(Received for publication, January 22, 1997)
From the Oral and Pharyngeal Cancer Branch, NIDR, National
Institutes of Health, Bethesda, Maryland 20892-4330 and the
Department of Pathology, School of Medicine and
University Hospital, SUNY at Stony Brook, Stony Brook, New
York 11794
Stimulation of high affinity IgE Fc receptors
(FcRI) in basophils and mast cells activates the tyrosine kinases
Lyn and Syk and causes the tyrosine phosphorylation of phospholipase
C-
, resulting in the Ca2+- and protein kinase
C-dependent secretion of inflammatory mediators. Concomitantly, Fc
RI stimulation initiates a number of signaling events resulting in the activation of mitogen-activated protein kinase
(MAPK) and c-Jun NH2-terminal kinase (JNK), which, in turn, regulate nuclear responses, including cytokine gene expression. To
dissect the signaling pathway(s) linking Fc
RI to MAPK and JNK, we
reconstructed their respective biochemical routes by expression of a
chimeric interleukin-2 receptor
subunit (Tac)-Fc
RI
chain (Tac
) in COS-7 cells. Cross-linking of Tac
did not affect MAPK in
COS-7 cells, but when coexpressed with the tyrosine kinase Syk, Tac
stimulation potently induced Syk and Shc tyrosine phosphorylation and
MAPK activation. In contrast, Tac
did not signal JNK activation, even when coexpressed with Syk. Ectopic expression of a
hematopoietic-specific guanine nucleotide exchange factor (GEF), Vav,
reconstituted the Tac
-induced, Syk- and Rac1-dependent
JNK activation; and tyrosine-phosphorylation of Vav by Syk stimulated
its GEF activity for Rac1. Thus, these data strongly suggest that Vav
plays a critical role linking Fc
RI and Syk to the Rac1-JNK pathway.
Furthermore, these findings define a novel signal transduction pathway
involving a multimeric cell surface receptor acting on a cytosolic
tyrosine kinase, which, in turn, phosphorylates a GEF, thereby
regulating its activity toward a small GTP-binding protein and
promoting the activation of a kinase cascade.
Activation of high affinity IgE Fc receptors (FcRI) in
basophils and mast cells induces the rapid release of histamine and other inflammatory mediators from secretory granules, and initiates a
cascade of signal transduction events leading to enhanced production and secretion of various biologically active cytokines (1). One of the
earliest events induced upon Fc
RI aggregation is the activation of
the nonreceptor tyrosine kinases Lyn and Syk, and the tyrosine
phosphorylation of cytoplasmic molecules, including phospholipase C-
(2). Phosphorylated phospholipase C-
hydrolyses phosphatidylinositol
4,5-bisphosphate and liberates inositol 1,4,5-trisphosphate and
diacylglycerol, which mobilizes Ca2+ from intracellular and
extracellular sources and activates protein kinase C (3), respectively.
Whereas these second-messenger generating systems appear to be
sufficient for the Fc
RI-mediated secretory response (4), how signals
initiated by Fc
RI aggregation at the plasma membrane are transmitted
to the nucleus thereby controlling cytokine gene expression is much
less understood.
Recently, it has been shown that stimulation of FcRI in mast cell
lines, such as RBL-2H3 cells, leads to the activation of members of the
mitogen-activated protein kinase (MAPK)1
superfamily of serine-threonine kinases. The function of these enzymes
is to convert extracellular stimuli to intracellular signals which, in
turn, participate in gene expression regulation. In particular,
engagement of Fc
RI receptors in mast cell lines has been shown to
result in the activation of MAPK and JNK (5, 6). In this regard,
recently available evidence suggests that engagement of Fc
RI with
antigen leads to the increased tyrosine phosphorylation of Shc and the
association of Shc with Grb2, thus resulting in the recruitment of Sos
and the stimulation of the Ras-MAPK pathway. Furthermore, Shc
phosphorylation and MAPK activation was shown to be diminished upon
overexpression of a dominant negative mutant of Syk, thus suggesting a
central role for this kinase in the biochemical route communicating
Fc
RI to MAPK (5). In contrast, how Fc
RI stimulation activates JNK
is still unknown.
