Article

Grb2 controls phosphorylation of FGFR2 by inhibiting receptor kinase and Shp2 phosphatase activity

Department of Biochemistry and Molecular Biology and Center for Biomolecular Structure and Function, University of Texas MD Anderson Cancer Center, Houston, TX 77030.
The Journal of Cell Biology (Impact Factor: 9.83). 02/2013; 200(4):493-504. DOI: 10.1083/jcb.201204106
Source: PubMed
ABSTRACT
Constitutive receptor tyrosine kinase phosphorylation requires regulation of kinase and phosphatase activity to prevent aberrant signal transduction. A dynamic mechanism is described here in which the adaptor protein, growth factor receptor-bound protein 2 (Grb2), controls fibroblast growth factor receptor 2 (FGFR2) signaling by regulating receptor kinase and SH2 domain-containing protein tyrosine phosphatase 2 (Shp2) phosphatase activity in the absence of extracellular stimulation. FGFR2 cycles between its kinase-active, partially phosphorylated, nonsignaling state and its Shp2-dephosphorylated state. Concurrently, Shp2 cycles between its FGFR2-phosphorylated and dephosphorylated forms. Both reciprocal activities of FGFR2 and Shp2 were inhibited by binding of Grb2 to the receptor. Phosphorylation of Grb2 by FGFR2 abrogated its binding to the receptor, resulting in up-regulation of both FGFR2's kinase and Shp2's phosphatase activity. Dephosphorylation of Grb2 by Shp2 rescued the FGFR2-Grb2 complex. This cycling of enzymatic activity results in a homeostatic, signaling-incompetent state. Growth factor binding perturbs this background cycling, promoting increased FGFR2 phosphorylation and kinase activity, Grb2 dissociation, and downstream signaling. Grb2 therefore exerts constitutive control over the mutually dependent activities of FGFR2 and Shp2.

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J. Cell Biol. Vol. 200 No. 4 493–504
www.jcb.org/cgi/doi/10.1083/jcb.201204106
JCB 493
Correspondence to John E. Ladbury: jeladbury@mdanderson.org; or Zamal
Ahmed: zahmed@mdanderson.org
Abbreviations used in this paper: C-SH3, C-terminal SH3 domain; ERK, extra-
cellular signal–regulated kinase; FGFR2, fibroblast growth factor receptor 2; FLIM,
fluorescence lifetime imaging microscopy; FRET, fluorescence resonance energy
transfer; FRS2, FGFR substrate 2; Grb2, growth factor receptor–bound protein 2;
MAP, mitogen-activated protein; RTK, receptor tyrosine kinase; Shp2, SH2 domain–
containing protein tyrosine phosphatase 2.
Introduction
Even in the absence of extracellular stimulation, receptor tyro-
sine kinase (RTK) phosphorylation is continuously turned over
in eukaryotic cells (Kleiman et al., 2011). Uncontrolled kinase
and/or phosphatase activity leads to aberrant signal transduction,
thus regulation of the opposing catalytic functions is required to
ensure that downstream response only occurs when an appro-
priate extracellular-stimulating ligand binds. Regulation of the
RTK broblast growth factor receptor 2 (FGFR2) and the SH2
domain–containing protein tyrosine phosphatase 2 (Shp2) is con-
trolled by the growth factor receptor–bound protein 2 (Grb2).
Previously, we observed that cells stably expressing
FGFR2 exhibit elevated receptor phosphorylation in the basal
state (Ahmed et al., 2008; Schüller et al., 2008). In the absence
of extracellular growth factor stimulus, Grb2 binds to FGFR2
via its C-terminal SH3 domain (C-SH3; Ahmed et al., 2010;
Lin et al., 2012). Grb2 is able to form a dimer and recruit two
receptor molecules into a heterotetramer. In this state at least
the two activation loop tyrosine residues (Y653 and Y654)
of FGFR2 are phosphorylated, but no downstream mitogen-
activated protein (MAP) kinase signaling is observed (Lin et al.,
2012). On engagement of the growth factor by FGFR2, recep-
tor dimerization is stabilized and autophosphorylation is up-
regulated. Grb2 is phosphorylated on a tyrosine residue (Y209)
by the fully active FGFR2, which results in dissociation from
the complex with the receptor. Release of the interaction with
Grb2 permits the FGFR2 kinase domain to access additional
tyrosine residues on the receptor, and to recruit downstream
effector proteins required for signal transduction. We also dem-
onstrated in vitro that Grb2 was able to inhibit Shp2-mediated
FGFR2 activation loop tyrosine dephosphorylation (Ahmed
et al., 2010). Grb2 therefore exerts pivotal control of receptor
C
onstitutive receptor tyrosine kinase phosphorylation
requires regulation of kinase and phosphatase
activity to prevent aberrant signal transduction.
