From the Programe in Molecular Biology and Cancer,
Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto,
Ontario M5G 1X5 and the
Department of Molecular and Medical
Genetics, University of Toronto, Toronto, Ontario M5G 1A8,
Canada
Received for publication, September 3, 2002, and in revised form, December 2, 2002
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ABSTRACT |
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Signaling by the Eph family of receptor
tyrosine kinases (RTKs) is complex, because they can interact with a
variety of intracellular targets, and can potentially induce distinct
responses in different cell types. In NG108 neuronal cells, activated
EphB2 recruits p120RasGAP, in a fashion that is associated with
down-regulation of the Ras-Erk mitogen-activated kinase (MAPK) pathway
and neurite retraction. To pursue the role of the Ras-MAPK pathway in
EphB2-mediated growth cone collapse, and to explore the biochemical and
biological functions of Eph receptors, we sought to re-engineer the
signaling properties of EphB2 by manipulating its regulatory motifs and SH2 binding sites. An EphB2 mutant that retained juxtamembrane (JM)
RasGAP binding sites but incorporated a Grb2 binding motif at an
alternate RasGAP binding site within the kinase domain had little
effect on basal Erk MAPK activation. In contrast, elimination of all
RasGAP binding sites, accompanied by the addition of a Grb2 binding
site within the kinase domain, led to an increase in phospho-Erk levels
in NG108 cells following ephrin-B1 stimulation. Functional assays
indicated a correlation between neurite retraction and the ability of
the EphB2 mutants to down-regulate Ras-Erk MAPK signaling. These data
suggest that EphB2 can be designed to repress, stabilize, or activate
the Ras-Erk MAPK pathway by the manipulation of RasGAP and Grb2 SH2
domain binding sites and support the notion that Erk MAPK regulation
plays a significant role in axon guidance. The behavior of EphB2
variants with mutations in the JM region and kinase domains suggests an
intricate pattern of regulation and target recognition by Eph receptors.
The mechanisms by which signals are conveyed from receptor
tyrosine kinases (RTKs)1 at
the plasma membrane to their intracellular targets in the cytoplasm and
nucleus have been extensively explored (1-3). Ligand-induced autophosphorylation in the activation segment of the kinase domain, as
well as phosphorylation of juxtamembrane (JM) tyrosines in RTKs such as
Eph receptors, induce conformational changes that stimulate the
activity of the kinase domain (4-6). Furthermore, receptor tyrosine
phosphorylation, usually within non-catalytic sequences flanking the
kinase domain, creates docking sites for proteins with SH2 or PTB
domains, leading to the recruitment of cytoplasmic targets that
regulate downstream signaling (3, 7).
This scheme is evident in the ability of RTKs to recruit negative or
positive regulators of the Ras GTPase, such as the p120 Ras
GTPase-activating protein (RasGAP) or Grb2 (8). RasGAP, which
stimulates the hydrolysis of Ras-bound GTP, is a modular polypeptide
with a C-terminal catalytic domain that down-regulates Ras, linked to
two SH2 domains (surrounding an SH3 domain) at the N terminus (9, 10).
In contrast, Grb2 is an adaptor comprised of a central SH2 domain
flanked by two SH3 domains that bind to targets with proline-rich
motifs (11-13). In particular, through recruitment of the Ras guanine
nucleotide exchange factor Sos1, Grb2 directs phosphotyrosine-induced
exchange of GDP for GTP on Ras, thereby stimulating the Erk MAPK
pathway (3). RasGAP and Grb2 are therefore SH2-containing proteins with
opposing effects on the state of Ras activation, and data from both
invertebrate and mammalian systems suggest that RTKs potentially employ
Grb2 and RasGAP in combination to achieve sophisticated regulation of
the Ras signaling pathway (14, 15).
SH2 domains generally bind short phosphotyrosine-containing peptide
sequences but differ in their ability to recognize residues C-terminal
to the phosphotyrosine (7, 16, 17). For example, the Grb2 SH2 domain
binds preferentially to pTyr-X-Asn motifs (17),
whereas the RasGAP SH2 domains binds optimally to
pTyr-X-X-Pro motifs (18). We have employed these
observations as the basis to explore signaling by EphB2, a member of
the predominant family of mammalian RTKs. Eph receptors are activated
by their association with cell surface ligands, ephrins, and regulate
biological events involving cell-cell interactions, including axon
guidance, boundary formation, neural crest migration, angiogenesis, and
synaptic function (19-23). In their cytoplasmic regions, Eph receptors
have a JM sequence, followed by the kinase domain, a SAM domain, and a
C-terminal PDZ domain binding motif. Activated receptors become phosphorylated on multiple tyrosine residues, including a tyrosine in
the activation loop of the kinase domain and two conserved tyrosines in
the JM region that regulate kinase activity (24-27). Phosphorylated
tyrosines in the JM region and elsewhere can then directly engage
cytoplasmic targets. In addition, the receptor can potentially
phosphorylate docking proteins, such as p62Dok-1, with the ability to
engage signaling proteins (18). Of interest, in contrast to other RTKs,
which generally stimulate Ras, activation of EphB2 (28) and EphA2 (29)
down-regulates the Ras-MAPK pathway in specific mammalian cell types
(i.e. neuronal and endothelial cells). In the context of
EphB2, RasGAP recruitment appears to inhibit Ras activation, which then
leads to down-regulation of the MAPK pathway (28). Interfering with Ras
down-regulation, either by expression of a dominant-negative form of
RasGAP or a constitutively activated Ras variant, suppresses the
ability of ephrin-B1 to induce neurite retraction in NG108 neuronal
cells expressing EphB2.
