as described in B. Signal intensities were determined by use of the BioImage
system (Millipore), and results are presented as percentage of maximal
binding determined for the Nef peptide encompassing residues
66-80. D, as B and C except that
immobilized Nef peptide (amino acids 66-80) was used to
precipitate the indicated soluble GST recombinant proteins. Results,
determined as in C, are expressed as arbitrary units of
optical density.
Figure 4:
In vitro physical and functional
interaction of HIV-1 Nef with Lck. A, kinase-active
recombinant GST-Lck fusion protein (100 ng) was incubated in 1% Brij 96
buffer for 2 h at 4 °C with 1 µg of either Nef or
p50NF-
B
immobilized on Ni
-agarose
beads. After precipitation and extensive washing, kinase activity that
precipitated with immobilized recombinant proteins was determined by in vitro kinase assay in the presence of
[
-
P]ATP followed by SDS-PAGE fractionation
and autoradiography. The GST-Lck and GST recombinant proteins were
directly tested as controls (Lanes 1 and 4,
respectively). Lane 2, Nef
precipitates; lane
3, p50NF-
B
precipitates. B, as A, except that precipitates were analyzed by immunoblotting as
indicated. Immobilized Nef
beads were used to precipitate
different amounts of GST or GST-Lck recombinant proteins (lanes
1-5 ), and precipitates were separated by SDS-PAGE and
subsequently analyzed by Lck immunoblotting (upper left panel, lanes 1-5). Filters were then stripped and reprobed by
GST immunoblotting (lower left panel, lanes
1-5). Conversely, soluble Nef
or
p50NF-
B
recombinant proteins were incubated in Brij
96 buffer with the GST-Lck fusion protein and immunoprecipitated by use
of an Lck polyclonal antibody directed against the unique region of Lck (lanes 6-7). SDS-PAGE-fractionated proteins were probed
by Nef immunoblotting (upper right panel, lanes
6-7), and after stripping of the filter, by GST
immunoblotting (lower right panel, lanes 6-7).
GST-Lck recombinant protein was directly loaded as a control (Lane
1). Lanes 2 and 3, 0.2 and 1 µg of GST
protein, respectively; lanes 4 and 5, 0.2 and 1
µg GST-Lck fusion protein, respectively; lane 6,
p50NF-
B
recombinant protein; lane 7,
Nef
recombinant protein. Note that the Nef
protein migrates in SDS-PAGE with an apparent molecular mass of
33-34 kDa according to the addition of the six histidine residues
to the Nef primary sequences. C, GST-Lck or GST recombinant
proteins were incubated with Nef
or p50Rel
as
indicated and in identical conditions as described for A,
except that kinase activity present in each sample was directly
determined by in vitro kinase assay without
precipitation.
The GST-Lck kinase activity
precipitated by immobilized-Nef was very weak, suggesting
that Lck might be affected by Nef. To test this hypothesis, GST-Lck was
incubated with soluble Nef
and simultaneously assayed by in vitro kinase assay. A 9-fold reduction of recombinant Lck
kinase activity was found in the presence of Nef
relative
to the GST-Lck activity determined in the presence of
p50NF-
B
(Fig. 4C, compare lanes 3 and 5). This effect was dose-dependent (IC
= 585 ng), the range of specificity being comprised
between 0 and 1 µg of Nef recombinant protein, and was prevented by
the addition of a Nef monoclonal antibody (data not shown).
Interestingly, an Lck-independent phosphorylated band corresponding to
Nef
was also detected (Fig. 4C, lanes
4 and 5). This band may result from the previously
reported autophosphorylation activity of Nef(55) , although
conflicting results have been described about such
activity(79) . Alternatively, this band may be due to
phosphorylation of Nef by a cellular protein kinase of SF9 cells, which
binds to and copurifies with Nef.
Figure 5:
In vivo physical and functional
interaction of HIV-1 Nef with Lck. A and B, cells
(5.10 cells/ml) were incubated for 15 min at 4 °C with
magnetic beads coated with CD4 receptor-specific monoclonal antibodies.
