(Received for publication, September 18, 1995; and in revised form, January 12, 1996)
From the
nef is a human immunodeficiency virus (HIV) gene
encoding a 27-kDa myristoylated protein with structural features of a
signal transducing molecule, but whose functions are largely unknown.
We studied the interactions of Nef with the signal transduction
pathways triggered by the platelet-derived growth factor (PDGF)
receptor. The association of phosphatidylinositol (PI) 3-kinase with
the activated receptor was severely impaired by nef expression. Conversely, PDGF-induced receptor tyrosine
phosphorylation, binding to phospholipase C- and to Ras-GAP were
not modified. Microtubule-associated protein kinase activation and
intracellular calcium influx in response to PDGF were either unaffected
or only slightly enhanced. Nef significantly reduced the proliferative
response to the growth factor, while the chemotactic response was
unchanged. These data show that Nef affects selectively the PI 3-kinase
signaling pathway and suggest that this interference results in some of
the HIV adverse effects on host cell functions.
Human HIV1 ()is a complex retrovirus containing
several genes which regulate viral replication and gene expression. nef is one of the seven nonstructural genes highly conserved
in HIV2, and SIV (for a review, see (1) ). Its expression is
critical for maintenance of high viral titer in vivo and for
disease progression in primates(2) . In vitro, nef is required for optimal viral replication in growth-stimulated
PBLs(3, 4, 5) . nef encodes a 27-kDa
N-terminal myristoylated protein which is localized at the plasma
membrane(1) . Myristoylation is an absolute requirement for
biological activity(6, 7, 8, 9) .
Nef features multiple phosphorylation sites and has been reported to
associate with an intracellular serine kinase(10) .
Furthermore, it contains a proline-rich region that binds at high
affinity the SH3 domains of the cytoplasmic tyrosine kinase hck(11) .
Together these data suggest that Nef may regulate viral growth and affect host cell function by interfering with signaling pathways. Indeed, (i) purified Nef protein microinjected in peripheral blood lymphocytes inhibits the proliferative response to IL-2(9) ; (ii) constitutive expression of nef inhibits signaling by IL-2 and TCR in leukemic cells (9, 12, 13, 14, 15) and by growth factors in murine fibroblasts(16) ; (iii) transgenic expression in thymocytes interferes with TCR signaling and perturbs their development(8, 17) . However, the biochemical mechanisms underlying such interference still wait to be elucidated. Herein, we investigated the interactions between the Nef protein and the signaling pathways triggered by the PDGF receptor, which are among the best characterized(18) .
Figure 1: Expression of Nef protein and PDGF receptor in NIH-3T3 fibroblasts. Control NIH-3T3 fibroblasts (line 2.0) and stable transfectants (lines 3.0 and 3.9), either unstimulated(-) or treated with PDGF-BB 100 ng/ml (+) for 15 min, were solubilized, separated by SDS-polyacrylamide gel electrophoresis, and analyzed by Western blot with anti-Nef (A), anti-PDGF receptor (B), and anti-phosphotyrosine antibodies (C).
In cells expressing either low or high levels of Nef, the amount of PDGF receptors was unchanged (Fig. 1B). Moreover, upon ligand stimulation, the receptor was tyrosine-phosphorylated to the same extent (Fig. 1C). Nef did not affect the kinetics of the receptor kinase in response to varying concentrations of ligand (data not shown). These data indicate that Nef does not interfere with the cell surface expression or the tyrosine kinase activity of the PDGF receptor.
Figure 2:
Effects of nef expression on
association of PI 3-kinase, PLC-, and Ras-GAP to the
PDGF-receptor. Control (line 2.0) or nef-expressing cells (lines 3.0 and 3.9), unstimulated(-) or
stimulated with 100 ng/ml PDGF-BB (+), were lysed and
immunoprecipitated with anti-PDGF receptor antibodies. The
immunocomplexes were analyzed for PI 3-kinase enzymatic activity (A) or by Western blot with anti-p85 (B),
anti-PLC-
(C), and anti-Ras-GAP (D)
antibodies.
The possibility that Nef inhibits PI 3-kinase/receptor association by direct binding to p85 or to the receptor was ruled out by measuring PI 3-kinase activity in anti-Nef immunoprecipitates and by probing anti-p85 or anti-PDGF receptor immunoprecipitates with anti-Nef antibodies (data not shown). Moreover, no evidence of Nef tyrosine phosphorylation upon PDGF stimulation was obtained (data not shown).
In order to establish whether the Nef inhibition on PI 3-kinase
association is a selective event or a consequence of a general
interference with the binding between the receptor and cytosolic
transducers, PLC- and Ras-GAP were studied. After ligand
stimulation, both transducers did bind to the PDGF receptor with the
same efficiency in cells either mock-transfected or expressing low or
high amounts of Nef (Fig. 2, C and D).