In this study, we demonstrate that mutating the previously identified
RasGAP binding sites in the EphB2 JM region is not sufficient to switch
the EphB2 signal from down-regulation to up-regulation of the MAPK
signaling. We identify a novel binding site for RasGAP and show that
introduction of a Grb2 binding sequence in place of this second RasGAP
binding site within the kinase domain stabilizes phospho-Erk levels in
ephrin-B1-stimulated cells. Complete abrogation of RasGAP binding
coupled with recruitment of Grb2, leads to an up-regulation of
phospho-Erk, and a block in the ability of EphB2 to induce neurite
retraction. These results demonstrate that it is possible to rewire RTK
signaling to the Ras-MAPK pathway by the rational design of mutant
receptors with modified sites for protein-protein interactions. Our
results are consistent with an intricate regulation of EphB2 through
the combined actions of the JM region and kinase domain.
DNA Constructs, Mutagenesis, and Reagents--
Full-length
murine EphB2 cDNA was cloned into the mammalian expression vector
pcDNA3 (18). Mutagenesis of EphB2 was performed using the
QuikChange system (Stratagene). Green fluorescent protein-tagged EphB2
(EphB2-GFP) was constructed by inserting the whole length of GFP
cDNA into ApaI site in the C-terminal of EphB2 between Arg983 and Ala984. For glutathione
S-transferase (GST) fusion constructs, cDNA sequences corresponding to the mouse Grb2 SH2 domain (residues 79-160)
and human RasGAP SH2 domain (residues 169-470) were cloned into pGEX
vector (Amersham Biosciences) and expressed as GST fusion proteins.
For immunoprecipitation and Western blotting, anti-EphB2 (18, 30) and
anti-HA mouse monoclonal (12CA5) (31) antibodies have been described
previously. Anti-Grb2 and anti-phospho-Erk1/2 antibodies were purchased
from Transduction Laboratories and Cell Signaling Technologies,
respectively. Other antibodies were purchased from Santa Cruz Biotechnology.
Cell Culture, DNA Transfection, and EphB2
Stimulation--
NG108-15 (NG108) cells were routinely cultured in
Dulbecco's modified Eagle's medium containing 10% fetal bovine serum
and 1× hypoxanthine-aminopterin-thymidine (Invitrogen). Stable cell lines in NG108 cells were produced as previously described (18, 28).
Briefly, parental NG108 cells were transfected with 20 µg of cDNA
using the calcium phosphate precipitation method (32). Cells were then
grown in the presence of 400 µg/ml G418 (Invitrogen) to select stable
transfectants. Human embryonic kidney 293 (HEK-293) cells were
maintained in Dulbecco's modified Eagle's medium with 10% fetal calf
serum. 20 µg of plasmid DNA was used to transfect 2 × 106 HEK-293 cells/100-mm plate by the calcium phosphate
precipitation method (32). Control cells were treated in the same way
but with no DNA or with expression vector DNA only. Stimulation of EphB2-expressing cells was carried out as previously described (18, 28)
with 2 µg/ml aggregated Ephrin-B1Fc. For Erk activity studies, EphB2
cells were seeded into six-well plates 24 h prior to stimulation
at 3 × 105 cells/well.
Immunoprecipitation, GST Fusion Protein Pull-down, and Western
Blotting--
Unless otherwise indicated, NG108 cells were
serum-starved overnight in DMEM containing 1×
hypoxanthine-aminopterin-thymidine. For Erk and anti-phosphotyrosine
(anti-pTyr) Western blots, cells were stimulated with Ephrin-B1Fc as
indicated and lysed directly in 2× SDS loading buffer. For
immunoprecipitation experiments, cells were rinsed twice in ice-cold
phosphate-buffered saline and routinely lysed in PLC lysis buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 10% glycerol,
1% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA, 10 mM NaPPi, 100 mM
NaF, 1 mM vanadate, and a mixture of protease inhibitors)
as previous described (18, 30). Protein concentrations of cleared
lysates were determined using a BCA assay (Pierce). Proteins were
immunoprecipitated for 1-2 h at 4 °C, and beads were routinely
washed three times in HNTG buffer (20 mM Hepes, pH 7.5, 10% glycerol, 0.1% Triton X-100, and 150 mM NaCl).
Proteins were separated on SDS-PAGE gels and transferred to an
Immobilon-P membrane (Millipore). Membranes were blocked in TBST
containing 5% bovine serum albumin for anti-phosphotyrosine blots or
5% skimmed milk for other blots, and immunoblotted as per standard
protocols. Primary antibodies were detected with anti-mouse- or
protein-A-horseradish peroxidase followed by treatment with Enhanced
Chemiluminescence (Pierce). For the GST fusion protein pull-down
experiment, pre-bound GST fusion protein beads (around 6 µg of
protein/ml) were used to pull down proteins from whole cell lysates in
PLC lysis buffer at 4 °C for 1 h and washed three times in HNTG buffer.
Peptide Spots Array Synthesis--
Peptide arrays were
constructed according to the Spots-synthesis method as previously
described (33). Acid-hardened cellulose membranes pre-derivatized with
polyethylene glycol (AbiMed, Langfield, Germany) were spotted with a
grid of Fmoc Phosphopeptide Mapping by Two-dimensional
Chromatography--
Wild type and mutant (JXGrb2) forms of
EphB2 were immunoprecipitated from transiently transfected NG108 cells,
radiolabeled by in vitro kinase assay with
[ Luciferase Assay--
HEK-293 cells were transiently transfected
with wild type EphB2, EphB2-EE, KINGrb2,
KIN667F, or JXEE-KINGrb2 in the
addition of pFA-Elk1 and pFR-Luciferase reporter constructs (Stratagene PathDetect® system), and pSV2- Neurite Retraction Assay--
NG108 cells were transiently
transfected by using the calcium phosphate precipitation method with
wild type or mutant EphB2-GFP. After 20 h, the transfected NG108
cells were differentiated by using 1 mM
N6,O2-dibutyryladenosine
3':5'-cycle monophosphate (Sigma) for 5 h. Cells with neurite
length of more than two times their cell body length were
stimulated with 2 µg/ml clustered ephrin-B1Fc and imaged every 1 min
for 20 min on an inverted Leica DM IRB fluorescence microscope equipped
with OpenLab software (Improvision Co., UK).