Bound cells were sorted with a magnet and disrupted in Brij 96 lysis
buffer. Immunocomplexes were harvested by centrifugation, washed, and
analyzed by CD4 and Lck immunoblotting or by in vitro kinase
assay, as indicated. Cell surface CD4-associated Lck kinase activity
was evaluated by the phosphorylation of the exogenous substrate enolase (A) or Lck auto-phosphorylation (B). Lane 1,
JH6.2 total cell lysate; Lanes 2 and 3, JH6.2 and JBru.2
cells, respectively. Integrated intensity of each signals was
determined by use of BioImage system (Millipore). C, cells
were either left unstimulated or stimulated by PMA and ionomycin to
increase nef expression, and lysed in Brij 96 buffer. 5
10
cells equivalent were immunoprecipitated with
the p56 Lck polyclonal antibody, fractionated on a 11% SDS-PAGE, and
immunoblotted with the Nef monoclonal antibody. Lane 1,
stimulated JH6.2 cells; lanes 2 and 3, unstimulated
and stimulated JBru.2 cells, respectively; lane 4, JBru.2
whole cell lysates (10
cell equivalent). Ig,
immunoglobulins from anti-Lck polyclonal antibody; KA, kinase
assay.
The interaction of Nef with Lck was further investigated by immunoprecipitation of cell-derived Lck from JBru.2 cells and Nef immunoblotting (Fig. 5C). A 29-kDa polypeptide co-migrating with Nef from whole cell lysates was detected in immunoprecipitates from induced nef-expressing cells (Fig. 5C, lane 3) and was absent in control cells (lane 1) but also barely undetectable in uninduced JBru.2 cells (lane 2). Densitometric determination indicated that this signal represented at least 2% of Nef protein present in whole cell lysate (lane 4).
Figure 6: Binding of GST-Lck SH2 fusion protein to phosphotyrosine-containing proteins from nef-expressing T-cells. Whole cell lysates from uninduced and PMA + ionomycin-induced JH6.2 and JBru.2 cells were obtained as described in the legend top Fig. 1and then precipitated with the GST-Lck SH2 recombinant fusion protein. After extensive washing, bound tyrosine-phosphorylated proteins were fractionated by 12% SDS-PAGE and analyzed by anti-phosphotyrosine immunoblotting. Lanes 1 and 2, unstimulated cells; lanes 3 and 4, induced cells.
HIV-1 Nef interacts with various cellular proteins(35, 36, 37, 54) , hence indicating the presence, within its primary sequence, of domains that direct protein-protein interactions. Indeed, Nef contains a proline-rich region (38, 39) that corresponds to the minimal consensus motif required for interaction with SH3 domains. The importance of this domain was outlined by the recent finding that mutations of the proline residues within this motif abolish the ability of Nef to enhance viral growth in infected peripheral blood mononuclear cell cultures(39) . Also, the proline-rich motif of Nef was shown to interact with Hck (39) , an Src-like tyrosine kinase whose expression is restricted to monocytes. While cells from the monocytic lineage clearly represent an important reservoir for HIV-1 infection, CD4+ T lymphocytes also constitute major targets for this virus. The interaction of Nef with monocyte-restricted Src-like tyrosine kinases suggested the possible interaction of Nef, in T-cells, with another Src-like tyrosine kinase, and in particular with the T-cell-restricted Lck. During the preparation of this manuscript, Greenway et al.(54) reported that recombinant GST-Nef fusion protein could precipitate Lck and CD4 from Jurkat T-cells. Also, when co-expressed in insect Sf9 cells, Nef and CD4 produced by recombinant baculoviruses could be co-immunoprecipitated(57) . These observations thus raised the following questions. (i) Does Nef bind directly to Lck, and does this interaction occur in vivo? (ii) If so, what are the molecular bases for this interaction? (iii) What are the consequences of the binding of Nef to Lck?
In this report we demonstrate that cell-derived Nef co-precipitates with recombinant GST-Lck fusion proteins. Conversely, an immobilized peptide encompassing the proline-rich motif of Nef precipitates the GST-Lck SH3 fusion protein. We also present evidence for an interaction between full-length recombinant Nef and Lck proteins. Together, these observations argue for the direct binding of Nef to Lck in vitro. The Nef-Lck interaction also occurs in intact cells as demonstrated in nef-transfected cells both by the co-immunoprecipitation of Nef with Lck and the down-regulation of the CD4-associated Lck kinase activity.