A dynamic mechanism is described here in which the
adaptor protein, growth factor receptor–bound protein 2
(Grb2), controls fibroblast growth factor receptor 2
(FGFR2) signaling by regulating receptor kinase and SH2
domain–containing protein tyrosine phosphatase 2 (Shp2)
phosphatase activity in the absence of extracellular
stimulation. FGFR2 cycles between its kinase-active, par-
tially phosphorylated, nonsignaling state and its Shp2-
dephosphorylated state. Concurrently, Shp2 cycles between
its FGFR2-phosphorylated and dephosphorylated forms.
Both reciprocal activities of FGFR2 and Shp2 were inhib-
ited by binding of Grb2 to the receptor. Phosphorylation
of Grb2 by FGFR2 abrogated its binding to the receptor,
resulting in up-regulation of both FGFR2s kinase and
Shp2’s phosphatase activity. Dephosphorylation of Grb2
by Shp2 rescued the FGFR2–Grb2 complex. This cycling
of enzymatic activity results in a homeostatic, signaling-
incompetent state. Growth factor binding perturbs this
background cycling, promoting increased FGFR2 phos-
phorylation and kinase activity, Grb2 dissociation, and
downstream signaling. Grb2 therefore exerts constitutive
control over the mutually dependent activities of FGFR2
and Shp2.
Grb2 controls phosphorylation of FGFR2 by inhibiting
receptor kinase and Shp2 phosphatase activity
Zamal Ahmed,
1
Chi-Chuan Lin,
1
Kin M. Suen,
1
Fernando A. Melo,
1
James A Levitt,
2
Klaus Suhling,
2
and John E. Ladbury
1
1
Department of Biochemistry and Molecular Biology and Center for Biomolecular Structure and Function, University of Texas MD Anderson Cancer Center, Houston, TX 77030
2
Department of Physics, Kings College London, London WC2R 2LS, England, UK
© 2013 Ahmed et al. This article is distributed under the terms of an Attribution–
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JCB • VOLUME 200 • NUMBER 4 • 2013 494
regulation and lead to abnormal receptor function and aberrant
downstream signaling. Thus, rather than being a passive adaptor
protein, Grb2 is an essential positive and negative regulator
of receptor phosphorylation and hence ultimately downstream
signal transduction.
Results
Shp2-mediated dephosphorylation of
FGFR2 is controlled by Grb2
In an earlier in vitro experiment we demonstrated that Shp2-
mediated dephosphorylation of the FGFR2 activation loop
tyrosine residues occurs only in the absence of Grb2 (Ahmed
et al., 2010). This nding was conrmed in a cellular context
by showing that knocking down the Grb2 concentration in
three different cell lines results in lower constitutive FGFR2 phos-
phorylation. Stable Grb2-depleted HEK293T cells (80%
knockdown) overexpressing wild-type FGFR2 with a C-terminal
GFP fusion tag (
WT
FGFR2-GFP) were generated using Grb2
shRNA (Fig. 1 A). In addition, we also generated stable Grb2-
depleted A431 (Fig. 1 B) and Rat-1 (Fig. 1 C) cells that express
only endogenous FGFR2. The basal level of phosphorylated
FGFR2 (pFGFR2) was compared between Grb2 knockdown
cells (Grb2i) and those infected with a scrambled control shRNA
(Ci). In HEK293T cells overexpressing
WT
FGFR2-GFP, Grb2
knockdown resulted in a decrease in the level of basal receptor
phosphorylation (Fig. 1 A). Similarly, knockdown of Grb2 in
A431 and Rat-1 cells also led to a measurable decrease in the
level of endogenous FGFR2 phosphorylation (Fig. 1, B and C).