Endogenous RasGAP Is Required for Down-regulation of the Erk MAPK
Pathway by Eph Receptor Signaling--
We have previously found that
activation of ectopically expressed EphB2 in NG108 neuronal cells leads
to a down-regulation of the Ras-MAPK pathway, in a fashion that is
attenuated by a dominant negative RasGAP polypeptide (33). To explore
whether this is a more general and physiological event, we have
investigated the phosphorylation of Erk MAPK in wild type mouse embryo
fibroblasts (MEFs) or MEFs derived from mouse embryos homozygous for a
null mutation in the gene for RasGAP. Stimulation of wild type MEFs with clustered Fc-ephrin-A1 to activate EphA2 endogenously expressed in
these fibroblasts led to down-regulation of Erk phosphorylation, whereas no change in Erk phosphorylation was detected in RasGAP Mutation of the EphB2 Juxtamembrane Region Reveals a Sequence
Dependence for Receptor Regulation and Phosphorylation and a Secondary
Binding Site for RasGAP--
To explore the determinants that regulate
the coupling of Eph receptors to RasGAP, and the Ras-MAPK pathway, we
turned to NG108 neuronal cells induced to express EphB2. Ephrin-B1
stimulation results in phosphorylation of EphB2 tyrosine residues,
including two conserved JM tyrosines
(Y604IDPFTY610EDP) (18, 25). In their
unphosphorylated form, these JM tyrosines suppress Eph kinase activity
(6, 24, 27); this inhibition is relieved by autophosphorylation of the
JM motifs, which can subsequently serve as docking sites for SH2 domain
containing proteins, including RasGAP (18). RasGAP recruitment to EphB2 is associated with the down-regulation of Erk MAPK activity and may
play a significant role in the axon guidance function of EphB2 (28). We
hypothesized that substitution of the RasGAP SH2 domain binding sites
in the JM region with known Grb2 binding motifs, the sequence
Y604VNVFTY610VNV (see Fig. 5A),
might lead to recruitment of Grb2 to the JM region of the resulting
JXGrb2 mutant EphB2 and induce up-regulation of MAPK
activity, an effect opposite to that of wild type (WT) EphB2.
To study the Erk MAPK response to the EphB2 mutant JXGrb2,
we derived an NG108-15 (NG108) cell line stably expressing this mutant.
The parental NG108 cells do not express detectable EphB2 and do not
respond to ephrin-B1 stimulation. In contrast, ephrin-B1 stimulation of
NG-EphB2 cells, which stably express wild type EphB2 receptor, leads to
an increase in the tyrosine phosphorylation of EphB2 and cellular
proteins such as p62Dok-1 and induces neurite retraction (18) and
down-regulation of MAPK activity (28). Similarly, ephrin-B1 stimulation
of cells expressing JXGrb2 caused an increase in cellular
tyrosine phosphorylation, although it was attenuated relative to WT
EphB2 (Fig. 2A). We also
observed ligand-dependent association of the mutant
receptor with RasGAP in NG108 cells expressing JXGrb2 (Fig.
2C), albeit to a lesser extent than WT EphB2, and Erk1/2
dephosphorylation (Fig. 2B) in a manner similar to cells
expressing WT receptor. Interestingly, we were unable to detect
association of either the WT or JXGrb2 EphB2 with Grb2
following ephrin-B1 stimulation (Fig. 2C).
The observation that the JXGrb2 Eph receptor retains the
ability to recruit RasGAP raises the possibility that tyrosine residues at the mutated Y604VNVFTY610VNV motif in
JXGrb2 may still be able to preferentially recruit RasGAP
instead of Grb2, or the mutant receptor could directly or indirectly
associate with RasGAP at other sites. In addition, the JM tyrosine
residues in the JXGrb2 mutant may not be phosphorylated upon ephrin-B1 stimulation, and therefore binding sites for the Grb2
SH2 domain may never be generated in the JXGrb2 mutant. To test the first hypothesis, arrays of tyrosine-phosphorylated and unphosphorylated peptides encompassing all tyrosine residues in the
intracellular EphB2 region were synthesized directly on a membrane and
probed with a GST-Grb2 SH2 domain fusion protein (Fig.
3A). As controls, a
YVNV-containing phosphopeptide from Shc, which binds the Grb2 SH2
domain, and a YSVP-containing phosphopeptide from p190 RhoGAP, which
binds the RasGAP SH2 domains, were included. The GST-Grb2 SH2 fusion
protein bound specifically to the phosphorylated Shc peptide and to the
JXGrb2 and JX'Grb2 peptides (Fig.
3A) as anticipated but not to their non-phosphorylated
counterparts. A GST fusion protein containing both RasGAP SH2 domains
associated with the p190 RhoGAP phosphopeptide but was unable to
associate with either the JXGrb2 or JX'Grb2
peptide (Fig. 3A). These results suggest that the mutated
Y604VNVFTY610VNV motif in JXGrb2
should bind to Grb2 and not RasGAP, if it were accessible and
phosphorylated after ephrin-B1 stimulation. To investigate whether the
mutated tyrosine sites in JXGrb2 were indeed
phosphorylated, WT EphB2 and mutated JXGrb2 were
radiolabeled by using [ Introduction of a Grb2 Binding Site in the EphB2 Kinase Domain
Stabilizes Erk Activity--
To identify other potential RasGAP
binding sites in mutant EphB2, we screened a peptide array encompassing
all the intracellular phosphopeptides of EphB2 for alternative sites of
interaction with a GST fusion protein containing both RasGAP SH2
domains. This revealed another possible RasGAP SH2 binding site,
Tyr667, in the EphB2 kinase domain (Fig. 3A).