Using recombinant GST fusion
proteins, we found that the Nef-Lck interaction involves both SH2 and
SH3 domains of Lck, in a synergistic manner. The proline-rich motif
identified within Nef was initially proposed to bind selectively Hck
and Lyn, in a filter binding assay, and the Nef-PXXP peptide
bound neither to Lck nor to Fyn SH3 recombinant proteins under these
experimental conditions(39) . Using a similar assay, we
confirmed these observations (data not shown). However, we observed the
precipitation of cell-derived Nef by the GST-Lck SH3 fusion protein,
and conversely, a peptide encompassing the proline-rich domain of Nef
allowed the precipitation of soluble GST-Lck SH3 recombinant protein.
The involvement of SH3 binding to Nef was further supported by the
severalfold increased precipitation of Nef by GST Lck SH2+SH3
recombinant protein as compared to Nef precipitation by either SH2 or
SH3 isolated domains. Thus, although the proline-rich motif of Nef
might display a higher affinity for Hck and Lyn SH3 domains in filter
binding assays(39) , it clearly cooperates with additional
domains within Nef to allow its binding to the Lck SH2-SH3 recombinant
protein. This cooperation presumably resulted from the previously
reported coordinated interplay between SH2 and SH3 domains of Src
family kinases(58, 59) . Indeed, occupancy of one
domain may influence accessibility of the other(58) .
Similarly, the binding of Tip, a Herpesvirus saimiri product that
associates with Lck (80) was recently shown to contain at least
two Lck-binding motifs(81) . One of these motifs is a
proline-rich domain similar to that of Nef and is required but not
sufficient for the binding of Tip to Lck(81) . SH2 domains are
implicated in mediating protein-protein interactions by binding to
tyrosine-phosphorylated proteins(60, 61) .
Interestingly, we observed that the specific precipitation of Nef by
the GST-Lck SH2 recombinant protein, that was prevented by the presence
of a tyrosine-phosphorylated specific peptide substrate of Lck SH2.
Together with the anti-phosphotyrosine immunoreactivity of Nef
immunoprecipitates, these results strongly argue for the in vivo phosphorylation of Nef on tyrosine residue(s) and for its
involvement in Lck SH2 binding. Densitometric determination evidenced
that the GST-Lck SH2 domain precipitated at least 2% of the Nef protein
present in whole cell lysates, hence indicating the proportion of in vivo tyrosine-phosphorylated Nef. Recently, nef alleles products from SIV viruses were similarly found to be
phosphorylated on tyrosine residue(s) when co-expressed with Src in
COS-1 cells(82) . The tyrosine residue(s) proposed to be
phosphorylated in SIV Nef are not present, however, in HIV-1,
suggesting that although the nef gene from these viruses might
have similar functions, different mechanisms have been selected in
order to reach it. Inspection of the HIV-1 NefBru primary sequence
identified seven tyrosine residues, among which at least five are
conserved features of different HIV-1 isolates, but also of HIV-2 and
SIV Nef proteins. The optimal binding sequence of Lck SH2 domains was
determined by use of a random library of tyrosine-phosphorylated
peptide and defined as pYEEI(60) . Such a consensus Lck-SH2
binding domain is not present in Nef. However, the possibility cannot
be excluded that Nef contains another phosphotyrosine-containing motif
that acts as such a substrate for Lck, as previously reported for c-Src
SH2 binding to platelet-derived growth factor receptor, or Lck binding
to ZAP-70 kinase(62) . Absence of significant precipitation of
Nef by phosphatidylinositol 3-OH-kinase p85, phospholipase C, and
even by Fyn SH2 domains, the latter belonging to the same tyrosine
kinase family as Lck, indicate the high specificity of this
Nef-phosphotyrosine for Lck. The kinase activity of the Src family
kinases is repressed when the carboxyl terminus tyrosine residue
(Tyr
in Lck) is
phosphorylated(40, 41, 42, 59) .