In this study, we thought to dissect the signaling pathway(s) linking
FcRI to MAPK and JNK by reconstructing their respective biochemical
routes upon ectopic expression of signaling molecules in COS-7 cells.
Using this experimental approach, we provide evidence that whereas Syk
and Shc connect Fc
RI to the Ras-MAPK pathway, signaling from Fc
RI
to JNK involves the tyrosine phosphorylation by Syk of a hematopoietic
specific guanine-nucleotide exchange factor, Vav, the exchange of GDP
for GTP-bound to Rac1, and the consequent stimulation of a kinase
cascade leading to JNK activation.
RBL-2H3 cells were grown in DMEM supplemented with 10% fetal bovine serum (FBS). Before cross-linking of IgE, cells were incubated overnight in DMEM containing 0.1% FBS. Sensitization with anti-trinitrophenyl (TNP) IgE ascites fluid (1:5,000) at 37 °C for 2 h and cross-linking with 0.1 µg/ml dinitrophenyl-coupled to human serum albumin were described previously (7).
COS-7 Cell Transfection and StimulationExpression plasmids (1 µg/plate) were transfected into subconfluent COS-7 cells by the DEAE-dextran technique (8), adjusting the total amount of DNA to 5 µg/plate with vector DNA (pcDNA3, Invitrogen) when necessary. Forty-eight hours later, cells were cultured overnight in DMEM containing 0.1% FBS. Cells were then left unstimulated or stimulated with EGF (100 ng/ml). Stimulation with antibodies to Tac was performed using 5 µg/ml of biotinylated monoclonal antibody to Tac, B1.49.9 (Amac). After washing with phosphate-buffered saline twice, cells were stimulated in serum-free medium containing 12 µg/ml of avidin (Sigma). After incubation for the times indicated, cells were lysed.
Immunoprecipitation, Immunoblotting, and in Vitro Kinase AssaysCell lysis, immunoprecipitation, immunoblotting, MAPK, and JNK assays were performed as described previously (8). Antiserum to MAPK and to Syk were purchased from Santa Cruz. Antibodies to Shc and to phosphotyrosine (anti-Tyr(P)) were purchased from Transduction Laboratories and ICN Biochemicals, respectively.
Expression PlasmidsSyk was cloned from a cDNA library prepared from purified human monocyte poly(A)+ mRNA templates by using a fragment of the porcine Syk cDNA (a gift from H. Yamamura) as a probe. An in frame BamHI site was generated immediately upstream of the initiation codon of Syk using polymerase chain reaction techniques and subcloned into pcDNA3. pcDNA3 Myr-Syk was generated by subcloning the Syk cDNA into pcDNA3-Myr (8).
Subcellular LocalizationpcDNA3 Myr-Syk was transfected into COS-7 cells. After 48 h, cells were lysed in a hypotonic buffer, and proteins were isolated as cytosolic and membrane fractions, as described (9). Each fraction was immunoprecipitated with antibodies to Src (Santa Cruz) and immunoblotted with antiserum to Syk (Santa Cruz) and antibody to Tyr(P) (ICN).
In Vivo Nucleotide Labeling of GTP-binding ProteinsCOS-7 cells were transfected using DEAE-dextran method, and cultured for 48 h, serum-starved in phosphate-free DMEM for 18 h, labeled with [32P]orthophosphate (100 µCi/ml) for 1 h for [32P]GDP accumulation and for 6 h for [32P]GDP and [32P]GTP determinations. Cells were disrupted in 50 mM Tris-HCl (pH 7.5), 20 mM MgCl2, 150 mM NaCl, 0.5% Nonidet P-40, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 25 µg/ml leupeptin, and 25 µg/ml aprotinin. Lysates were immunoprecipitated with a monoclonal antibody to AU5 (Babco) for 1 h and immunocomplexes recovered using gamma-binding G-Sepharose beads (Pharmacia Biotech Inc.). Immunoprecipitates were washed twice in lysis buffer, twice in 50 mM Tris-HCl (pH 7.5), 20 mM MgCl2, 500 mM NaCl, and resuspended in 1 M KH2PO4, 5 mM EDTA (pH 8.0). Bound nucleotides were released by heating and fractionated using polyethyleneimine thin layer chromatography plates (J. T. Baker).