Tyr667 has been previously identified as a phosphorylation
site in vivo (25) and is on the surface of the kinase domain
(6). We therefore considered the possibility that the
Tyr667site might be a suitable candidate site for
introducing an ectopic Grb2-binding motif in EphB2. To test whether
such a mutant EphB2 (KINGrb2), engineered to contain the
Grb2-binding motif at this site (Y667VNV), can be
phosphorylated, we expressed a GST fusion protein containing the
intracellular domain of the KINGrb2 EphB2 mutant. The
GST-KINGrb2 fusion protein became tyrosine-phosphorylated, as detected by an anti-phosphotyrosine antibody (data not shown). The
GST-KINGrb2 protein was subsequently digested and analyzed by nanoelectrospray tandem mass spectrometry to map the phosphorylation sites. Ion peaks corresponding to the Tyr667-containing
phosphopeptide (SGpY667VNVRQ) were identified by sequencing
and, thus, confirmed the specific phosphorylation of Tyr667
in mutant KINGrb2 (data not shown). These results suggest
that the KINGrb2 binding site can be phosphorylated
in vitro and may thus be able to recruit Grb2 in
vivo.
To test whether the mutant KINGrb2 receptor can bind to
Grb2 and thereby influence Erk MAPK activity in vivo, we
made an NG108 cell line stably expressing KINGrb2.
Stimulation of cells expressing WT or KINGrb2 EphB2 caused
an increase in cellular tyrosine phosphorylation in both cases; the
KINGrb2 receptor itself was inducibly
tyrosine-phosphorylated, although to a lesser extent than WT (Fig.
4, A and C).
However, although we observed down-regulation of MAPK activity in WT
EphB2 cells, cells expressing KINGrb2 retained a constant
level of phosphorylated Erk1/2 over time (Fig. 4B).
Immunoprecipitation of EphB2 and blotting for either RasGAP or Grb2
revealed that, like WT EphB2, the KINGrb2 mutant was able
to associate with RasGAP, but in contrast to the WT receptor,
KINGrb2 also recruited endogenous Grb2 in a
ligand-dependent manner (Fig. 4C). Thus, the
mutant EphB2 receptor, KINGrb2, is able to bind to Grb2 and
to neutralize the down-regulation of MAPK activity observed in response
to WT EphB2, suggesting that the positive effect of Grb2 may balance
the inhibitory function of RasGAP. However, we could not immediately
exclude the possibility that the stable MAPK activity in cells
expressing the KINGrb2 mutant after ephrin stimulation was
due to a lower level of receptor activation, rather than Grb2
recruitment. To address this issue, we sought to engineer an EphB2
receptor that could up-regulate MAPK activity through Grb2
recruitment.
Engineering an EphB2 Variant That Up-regulates Erk MAPK Activity in
NG108 Cells--
We speculated that the inability of the
KINGrb2 mutant to stimulate Erk activation might be due to
its association with RasGAP as well as Grb2 and, therefore, sought to
engineer a receptor that lacks RasGAP binding sites but possesses a
phosphorylated Grb2 binding motif. To this end, a series of mutations
were introduced into EphB2 at tyrosine phosphorylation sites in the JM
sequence, the kinase domain (Tyr667), and SAM domain
(Tyr938), either singly or in combination, as shown in Fig.
5A. Tyr938 in the
EphB2 SAM domain is conserved across all Eph receptors except EphA3 and
has been implicated in binding to the Grb10 adaptor protein (35). WT
and mutant EphB2 receptors were transiently expressed in 293 cells and
analyzed for tyrosine phosphorylation (Fig. 5B). EphB2
mutants became tyrosine-phosphorylated to varying degrees, with the
exception of JXFF, in which the two JM tyrosines are
changed to phenylalanine, thus locking the receptor in its autoinhibited state (6, 18). The phosphorylation of mutants with tandem
Grb2 binding motifs in the JM region, and a YVNV site in either the
kinase or SAM domain (JXGrb2-KINGrb2 and
JXGrb2-SAMGrb2) was defective (Fig.
5B, upper panel), and indeed a GST-Grb2 SH2 fusion failed to bind JXGrb2-KINGrb2 or
JXGrb2-SAMGrb2 (Fig. 5C). We
therefore sought to eliminate RasGAP binding in the JM region without
significantly altering the surrounding amino acid sequence (24, 28). To
this end, we replaced the JM tyrosines with glutamates, which preserve
the ability of the receptor to be activated but do not provide SH2
binding sites (27), and introduced a Grb2 binding motif into the kinase
domain at Tyr667. We anticipated that this mutant
(JXEE-KINGrb2) might retain kinase activity and
bind selectively to Grb2 but not to RasGAP. Indeed, the
JXEE-KINGrb2 receptor was
tyrosine-phosphorylated in ephrin-B1-stimulated NG108 cells (Fig.
5B).
To test binding of the various EphB2 mutants to Grb2 or RasGAP, we used
GST fusion proteins containing the SH2 domains of Grb2 or RasGAP in
pull-down experiments with EphB2 mutants from whole cell lysates (Fig.
5C). The GST-Grb2-SH2 fusion protein bound to
KINGrb2, but not the corresponding mutant with
Tyr667 replaced with Phe (KINF-Grb2),
indicating that the association of KINGrb2 with Grb2
depends on Tyr667. As anticipated, both KINGrb2
and KINF-Grb2 bound the RasGAP SH2 domains, consistent with
the presence of the JM RasGAP binding sites in these mutants.
Significantly, the double-mutant JXEE-KINGrb2 bound the Grb2 SH2 domain but not the RasGAP SH2 domains (Fig. 5C). A similar Grb2 binding profile was observed when EphB2
was co-expressed with HA-tagged, full-length Grb2 (Fig. 5D),
indicating that Grb2 can associate with both KINGrb2 and
JXEE-KINGrb2 in vivo.
To test the effect of the JXEE-KINGrb2 EphB2 on
cellular signaling pathways, we constructed stable NG108 lines
expressing this mutant receptor. Stimulation of cells expressing
JXEE-KINGrb2 with ephrin-B1 induced tyrosine
phosphorylation, albeit with slower kinetics than WT EphB2 (Fig.
6A), and association of the
mutant receptor with Grb2 (Fig. 6C) but not RasGAP (data not
shown). To determine the effect of JXEE-KINGrb2
on Erk activation, cells expressing WT and mutant EphB2 were stimulated
with pre-clustered ephrin-B1, and lysates were probed with phospho-Erk
antibodies. Stimulation of WT EphB2 led to attenuation of pErk levels,
whereas stimulation of the JXEE-KINGrb2 variant
produced a time-dependent, biphasic increase in phospho-Erk
(Fig. 6B), suggesting that this mutant can up-regulate
signaling through the Erk MAPK pathway.