This phosphorylation creates a binding site for the SH2 domain and
results in intramolecular interactions that are thought to lock the
protein in an inactive conformation. Among the various conserved
tyrosine residues identified within Nef, residue Tyr
(YXPXP) shares an intriguing similarity with
the regulatory Tyr
motif from Lck
(YXPXP). Strikingly, the YXPXP
motif is uniquely shared by Lck, while Fyn and Src have a shorter motif
(YXP) also found in Nef (Tyr
). The respective
contribution of these various tyrosine residues in both Nef
phosphorylation and binding to Lck is currently investigated.
Triggering of the T-cell receptor results in the tyrosine phosphorylation of many cytoplasmic and membrane effectors that appear to play an important role in the transcriptional activation of IL-2. Tyrosine phosphorylation is an obligatory event for IL-2 production (63) , and various protein tyrosine kinases have been identified in this process, including Lck (for a review, see (64) ). Interestingly, the tyrosine phosphorylation of several proteins upon TcR triggering is selectively affected in cells expressing a CD8-Nef chimeric protein (29) . In particular, the tyrosine phosphorylation of p36, p48, and at least two additional proteins with molecular mass in the range of 70-80 KDa were reported to be specific targets of Nef. Identification of these cellular proteins is critical in the outstanding comprehension of Nef-mediated T-cell defects. Here, we identified several Nef-sensitive cellular tyrosine-phosphorylated proteins as substrates of Lck SH2 domain. The defective tyrosine phosphorylation pattern observed in the present report by use of the recombinant GST-Lck SH2 fusion protein is very similar to that identified in the CD8-Nef expressing cells. Together with the observation that the transcriptional induction of an IL-2 promoter reporter construct is specifically decreased 8-fold in nef transfected cells, these results suggest a role for the Nef-Lck interaction in perturbation of both tyrosine phosphorylation and IL-2 induction. The function of Lck in T-cell signaling implicates both its catalytic activity and binding to tyrosine-phosphorylated substrate through its SH2 domain(65, 66, 67) . In particular, the CD4-associated Lck is thought to play an important role upon the MHC-restricted recognition of antigenic peptide by the TcR(68) . Here, we found that Nef affects the CD4-associated Lck kinase activity purified from nef transfected cells by more than 50% and decreased the kinase activity of recombinant Lck in vitro 9-fold. As both intact SH2 and SH3 domains are required for Lck catalytic activity, we propose a mechanism by which the impaired Lck kinase activity induced by Nef might result from an inappropriate folding of the kinase in the Lck-Nef complex or from an active repression mediated by occupancy of both SH2 and SH3 domains by Nef. As a consequence, the tyrosine phosphorylation of Lck substrates would be affected (see Fig. 7for the proposed model).
Figure 7: Interaction of Nef and Lck. A general model is proposed that involves multiple consequences on cell functions and viral replication.
The multiple role played by Lck in T-cells suggests foreseeable consequences of the Nef-Lck interaction both on T-cell functions and viral replication. The turnover of CD4 membrane expression is highly dependent on Lck interaction(45) . It is tempting to speculate that the interaction of Nef with Lck might influence the rate of CD4 internalization. Indeed, the Lck binding domain within CD4 is required for Nef-mediated CD4 down-regulation(69) . However, while the Nef-Lck interaction might at least participate in CD4 down-modulation in Lck-positive cells, the molecular basis of CD4 down-regulation by Nef in Lck-negative cells remains to be elucidated. The CD4 cytoplasmic domain also appears to play a critical role during the early stages of HIV infection(51) . The CD4-associated Lck has been proposed to provide a transduction signal that might influence viral transcription. Interaction of Nef with this process would in turn influence the viral replication rate. Indeed, by reducing both Lck enzymatic activity and affinity for tyrosine-phosphorylated substrates, Nef might influence the outcome of T-cell activation while promoting cellular factors needed for viral replication. The recent finding that Nef also binds to another Src family kinase in monocytic cells, namely Hck, further indicates the physiological relevance of such interactions in the HIV life cycle and makes the molecular basis of the Nef-Lck binding an important target for therapeutic strategies.