Figure 3: Effects of nef expression on PDGF-induced intracellular calcium increase and MAP kinase activation. A, intracellular calcium concentration in response to PDGF stimulation, in control (line 2.0) and nef-expressing cells (line 3.9). The ligand concentrations are indicated. B, Western blot with anti-MAP kinase antibodies on whole cell lysates from control (line 2.0) or nef-expressing cells (lines 3.0 and 3.9), unstimulated(-), or stimulated with 100 ng/ml PDGF-BB (+).
PDGF also triggers an intracellular serine kinase cascade leading to activation of MAP kinase. This activation follows phosphorylation of MAP kinase at serine and tyrosine residues, which results in a mobility shift that can be observed in Western blots(24) . Expression of nef did not interfere with MAP kinase activation in all cell lines tested (Fig. 3B). A modest enhancement of MAP kinase phosphorylation in resting cells was observed.
Figure 4: Effects of nef expression on PDGF-induced proliferation and chemotaxis. PDGF-induced proliferation (A) and chemotactic migration (B) of control (line 2.0) and nef-expressing cells (lines 3.0 and 3.9). Cells were incubated in DMEM/1% FCS, either alone (open bar) or containing PDGF (shaded bar = 10 ng/ml, filled bar = 100 ng/ml) or in DMEM/10% FCS (hatched bar). Values are expressed as fold increase of cells/field (A) or of absorbance at 590 nm (B). In A, the value 1 represents 65, 70, and 74 cells/field for 2.0, 3.0, and 3.9 cells, respectively. Each value is an average of quadruplicates.
This work shows that the HIV protein Nef interferes with signaling pathways in a model system generated by stable transfection of the nef gene in NIH-3T3 fibroblasts. Nef protein inhibits the complex formation between PI 3-kinase and the tyrosine-phosphorylated PDGF receptor. The biochemical mechanisms underlying such inhibition are unclear. Nef may compete with p85 (the docking subunit of PI 3-kinase) for the binding with specific phosphorylated tyrosines of the receptor(18) . On the other hand, through its proline-rich motif Nef might bind the p85 SH3 domain. However, these hypotheses were not supported by co-precipitation experiments. Alternatively, as Nef is involved in a specific endocytic cellular pathway(1) , it may sequester PI 3-kinase, making the enzyme unavailable for receptor binding. Finally, the viral protein may inhibit the interaction, by modifying either the PDGF receptor or the PI 3-kinase. PDGF receptor phosphorylation on tyrosine was unaffected. Nef, however, has been reported to form a complex with a cellular serine kinase(10) , and serine phosphorylation leads to negative modulation of PI 3-kinase (26) .
Whatever the
mechanism, the effect of Nef on PI 3-kinase is selective, as the
association between the PDGF receptor and PLC- or Ras-GAP was not
perturbed. Similarly, no significant alterations were observed when
downstream events of the signaling cascade, such as Ca
increase and MAP kinase activation, were analyzed. These results
do not confirm a previous study reporting Nef-mediated inhibition of
intracellular calcium mobilization in PDGF or bombesin-stimulated
fibroblasts; intriguingly, no inhibition of PLC-mediated PIP
hydrolysis was detected(16) . The authors suggest that
Nef may act on IP
receptor regulation or expression, rather
than directly on signal transduction. The conflicting results reported
here may depend on the different Nef alleles transfected. Indeed,
either increased or decreased calcium responses were observed in murine
T cells expressing different Nef alleles(8, 13) .
In NIH-3T3 fibroblasts, PDGF-BB elicits a dual biological response,
stimulating cell growth and chemotaxis(25) . It is known that
the signaling pathways involved are distinct, although partially
overlapping(27) . We show that Nef selectively inhibits
PDGF-dependent proliferation, without affecting chemotactic migration.
As activation of PI 3-kinase is essential for both responses, the
apparent paradox can be explained by a different threshold of PI
3-kinase activation required for proliferation or
chemotaxis(27, 28) . In nef transfectants,
the low signal transduced by the residual receptorPI 3-kinase
complex could be sufficient to elicit the chemotactic response. A
``threshold effect'' has also been invoked to explain how
epidermal growth factor and nerve growth factor elicit different
biological responses in PC12 cells by activating the same signaling
pathway to a different extent(29) . Recently, a rare
SIV-nef allele has been reported to act as an oncogene in
NIH-3T3 fibroblasts(30) . However, the growth-stimulating
properties of the SIV-nef allele depend on two closely spaced
tyrosine-containing specific sequences (31) which are not
present in HIV1-nef.
Recent evidence suggests that nef is a major determinant in AIDS pathogenesis, by enhancing viral replication in host cells stimulated by mitogens(1) . It is not know whether the uncoupling of PI 3-kinase from growth factor receptors is at all involved. However, it is intriguing to speculate that nef may promote viral growth by redirectioning proliferative signals, arisen from growth factor receptors, to the viral replication machinery. Indeed, the binding of Nef to SH3 domains is required to achieve optimal viral replication in infected cells(11) . Finally, besides affecting viral replication, nef may contribute to HIV-1 pathogenesis by altering T cell responses to antigenic stimulation(8, 13, 15) . PI 3-kinase activation is in fact involved in the CD28-mediated stimulation by antigen presenting cells(32) .