To pursue the effect of JXEE-KINGrb2 on MAPK
signaling, HEK 293 cells were transiently transfected with WT EphB2,
EphB2-EE, KINGrb2, KIN667F, or JXEE
-KINGrb2 as well as pFA2-Elk1, pFR-Luciferase, and
The Effect of EphB2 Mutants on Neurite Retraction--
To assess
the biological properties of these EphB2 mutants, we established a
neurite retraction assay measuring ephrin-induced changes of neurite
length in NG108 cells transiently transfected with WT or mutant forms
of EphB2, tagged at the C terminus with green fluorescent protein (GFP)
to visualize expressing cells. Ephrin-B1 stimulation of differentiated,
neurite-bearing NG-EphB2 cells leads to the reorganization of
polymerized actin structures and to neurite retraction (18, 28). When
transiently expressed in NG108 cells, WT EphB2-GFP was
tyrosine-phosphorylated following ephrin-B1 stimulation and was bound
by the RasGAP SH2 domains in vitro (data not shown),
indicating that it has similar biochemical features as native EphB2.
Transfected NG108 cells were therefore differentiated with cAMP,
stimulated for 20 min with ephrin-B1 (Fig.
7A), and examined for neurite
retraction in response to activation of WT or mutant EphB2-GFP
receptors. Only the green fluorescent cells with neurite lengths
more than two times their cell body length were included (Fig.
7B). After 20-min stimulation with 2 µg/ml ephrin-B1, the
neurites of cells expressing WT EphB2-GFP cells showed a strong
retraction to 23.1 ± 6.4% (n = 13) of their original length, whereas neurites of cells expressing the
constitutively autoinhibited receptor JXFF-GFP were not
significantly retracted (91.5 ± 5.6% (n = 10) of
the original neurite length). Cells expressing the
KINGrb2-GFP (83.7 ± 6.6%, n = 14) or
JXEE-KINGrb2-GFP (92.6 ± 3.7%,
n = 10) mutants exhibited a lack of neurite retraction similar to the JXFF-GFP-expressing cells (Fig.
7B). Thus, switching the binding and signaling properties of
EphB2 from repressing Erk1/2 activation in neuronal cells, to
stabilizing or stimulating this MAPK pathway, as in the case of
KINGrb2 or JXEE-KINGrb2, also leads
to a change in the receptor's biological activity in NG108 neuronal
cells. Together, these data suggest that EphB2 signaling can be altered
by the rational design of mutant receptors with modified regulatory
sites and SH2 binding motifs.
Activated RTKs can engage both positive regulators of the Ras-MAPK
pathway, such as the Grb2·Sos1 complex, and Ras inhibitors such as RasGAP. Although the primary effect of most RTKs is to enhance
Ras-GTP loading, stimulation of EphB2 or EphA2 down-regulates GTP-bound
Ras and the level of phospho-Erk1/2 in at least some cells (28, 29). To
probe EphB2 signaling, we have attempted to alter the binding
properties of EphB2 to favor recruitment of Grb2 as compared with
RasGAP. Surprisingly, YVNV Grb2 binding motifs incorporated in place of
the JM YXXP RasGAP binding sites were apparently not
phosphorylated, even though kinase activity of the mutant receptor was
induced following ephrin-B1 stimulation, most likely due to
autophosphorylation within the activation segment of the kinase domain
(24, 25). These substitutions therefore appear to interfere with the
inhibitory interaction of the JM sequence with the kinase domain and
with recognition of the JM tyrosines by the kinase domain active site.
These data suggest that the sequence, and possibly conformation, of the
JM sequence is important not only for repression of kinase activity but
also for the phosphorylation of the JM tyrosines induced by ephrin stimulation. Consistent with this interpretation, a
JXF-Grb2 mutant, in which the tyrosine residues in the JM
YVNV motifs of JXGrb2 were changed to phenylalanine,
behaved similarly to JXGrb2 (data not shown). Despite
lacking the RasGAP binding sites in the JM region, the
JXGrb2 mutant was effectively WT in activity, in the sense
that ephrin-B1 stimulation of JXGrb2 NG108 cells still led to RasGAP recruitment (albeit less strongly than WT EphB2) and attenuation of Erk1/2 phosphorylation (Fig. 2).
We have previously found that an EphB2 mutant (EphB2-EE) in which the
JM tyrosines are changed to glutamate also retained an ability to
associate with RasGAP (28), raising the possibility of a secondary
RasGAP binding site on EphB2. Peptide binding experiments demonstrated
that the RasGAP SH2 domains were unable to associate with the JM
peptides from either JXGrb2 or EphB2-EE but weakly associated with a peptide that includes phosphorylated
Tyr667 in the kinase domain (Fig. 3A). In the
crystal structure of the EphB2 kinase domain, Tyr667 is
exposed on the upper surface of the N-terminal lobe, situated in the
Multiple sites of interaction between two signaling proteins are quite
common. In the context of Eph receptors, Yu et al. (36) have
recently reported that EphB2 and the Abl/Arg tyrosine kinases can
interact by distinct mechanisms, at two independent sites on EphB2. Why
RasGAP and Abl/Arg engage in such complex binding interactions with
EphB2 remains to be established. Multiple binding sites may serve to
finely regulate the association of an individual target with EphB2 or
to allow the selective binding of specific proteins among multiple
possible targets. Peptide competition studies have demonstrated a
relatively weak binding affinity between a phosphorylated EphB2 JM
peptide and the N-terminal RasGAP SH2 domain (18). The presence of
multiple binding sites in EphB2 may therefore stabilize its
interactions with targets such as RasGAP.