It should be noted that in A431 cells the efciency of Grb2
knockdown was much higher than that in Rat-1 cells, which is
reected in the respective levels of receptor dephosphorylation
observed between the two cell lines. In addition to the experi-
ments in the Grb2 knockdown background, we used a HEK293T
cell line overexpressing the oncogenic S252W mutant FGFR2
(
S252W
FGFR2). This mutant receptor is incapable of binding to
Grb2 (Fig. S1 C). In the absence of the Grb2 interaction, basal
receptor phosphorylation is reduced in a similar way to that
seen in the Grb2i cells (Fig. S2 A). The observation of decreas-
ing pFGFR2 in all three Grb2 knockdown cell lines, and in the
cells expressing
S252W
FGFR2 is consistent with (1) the increased
access of active phosphatase to the receptor in the absence of
the inhibitory effect of Grb2, and/or (2) the loss of Grb2–FGFR2
heterotetramer formation reducing the propensity for basal re-
ceptor phosphorylation.
Knocking down of Shp2 in A431 (Shp2i, Fig. 1 B) in the
presence of Grb2 shows a small increase in receptor phosphory-
lation under nonstimulated conditions, suggesting that Shp2 is a
phosphatase for FGFR2 and that Grb2 inhibition of Shp2 activ-
ity in normal cells is not 100% effective. However, to conrm
that Shp2 is responsible for the dephosphorylation of FGFR2 in
the absence of Grb2, we measured the effect of a Shp2 inhibitor
(NSC87877) on the pFGFR2 level in HEK293T cells in which
Grb2 had been knocked down (Fig. 1 D). NSC87877 is a potent
inhibitor that is selective for Shp2 over other PTPs (except the
highly homologous Shp1; Chen et al., 2006). In Grb2i cells the
concentration of pFGFR2 was reduced, corroborating the data
phosphorylation–dephosphorylation; however, the mechanistic
details for this important, constitutive role remain elusive.
Ubiquitously expressed Grb2 forms a heterotetrameric com-
plex with FGFR2 but plays a more familiar role in linking RTKs
to the MAP kinase signaling pathway (Lowenstein et al., 1992;
Chardin et al., 1993; Rozakis-Adcock et al., 1993). Grb2 largely
consists of a central SH2 domain sandwiched between N- and
C-terminal SH3 domains. Grb2 is a highly abundant protein and
is able to interact with numerous cellular phospho- and nonphos-
phoproteins through its SH2 and SH3 domains, respectively.
Somatic mutations in FGFR2 have been associated with
a number of human cancers (Jang et al., 2001; Pollock et al.,
2007; Dutt et al., 2008; Byron et al., 2010), whereas missense
germline mutations of the fgfr2 gene are seen in congenital
skeletal disorders (Wilkie et al., 1995; Johnson et al., 2000;
Yu et al., 2000; Goriely et al., 2010; Turner and Grose, 2010).
Alternative gene-splicing events provide numerous structural
variants of FGFR2. C-terminal sequence splicing provides a
major group of FGFR2 isoforms. Variants that result in dele-
tions of the C-terminal sequence show enhanced transform-
ing activity (Cha et al., 2008) and are expressed in increased
amounts in gastric, bladder, and stomach cancer cell lines
(Hattori et al., 1990, 1996; Itoh, et al., 1994; Ishii et al., 1995)
and in a majority of human breast carcinoma cells (Cha et al.,
2009). Furthermore, point mutations in the C-terminal region
of FGFR2 have recently been linked with melanoma (Gartside
et al., 2009). The C terminus of FGFR2 harbors numerous
sites for the recruitment of downstream signaling effector pro-
teins, thus perturbation of this region of FGFR2 can contribute
to oncogenesis.