To begin dissecting the signaling pathway(s) linking FcRI to
MAPK and JNK, we initially studied the temporal relationship between
MAPK and JNK activation in RBL-2H3 cells. As expected, engagement of
Fc
RI by addition of dinitrophenyl (DNP) coupled to human serum
albumin to anti-TNP IgE-primed RBL-2H3 cells potently activated MAPK
and JNK; however, each followed a distinct temporal pattern (Fig.
1, A and B). These data suggested
that MAPK and JNK might be activated by different signaling pathways.
For MAPK, Fc
RI cross-linking is known to activate the nonreceptor
tyrosine kinase Syk, and it has been suggested recently that Syk
phosphorylates the adapter protein Shc, thereby stimulating the
Ras-MAPK pathway through Grb2 and Sos (10). Consistent with that, we
observed that in RBL-2H3 cells Fc
RI activation leads to the rapid
tyrosine phosphorylation of Syk and the adapter protein Shc, following a time course similar to that of MAPK stimulation (Fig. 1, C
and D).
FcRI is a multimeric receptor containing a single
and
subunit and a homodimer of
subunits (11). Both
and
chains exhibit a structural motif termed ITAM, for immunoreceptor
tyrosine-based activation motif (12), which participate in the
recruitment of cytoplasmic tyrosine kinases and in the consequent
tyrosine phosphorylation of their downstream targets (1). Studies with chimeric molecules containing the extracellular and transmembrane domains of the interleukin-2 receptor
subunit (Tac) fused to the
cytosolic domain of
(Tac
) and
(Tac
) chains of Fc
RI have helped simplify the analysis of early signaling events provoked by
Fc
RI activation (13). When expressed in RBL-2H3 cells, cross-linking of the Tac
chimera is sufficient to mimic the majority of the biochemical and biological responses triggered by Fc
RI stimulation. In contrast, cross-linking of Tac
does not appear to elicit
signaling responses (13). Therefore, to investigate whether activation of Tac
is sufficient to activate Syk, both were expressed in COS-7
cells, which lack endogenous Fc
RI or Syk (see below). Transfected Tac
was efficiently expressed, as judged by immunofluorescence labeling techniques (data not shown). Cross-linking of Tac
chimeras with biotinylated anti-Tac antibodies followed by streptavidin induced
the rapid tyrosine phosphorylation of a coexpressed epitope-tagged Syk
(Fig. 2A). When coexpressed with an
epitope-tagged form of Shc, cross-linking of Tac
induced only a
limited increase in Shc tyrosine phosphorylation (Fig. 2B).
However, when Syk was coexpressed, Tac
engagement provoked a rapid
and substantial increase in Shc tyrosine phosphorylation (Fig.
2B). Paralleling Shc phosphorylation, cross-linking of
Tac
induced a very poor MAPK response, but when coexpressed with
Syk, Tac
potently elevated the phosphorylating activity of MAPK to
an extent comparable with that elicited in response to EGF (Fig.
2C). Taken together, these results support a central role
for the
subunit of Fc
RI and Syk in signaling from IgE receptors
to the MAPK pathway. Surprisingly, however, cross-linking of Tac
chimeras did not result in JNK activation, even when coexpressed with
Syk. As a control, EGF effectively elevated JNK activity under
identical experimental conditions (Fig. 2C). Collectively,
these data established that coexpression of Tac
and Syk in COS-7
cells is sufficient to reconstitute the MAPK response to Fc
RI
stimulation, while suggesting that additional molecules not
endogenously expressed in COS-7 cells were necessary to link Fc
RI to
JNK.
Whereas Ras controls the activation of MAPK, we and others have
recently observed that two members of the Rho family of small GTP-binding proteins, Rac1 and Cdc42, regulate JNK activity (8). Although most molecules connecting Syk to Ras, including Shc, Grb2, and
Sos, are ubiquitously expressed, guanine nucleotide exchange factors
(GEFs) for Rho, Rac1, and Cdc42 exhibit a very restricted cell type and
tissue distribution (14). Thus, we hypothesized that COS-7 cells might
lack an exchange factor acting downstream from Syk in the Rac/Cdc42-JNK
pathway. In this regard, as recently shown by others (5), FcRI
activation in RBL-2H3 cells induces the rapid and prolonged tyrosine
phosphorylation of the Vav proto-oncogene product (Vav) (Fig.