The unusual ability of Eph receptors to antagonize Erk MAPK activation
has provided an opportunity to engineer EphB2 variants in which
biochemical signaling to the Ras-MAPK pathway is switched through the
incorporation of a Grb2 binding site at Tyr667, such that
ephrin stimulation induces Erk activation. The ability of
this mutant (JXEEKINGrb2) to up-regulate
Erk phosphorylation in a ligand-inducible fashion was dependent on the
ablation of RasGAP binding sites in the JM region (while at the same
time suppressing the inhibitory effect of the JM sequence on kinase activity). Indeed a mutant with both Grb2 and RasGAP binding sites (KINGrb2) was neutral in its effects on Erk
phosphorylation. These data suggest that an Eph receptor's effects on
the Erk MAPK pathway can be titrated from inhibitory to neutral to
stimulatory by the relative incorporation of RasGAP and Grb2 binding
sites. The switch in the biochemical output of the
JXEE-KINGrb2 mutant is accompanied by a loss of
the ephrin-B1-induced neurite retraction observed in NG108 cells
expressing WT EphB2. These data are consistent with the possibility
that down-regulation of the Ras-MAPK pathway is involved in the process
of neurite retraction induced by EphB2 (28); indeed, these observations
may have a more general significance, because neuronal outgrowth
induced by netrin-1 signaling through the attractive DCC receptor
appears dependent on MAPK activation and is antagonized by inhibition
of the MAPK pathway (37).
RTKs have generally been viewed as being regulated by
autophosphorylation within their kinase domains and forming docking sites for cytoplasmic targets within non-catalytic regions of the
receptor. Although this scheme is generally applicable, it is apparent
that Eph receptors are organized in a somewhat more complex fashion, in
which the non-catalytic JM region can both regulate kinase activity and
bind SH2-containing targets. The importance of the sequence and,
likely, the conformation of the JM region in integrating Eph receptor
signaling are revealed by the observation that altering the sequence
C-terminal to the JM tyrosines interferes with both its regulatory
properties, as well as its ability to become phosphorylated and bind
cytoplasmic polypeptides. Furthermore, we have identified a site within
the kinase domain itself, Tyr667, which has the potential
to act as a docking site for SH2 proteins. Although the physiological
importance and general applicability of this observation remains to be
fully explored, it emphasizes that RTK targets may potentially interact
with the kinase domain at sequences removed from the active site. In a
similar fashion, the kinase domain of the serine/threonine-specific
protein kinase glycogen synthase kinase 3
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-alanine (Bachem) prior to peptide synthesis. Standard
Fmoc chemistry was used throughout. Fmoc-protected and -activated amino
acids were spotted in high density 24- × 18-spot arrays on 130- × 90-mm membranes using an AbiMed ASP422 robot. All washing, Fmoc, and
side-chain deprotection steps were done manually in polypropylene
containers. The amino acids were used at a concentration of 0.25 M and were twice spotted at a volume of 0.2 µl for each
coupling reaction. The peptides covering all tyrosine residues in the
EphB2 intracellular region were synthesized (see sequence in
Fig. 3 below). Following peptide synthesis and side-chain deprotection,
membranes were blocked overnight in 5% skim milk. Purified GST or GST
fusion proteins were added at 0.1 µM in TBS and incubated
for 1 h at 4 °C. Membranes were washed three times in TBS and
incubated with horseradish peroxidase-conjugated anti-GST antibody for
1 h in TBS. Detection was by SuperSignal enhanced
chemiluminescence (Pierce).
-32P]ATP, and digested with trypsin. The digests were
resolved in two dimensions on 100-µm × 20-cm × 20-cm
thin-layer cellulose plates by electrophoresis followed by ascending
chromatography. Electrophoresis was performed at pH 1.9 in 50:156:1794
88% formic acid/glacial acetic acid/water for 40 min at 1000 V on a
Hunter HTLE-7000 thin layer electrophoresis system, and ascending
chromatography was carried out in 750:500:150:600
n-butanol/pyridine/glacial acetic acid/water. The plates
were exposed to x-ray film for indicated days at
70 °C.
-galactosidase. At 48 h
post-transfection, the cells were lysed with reporter lysis buffer
(Promega Corp.). The luciferase activity in lysates was determined by a
microplate luminometer LB 96V (EG&G Berthold, Germany) and Luciferin
reagent (Promega Corp.).
-Galactosidase assays were performed as per the standard protocol (34).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
cells. These data suggest that the ability of endogenous Eph receptors
to down-regulate the Erk MAPK pathway depends on the expression of
RasGAP (Fig. 1).
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Fig. 1.
Endogenous RasGAP is required for
down-regulation of Erk MAPK phosphorylation in response to EphA2.
MEF cells that are WT and null for RasGAP were stimulated with 4 µg/ml Fc-ephrin-A1 for the indicated time points and lysed directly
in sample buffer. Lysates were resolved by SDS-PAGE and probed for
phosphorylated Erk 1/2 (upper panel) or total Erk
(bottom panel).
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Fig. 2.
Response of EphB2 wild type
(WT) and a mutant containing a tandem potential Grb2
SH2 binding motif in the JM region (JXGrb2) to ephrin
stimulation. NG108 cells were stably transfected with
pcDNA-EphB2 or pcDNA-EphB2-JXGrb2, and individual
G418-resistant clones were isolated. A and B,
parental, EphB2-, and JXGrb2-expressing NG108 cells were
serum-starved overnight and challenged with 2 µg/ml clustered
Ephrin-B1Fc for the indicated time points and lysed directly in 2×
SDS-PAGE sample buffer. The lysates were electrophoresed, blotted and
probed with antibodies against phosphorylated tyrosine (A,
top panel) or phosphorylated Erk1/2 (B, top
panel). The blots were stripped and reprobed for EphB2 receptor
and total Erk1/2 (bottom panels, A and
B, respectively). C, serum-starved parental,
EphB2-, and JXGrb2-expressing NG108 cells were incubated in
the presence or absence of 2 µg/ml aggregated ephrin-B1 for 20 min
and lysed in PLC lysis buffer. EphB2 immunoprecipitates were separated
by SDS-PAGE, the upper portion of the blot was probed with
anti-pTyr antibody (upper panel) and reprobed with
anti-EphB2 (second panel) and anti-p120-RasGAP (third
panel) antibodies. The lower portion of the blot was
probed with anti-Grb2 antibody (bottom panel).