Shp2
(also known as protein tyrosine phosphatase non-
receptor type 11 [PTPN11]) can be recruited by several RTKs
either directly, or indirectly via auxiliary adaptor proteins
(Freeman et al., 1992; Feng et al., 1993; Barford and Neel., 1998;
Grossmann et al., 2010). Its phosphatase activity is thus impor-
tant in regulation of intracellular signaling activity (Holgado-
Madruga et al., 1996; Kouhara et al., 1997; Neel et al., 2010). Shp2
is auto-inhibited by an intramolecular SH2 domain–mediated
interaction, displacement of which is necessary to promote
catalytic activity (Hof et al., 1998). Although there is some con-
jecture in the eld, it has been reported that activated RTKs
phosphorylate tyrosine 542 (Y542) on Shp2, which appears to
enhance phosphatase activity (Lu et al., 2001; Araki et al., 2003;
Keilhack et al., 2005).
Here we provide the mechanism for the control by Grb2
of FGFR2 kinase and the Shp2 phosphatase activity in the ab-
sence of extracellular stimulation. Grb2 plays the pivotal role in
regulating the level of FGFR2 phosphorylation through con-
trolled inhibition of (1) the kinase activity of the receptor neces-
sary for full autophosphorylation and signal transduction (Lin
et al., 2012); (2) the kinase activity of the receptor required to
phosphorylate Shp2; and (3) the phosphatase activity of Shp2
directed at FGFR2. Our data demonstrate the mutual depen-
dency of FGFR2 and Shp2 on Grb2 to dictate their respective
cellular activity and maintain homeostasis of receptor phos-
phorylation in the nonstimulated state. Perturbation of this con-
trol in the nonstimulated cell can disrupt appropriate FGFR2
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495Grb2 controls FGFR2 and Shp2 activity • Ahmed et al.
Colocalization and binding were imaged under serum-starved
conditions (Fig. 2, A–D). As expected, in nonstimulated cells
FGFR2-GFP is localized primarily in the plasma membrane,
whereas Shp2-RFP displays a diffused distribution (Fig. 2,
A–D). In both the wild-type Ci control cells and Grb2i cells no
signicant interaction between FGFR2-GFP and Shp2-RFP
was seen (Fig. 2, A and B, basal), as shown by the population
distribution of the uorescence lifetimes being largely at longer
lifetimes (right shifted) than that of isolated GFP at 2.0 ns (indi-
cated by the vertical line through the right-hand panels), i.e.,
there
is no evidence of FRET between the GFP and RFP uor-
ophores. Because the FGFR2–Shp2 interaction might only
occur on transient catalytic turnover of receptor substrate phos-
photyrosine residues, we repeated the FLIM experiments in cells
transfected with the Shp2 C459S “substrate-trapping” mutant,
the catalytic activity of which is compromised, and the phos-
phatase domain interacts with the substrate in an equilibrium-
binding mode only (Agazie and Hayman, 2003; Blanchetot
et al., 2005). Again we see no signicant evidence of binding
between receptor and phosphatase in the presence of Grb2
(Fig. 2 C). However, in the absence of Grb2, binding of Shp2
C459S mutant to FGFR2 can be observed (Fig. 2 D; peak in
right-hand panel shifts to left of the vertical line, i.e., to shorter
lifetimes resulting from FRET between GFP and RFP). These
data support a model in which the presence of Grb2 inhibits
direct binding of Shp2 to the receptor. The presence of Grb2
bound to the C terminus of FGFR2 may sterically hinder access
of the phosphatase to its cognate site, and/or inhibit the recep-
tor kinase activity toward a tyrosine residue(s) required for
Shp2 recognition.
By way of comparison, we show that on stimulation of
FGFR2 with FGF9 the population of Shp2 and FGFR2 complex
increases signicantly (Fig. 2, E and F; see population shifts to
shorter lifetimes in right-hand panels). This is consistent with
the model in which stimulation and up-regulation of kinase
activity of FGFR2 results in dissociation of Grb2 (Lin et al.,
2012), promoting formation of the Shp2–FGFR2 complex.
FGFR2-mediated phosphorylation of Shp2
is controlled by Grb2
Phosphatase activity has been reported to be enhanced by tyrosyl
phosphorylation on Shp2. Shp2 has two C-terminally located
tyrosine residues (Y542 and Y580) that have been implicated
in the mechanism for release of the auto-inhibited state of the
phosphatase (Lu et al., 2001). It has been suggested that phos-
phorylation of one (or both) of these tyrosines is important in
Shp2 activation, although there has been no clear demonstration
that tyrosyl phosphorylation of Shp2 is necessary to initiate sig-
naling. However, phosphorylation of Shp2 does appear to be in-
volved in effecting downstream extracellular signal–regulated
kinase (ERK) activation (Araki et al., 2003).