3A), which is preferentially expressed in
cells of the hematopoietic lineage. Moreover, Vav exhibits structural
motifs frequently found in GEFs for small GTP-binding proteins of the
Ras and Rho families (14), and we have shown recently that truncated,
oncogenically active forms of Vav (Onco-Vav), can potently activate
JNK, but not MAPK, acting on a Rac-1-dependent signaling
pathway (15). These results prompted us to explore the possibility that
wild-type Vav serves as a link between Fc
RI and the Rac-1-JNK
pathway.
Expression of Vav alone (15) or together with the Tac chimera failed
to induce JNK activation (Fig. 3B), and cross-linking of
Tac
failed to induce Vav tyrosine phosphorylation when coexpressed in COS-7 cells (Fig. 3B). However, when Tac
, Syk and Vav
were each simultaneously coexpressed in these cells, Tac
aggregation resulted in enhanced Vav tyrosine phosphorylation and a remarkable activation of JNK. These data together with results obtained in RBL-2H3
cells demonstrate the importance of Vav in signaling from Fc
RI/Syk
to JNK.
We next asked whether recruitment of Syk to the plasma membrane upon
aggregation of FcRI or cross-linking of Tac
chimeric molecules is
the determining step initiating activity of Syk downstream signaling
pathways. To that end, we examined the ability of a membrane-targeted
form of Syk to bypass the requirement of Tac
engagement for
signaling to the MAPK and JNK pathway. A chimeric protein containing
the NH2-terminal myristoylation signal of Src fused to Syk
(Myr-Syk), localized to the plasma membrane when expressed in COS-7
cells, rather than exhibiting the typical cytosolic location of
wild-type Syk (Ref. 16 and data not shown). Furthermore, this
membrane-targeted form of Syk was heavily tyrosine-phosphorylated (Fig.
3C), and its expression was sufficient to elevate the
activity of a cotransfected epitope-tagged MAPK (Fig. 3D).
However, Myr-Syk alone did not enhance JNK activity but, when
cotransfected with Vav, it effectively induced the tyrosine
phosphorylation of Vav (not shown) and potently activated the JNK
pathway, to an extent comparable with that provoked by expression of
the fully active, transforming vav oncogene (Fig.
3D). These data indicate that once Syk is activated upon
recruitment to the plasma membrane, no other Fc
RI-associated kinases
are required to signal to MAPK or to activate JNK in a
Vav-dependent manner.
We have reported recently that JNK activation by Onco-Vav can be
blocked by expression of a dominant negative mutant of Rac-1, N17 Rac-1
(15), thereby inferring that Onco-Vav acts as a GEF for Rac-1. In view
of those results and our present data, we next asked whether expression
of Vav proteins could promote guanine nucleotide exchange on Rac1
in vivo. In this regard, the high intrinsic GTPase activity
of Rho, Rac1, and Cdc42 has prevented the detection in living cells of
their corresponding GTP-bound forms (17). Thus, for these experiments
we took advantage of a recently described technique that uses the
levels of 32P-labeled GDP bound to these small GTPases
after a brief exposure to [32P]orthophosphate-containing
medium as an approach to evaluate their nucleotide exchange in
vivo. Initially, we expressed in COS-7 cells AU5-epitope-tagged
Ha-Ras, RhoA, Rac1, and Cdc42 (18, 19), together with empty expression
vector (control), a membrane-targeted form of the catalytic domain of
Sos (Myr-Sos) (8), or Onco-Vav (Fig. 4A). All
tagged small GTP-binding proteins were efficiently expressed, as judged
by Western blotting with the anti-epitope antibody. Furthermore, when
transfected cells were starved and then cultured for a short period of
time in the presence of [32P]orthophosphate, each small
GTPase incorporated labeled GDP, as determined by thin layer
chromatography analysis of anti-AU5 immunoprecipitates. Under these
experimental conditions, no labeled nucleotides were observed in
mock-transfected cells (not shown), and Myr-Sos consistently enhanced
2-3-fold the level of radioactive GDP bound to Ras, without displaying
any demonstrable effect on the other small GTP-binding proteins (Fig.