WCL, whole cell lysate.
-32P]ATP in an in
vitro kinase assay, digested with trypsin, and analyzed by
two-dimensional tryptic phosphopeptide mapping. The resulting maps
indicated that the most prominent phosphopeptide present in WT EphB2,
likely corresponding to the phosphorylated JM site (24, 25), is missing
from the mutant JXGrb2 (Fig. 3B). Taken
together, these results indicate that the JXGrb2 mutant retains an ability to down-regulate Erk1/2 activity, and to recruit RasGAP, suggesting the presence of a secondary binding site for RasGAP.
In addition, the JXGrb2 mutant was unable to recruit Grb2, apparently due to a lack of phosphorylation at the modified JM region.
Because the JXGrb2 mutant receptor was still activated by
ephrin-B1 stimulation, these results indicate that mutation of the
residues C-terminal to the JM tyrosines interferes with both the
inhibitory activity of the JM sequence as well as its ability to become
phosphorylated upon receptor activation.
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Fig. 3.
Phosphorylated JXGrb2 peptide
binds with GST-Grb2 SH2 in vitro, but the JM tyrosine
residues in JXGrb2 are unphosphorylated in
vivo. A, arrays of tyrosine-phosphorylated
(upper row) and unphosphorylated (bottom row)
peptides encompassing all tyrosine residues in the intracellular
portion of EphB2 were synthesized directly on membrane. SHC, GAP190,
JXGrb2, and JX'Grb2 peptides contain the Grb2
SH2 binding motif of SHC, the RasGAP SH2 binding motif of p190-RhoGAP,
and the two engineered Grb2 SH2 binding motifs of the
JXGrb2 EphB2 mutants, respectively. Peptide sequences are
also listed. Membranes were incubated with 0.1 µM
purified GST fusion proteins, followed by detection with horseradish
peroxidase-anti-GST antibody. B, tryptic phosphopeptide maps
of wild type (WT) and mutant JXGrb2 of EphB2.
Wild type and mutant JXGrb2 receptors expressed in 293 cells were immunoprecipitated, autophosphorylated with
[ -32P]ATP, and digested with trypsin, and the
resulting phosphopeptides were separated in two dimensions and exposed
to film for 2 days at
70 °C for the WT and for 2 weeks at
70 °C for JXGrb2 EphB2 mutant.
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Fig. 4.
Response of EphB2 wild type
(WT) and a mutant containing a potential Grb2 SH2
binding motif in the kinase domain (KINGrb2) to ephrin
stimulation. NG108 cells were stably transfected with
pcDNA-EphB2 or pcDNA-EphB2- KINGrb2. A
and B, parental, EphB2-, and KINGrb2-expressing
NG108 cells were serum-starved overnight and challenged with 2 µg/ml
clustered ephrin-B1Fc for the indicated time points and lysed directly
in 2× SDS-PAGE sample buffer. The lysates were electrophoresed and
blotted with antibodies against anti-pTyr (A, top
panel) or phosphorylated Erk1/2 (B, top
panel). The blots were stripped and reprobed for EphB2 receptor
and total Erk1/2 (bottom panels, A and
B, respectively). C, serum-starved EphB2- and
KINGrb2-expressing NG108 cells were incubated in the
presence or absence of 2 µg/ml aggregated ephrin-B1Fc for 20 min and
lysed in PLC lysis buffer. EphB2 immunoprecipitates were separated by
SDS-PAGE, and the upper portion of the blot was probed with anti-pTyr
antibody (upper panel) and reprobed with anti-EphB2
(second panel) or anti-RasGAP (third panel)
antibodies. The lower portion of the blot was probed with anti-Grb2
antibody (bottom panel).
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Fig. 5.
The association of RasGAP and Grb2 with
different mutant EphB2 receptors. A, schematic overview
of different EphB2 mutant constructs. B, tyrosine
phosphorylation of different EphB2 mutants. HEK-293 cells were
transiently transfected with pcDNA-EphB2 or different mutant
constructs as indicated. At 48 h post-transfection, the cells were
lysed in PLC lysis buffer, and the lysates were subjected to
immunoprecipitation with anti-EphB2 antibody, electrophoresed, and
blotted with anti-pTyr antibody (top panel). Immunoblots
were subsequently reprobed for EphB2 receptor (bottom
panels). C, GST fusion protein pull-down assays of
EphB2 mutants. Sepharose beads, containing 6 µg/ml of the GST-Grb2
SH2 domain (upper panel) or the GST-GAP-both SH2 domain
(bottom panel) fusion proteins, were used to pull down WT or
mutant EphB2 from transiently transfected HEK-293 cells. The pull-down
products were electrophoresed and blotted with anti-EphB2 antibody.
D, co-immunoprecipitation of Grb2 with different EphB2
mutants. HA-tagged Grb2 and different EphB2 mutants were co-transfected
in HEK-293 cells, and an anti-HA antibody was used to immunoprecipitate
Grb2. Immunoprecipitates were subsequently separated by SDS-PAGE and
probed with anti-EphB2 (upper panel) or anti-HA
(bottom panel) antibodies, respectively.
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Fig. 6.
Response of WT EphB2 wild type and a double
mutant containing two tyrosine residues changed to glutamic acid in the
JM region and an introduced Grb2 SH2 binding motif in the kinase domain
(JXEE-KINGrb2) to ephrin stimulation.
NG108 cells were stably transfected with pcDNA-EphB2 or
pcDNA-EphB2-JXEE-KINGrb2. A,
serum-starved EphB2- and
JXEE-KINGrb2-expressing NG108 cells were
stimulated by ephrin-B1Fc for 20 min and lysed in PLC lysis buffer.