RTKs have been shown to phosphorylate Shp2, although
a role for FGFR2 in this activity has not been reported. To dem-
onstrate that Shp2 is a substrate for FGFR2, HEK293T Ci serum-
starved cells were incubated with and without FGFR2 kinase
inhibitor (SU5402) for 2 h. Fig. 3 A shows in the Ci nonstimu-
lated cells there is a background level of phosphorylated Y542
from Fig. 1, A–C. Incubation with the Shp2 inhibitor resulted
in an increase in pFGFR2 to levels similar to those seen in Ci
cells (Fig. 1 D). Again in HEK293T cells overexpressing the
S252W
FGFR2 mutant receptor, which is unable to bind Grb2, in-
cubation with NSC87877 resulted in increased basal receptor
phosphorylation (Fig. S2, B–D). These data strongly suggest
that Grb2 constitutively inhibits the ability of Shp2 to dephos-
phorylate the receptor. Furthermore, it also infers that a basal
level of FGFR2 phosphorylation prevails even in the absence
of Grb2. Thus, it appears that in wild-type cells Grb2 serves to
stabilize and control this FGFR2 kinase activity through hetero-
tetramer formation.
Grb2 inhibits direct complex formation
between FGFR2 and Shp2
Having demonstrated that dephosphorylation of FGFR2 by Shp2
is impeded in the presence of Grb2, we investigated whether
Grb2 was inhibiting the formation of a direct equilibrium com-
plex between the receptor and phosphatase. Formation of a sta-
ble complex is likely to be important in directing phosphatase
activity toward FGFR2. To measure direct binding of Shp2 to
FGFR2 we used uorescence lifetime imaging microscopy
(FLIM) to detect stable complexes through uorescence reso-
nance energy transfer (FRET) between uorophore-tagged pro-
teins. RFP-tagged Shp2 was transfected into HEK293T Ci
and Grb2i cells that were stably overexpressing FGFR2-GFP.
Figure 1. Knockdown of Grb2 and Shp2 reveal that the level of
WT
FGFR2
phosphorylation is controlled by Grb2. (A) Total HEK293T cell lysates were
immunoblotted with anti-pFGFR antibody (top), and reprobed for total FGFR
(middle) and Grb2 (bottom). Anti-pFGFR2 antibody is specific for A loop
residues Y653 and Y654. (B) Cell lysates of overnight serum-staved stable
A431 cells containing control shRNA (Ci), Grb2-shRNA (Grb2i), or Shp2-
shRNA (Shp2i) were analyzed for FGFR2 phosphorylation as above. Only
the nonstimulated state is shown (i.e., each lane is duplicated). Numbers
on
pFGFR2 panel are normalized intensity pFGFR2/total FGFR2. (C) Analy-
sis of FGFR2 phosphorylation in Rat-1 fibroblast cells with control shRNA
(Ci) and Grb2-shRNA (Grb2i) as above. Only the nonstimulated state was
investigated (i.e., each lane is duplicated). (D) Inhibition of Shp2 in Grb2
knockdown cells restores basal receptor phosphorylation. Serum-starved
WT-Ci and WT-Grb2i cells were incubated with 50 µM NSC87877 for
4 h and the resultant cell lysates were analyzed by Western blotting with
anti-pFGFR2 antibody, Shp2 pY542-specific antibody, and anti-Grb2 anti-
body. The immunoblot was stripped and reprobed for total FGFR2 and
Shp2 as the loading control. The numbers on the pFGFR2 panel represent
normalized intensity pFGFR2/total FGFR2.