4A, left panel). As a control, we used the standard, more
prolonged incubation with [32P]orthophosphate containing
medium. Under those conditions, Myr-Sos induced a dramatic increase in
GTP-bound Ras (Fig. 4A, right panel). In contrast, under
either incubation time expression of Onco-Vav did not affect Ras, but
increased the level of labeled GDP bound to Rac1 more than 8-fold (Fig.
4A). Collectively, these results indicate that Onco-Vav can
promote guanine nucleotide exchange in vivo on Rac1.
Under identical experimental condition, neither wild-type Vav nor
Myr-Syk induced nucleotide exchange on Rac1 (Fig. 4B), which was consistent with the failure of each one alone to induce JNK activity (see above). However, when Myr-Syk was coexpressed with Vav,
we observed a dramatic increase in the incorporation of labeled GDP
into Rac1. These two observations, 1) potent JNK activation provoked by
coexpression of Myr-Syk together with Vav or upon cross-linking of
Tac when coexpressed with Syk and Vav and 2) Syk's ability to
effectively tyrosine-phosphorylate Vav in vivo, strongly
suggest that Syk-induced tyrosine phosphorylation of Vav increases its
GEF toward Rac1, leading to JNK activation. Consistent with this
conclusion, JNK stimulation induced by Tac
cross-linking in Tac
-,
Syk-, and Vav-transfected COS-7 cells was blocked by the dominant
negative mutant of Rac1, N17 rac1 (Fig. 4C). Moreover, we
have recently observed that tyrosine phosphorylation of purified Vav
protein dramatically enhances its GEF activity on bacterially expressed
Rac1 when analyzed in in vitro assays (19), further
supporting the emerging notion that Vav behaves as a tyrosine
phosphorylation-dependent GEF for Rac1.
A number of GEFs for small GTP-binding proteins of the Rho family have
been identified by virtue of their transforming potential in murine
fibroblasts (20). Nevertheless, the normal function of these GEFs, as
well as the molecular mechanisms controlling their enzymatic activity
in their natural setting, is still unknown. In this regard, our
findings provide solid evidence that whereas Onco-Vav is constitutively
active, wild-type Vav only promotes guanine nucleotide exchange in Rac1
upon activation of an upstream tyrosine kinase, Syk, and that Vav
function(s) in this setting are controlled by tyrosine phosphorylation.
Thus, these findings define a novel signal transduction pathway
involving a cell surface receptor activating a nonreceptor tyrosine
kinase, which, in turn, phosphorylates a GEF in tyrosine residues,
thereby regulating its activity toward a small GTP-binding protein and
promoting the activation of a kinase cascade. A schematic
representation of such a likely biochemical route, including,
sequentially, FcRI, Syk, Vav, Rac1, and its downstream target, JNK,
as well as the pathway connecting Syk to MAPK is depicted in Fig.
5.
Our present findings might also have important implications regarding
the functioning of other multimeric antigen receptors. As discussed
above, in mast cells accumulating evidence demonstrates that the subunit of Fc
RI signals Syk activation. The Fc
RI
chain is
functionally analogous to the
chain of the antigen T cell receptor,
and whereas Fc
RI
subunits recruit Syk, the T cell receptor
subunits interact with Zap70 (21, 22). Furthermore, T cell receptor and
B cell receptor activation both lead to Vav tyrosine phosphorylation
(23, 24) and JNK activation (25). Based upon our results, it is
predictable that Vav plays a common role in basophils, mast cells, T
cells, and B cells, linking multimeric antigen receptors and their
associated downstream nonreceptor tyrosine kinases to the Rac1-JNK
signaling pathway.
We thank Dr. Richard D. Klausner for
providing Tac cDNA and Dr. Hirohei Yamamura for providing the
fragment of the porcine Syk cDNA.