EphB2 immunoprecipitates were separated by SDS-PAGE, probed with
anti-pTyr antibody (upper panel), and reprobed with
anti-EphB2 (bottom panel) antibody. B, EphB2- and
JXEE-KINGrb2-expressing NG108 cells were
serum-starved overnight and challenged with ephrin-B1Fc for the
indicated time points and lysed directly in 2× SDS-PAGE sample buffer.
The lysates were electrophoresed and probed with antibody against
phosphorylated Erk1/2 (top panel) and reprobed for total
Erk1/2 (bottom panels). C, serum-starved EphB2-
and JXEE-KINGrb2-expressing NG108 cells were
stimulated by ephrin-B1Fc for 20 min and lysed in PLC lysis buffer.
Anti-Grb2 immunoprecipitates were separated by SDS-PAGE. The upper
portion of the blot was probed with anti-EphB2 antibody (upper
panel), and the lower portion of the blot was probed with
anti-Grb2 antibody (bottom panel). D, HEK-293T
cells were transiently transfected with wild type EphB2, EphB2-EE,
KINGrb2, KIN667F, or
JXEE-KINGrb2 in the addition of pFA-Elk1 and
pFR-Luciferase reporter constructs, and pSV2- -galactosidase. At
48 h post-transfection, the cells were washed and lysed. The
luciferase activity in lysates was determined using Promega's
luciferase assay system. The assay was repeated several times, and a
representative experiment is shown here.
-galactosidase reporter constructs. The pFA2-Elk1
trans-activator plasmid consists of the activation domain of
the Elk transcription activator fused with the DNA binding domain of
the yeast GAL4, and the pFR-Luc reporter plasmid contains a synthetic
promoter with five tandem repeats of the yeast GAL4 binding sites that control expression of the Photinus pyralis (American
firefly) luciferase gene (Stratagene PathDetect® system). Control
cells transfected with pcDNA, pFA-Elk1, and pFR-Luciferase reporter constructs had very low basal Elk1-induced luciferase activity (data
not shown). Overexpression of KINGrb2 and
JXEE-KINGrb2 resulted in a significant increase
of luciferase activity, as compared with WT EphB2 (Fig. 6D).
In addition, EphB2-EE and KIN667F expression resulted in
luciferase activity higher than WT EphB2, suggesting that loss of
RasGAP binding sites on EphB2 leads to an increase in MAPK activity. In
this assay the transfected receptors are autoactivated by
overexpression, and hence ephrin-B1 stimulation did not augment these
effects (data not shown). Collectively, these results indicate that
introduction of a Grb2 binding site into the EphB2 kinase domain can
recruit Grb2, and, in conjunction with ablation of the RasGAP binding
motifs, lead to ligand-dependent up-regulation of Erk MAPK
activity. We have therefore been able to switch EphB2 signaling to the
Erk MAPK pathway in neuronal cells from inhibitory to stimulatory by
manipulation of the receptor's regulatory sites and SH2 binding motifs.
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Fig. 7.
Neurite retraction assay in neuronal cells
expressing WT or mutant EphB2. A, images of
representative NG108 cells transiently transfected with
EphB2-GFP and stimulated with aggregated ephrin-B1Fc. NG108 cells were
transiently transfected with WT and mutant EphB2-GFP and were
differentiated by the addition of 1 mM cAMP for 5 h.
Transfectants were stimulated with 2 µg/ml clustered ephrin-B1Fc for
20 min, and cells with neurite length greater than twice the cell body
length were monitored for neurite retraction. B, relative
neurite length of WT and mutant EphB2 in response to ephrin-B1Fc. The
data are expressed as a percentage of the original neurite length
before the ligand stimulation. Each error bar represents the
mean and S.E. of more than 10 cell experiments for each
construct.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3-
C linker (6), and is therefore potentially accessible for
binding. Indeed we were able to abrogate RasGAP association with EphB2
only when both the JM motifs and the Tyr667 motif were
mutated in concert, as in the JXEE-KINGrb2
mutant (Fig. 5). Single substitutions in the JM region as in EphB2-EE (28) or JXGrb2 (Fig. 2) or in the kinase domain such as
KINGrb2 did not fully inhibit recruitment of RasGAP (Fig.
5), indicating EphB2 receptor may have multiple binding sites for
RasGAP. These suggestions are further supported by luciferase assay
results, which demonstrate that overexpression of EphB2 mutants lacking either of the RasGAP binding sites (EphB2-EE and KIN667F)
increased luciferase activity compared with WT EphB2. Introduction of a Grb2 binding site to these mutants, to make
JXEE-KINGrb2 and KINGrb2, respectively, led to further increases in luciferase activity (Fig.
7).
binds the
phosphoserine/threonine residues of primed substrates through a
positively charged pocket within the kinase domain (38). The complex
regulation and signaling properties of Eph receptors likely reflect
their sophisticated ability to control cell-cell interactions in both
embryonic and adult tissues.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Sacha J. Holland for early work on EphB2 receptor and EphB2 stable cell lines and members of the Pawson laboratory for many helpful discussions. We also thank Suzanne Del Rizzo for purification of Ephrin-B1Fc.
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FOOTNOTES |
---|
* This work was supported in part by grants from the Canadian Institutes of Health Research (CIHR) and the Ontario Research and Development Challenge Fund (to T. P.).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.
§ Both authors contributed equally to this work.
¶ Supported by a CIHR postdoctoral fellowship.
** Supported by a National Science and Engineering Research Council scholarship.
A Distinguished Scientist of the CIHR. To whom correspondence
should be addressed. Tel.: 416-586-8262; Fax: 416-586-8869; E-mail:
Pawson@mshri.on.ca.
Published, JBC Papers in Press, December 16, 2002, DOI 10.1074/jbc.M208972200
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ABBREVIATIONS |
---|
The abbreviations used are: RTK, receptor tyrosine kinase; JM, juxtamembrane; RasGAP, p120 Ras GTPase-activating protein; Erk, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; GFP, green fluorescence protein; GST, glutathione S-transferase; HA, hemagglutinin; PLC, phospholipase C; Fmoc, N-(9-fluorenyl)methoxycarbonyl; MEF, mouse embryo fibroblast; WT, wild type.
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