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JCB • VOLUME 200 • NUMBER 4 • 2013 496
FGFR2 kinase activity is switched on in the nonstimulated
state, as demonstrated by phosphorylation of activation loop
tyrosine residues Y653/Y654 (Fig. 1, A–C). Here we conrm
the occurrence of this activity under nonstimulated conditions
by the observation of a higher molecular weight band corre-
sponding to phosphorylated Grb2 (also described by Lin et al.,
2012) that is apparent in HEK293T cells overexpressing FGFR2
(Fig. 4 A). This phosphorylation of Grb2 can be attributed to
FGFR2 kinase activity because the higher molecular weight
band is absent in wild-type HEK293T cells, which do not ex-
press endogenous FGFR2 kinase. Thus, the phosphorylation
and subsequent dissociation of Grb2 can occur in the nonstimu-
lated state.
Accumulation of increasing cellular concentrations of
pGrb2 (which cannot bind receptor) would result in release of
the controlling inuence of Grb2 on FGFR2 kinase activity;
thus, dephosphorylation of Grb2 is required to maintain cellular
homeostasis in the nonstimulated state. Using an in vitro experi-
ment in which puried full-length wild-type Shp2 (
WT
Shp2)
was incubated with phosphorylated C-SH3 domain of Grb2 we
demonstrated that Shp2 can dephosphorylate Grb2 on residue
(pY542) on Shp2. In the presence of the FGFR2 inhibitor the
concentration of pY542 is reduced. This strongly suggests that
Shp2 is a substrate for basal FGFR2 kinase activity and thus the
receptor is able to enhance phosphatase activity. We have thus
identied a catalytic cycle around Shp2 phosphatase dephos-
phorylating FGFR2, and FGFR2 kinase phosphorylating Shp2.
To assess whether constitutive FGFR2 phosphorylation of
Shp2 is under the control Grb2, phosphorylation of Y542 on
Shp2 was investigated in the presence or absence of Grb2. In
A431-Grb2i cells the concentration of phosphorylated of Shp2
is elevated by 2.5-fold compared with A431-Ci cells in the
nonstimulated state (Fig. 3, B and C). The presence of Grb2
therefore has an inhibitory effect on FGFR2-derived phos-
phorylation of the phosphatase.
Shp2 binds to and dephosphorylates Grb2
Previously we reported that Grb2 can be phosphorylated by
FGFR2. Phosphorylation of Y209 in the C-terminal SH3 do-
main (C-SH3) of Grb2 in the interface with FGFR2 sterically
and/or electrostatically hinders complex formation, resulting in
the release of phosphorylated Grb2 (pGrb2; Lin et al., 2012).
Figure 2. Grb2 inhibits the interaction of Shp2 with FGFR2.
(A) FLIM analysis of the FRET between the FGFR2-GFP and
RFP-Shp2. In the control (WT-Ci) serum-starved cells no inter-
action between FGFR2 and Shp2 was observed in the basal
state. The mean FRET lifetime is 2.0 ns (line in right-hand
panel), which corresponds to the mean lifetime for isolated
GFP. No apparent interaction between FGFR2 and Shp2 in
WT-Grb2i cells (B), or WT-Ci cells transfected with the RFP-
tagged substrate-trapping C459S Shp2 mutant (C). Inter-
action between FGFR2 and Shp2 is observed in the Grb2i
the substrate-trapping C459S mutant (D). Stimulating cells that
contain
WT
Shp2 or C459S mutant Shp2 with FGF9 results in
clear binding between FGFR2 and Shp2 after 15 min (E and F),
respectively. Bar, 10 µm.
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497Grb2 controls FGFR2 and Shp2 activity • Ahmed et al.
level of interaction between Shp2 and Grb2 is observed,
as shown by slight left shift in peak position and overall distri-
bution under the curve compared with CFP alone (line drawn
at the lifetime of isolated CFP, 2.2 nm; see right-hand panels
of Fig. 5, A and B). The shift to shorter lifetimes is more pro-
nounced in the stimulated state, presumably due to the presence
of increased concentrations of phosphorylated Shp2 (Fig. 5 B).
The nonphosphorylatable Y542F Shp2 mutant showed less abil-
ity to bind Grb2, even under stimulated cellular conditions
(Fig. 5 C). This demonstrates that binding to, and hence dephos-
phorylation of Grb2 by Shp2 is, at least in part, mediated by the
binding of pY542. Interestingly, the substrate-trapping C459S
mutant shows signicant binding in the basal state (almost
the entire population of tagged protein is left shifted; Fig. 5 D).
Because the substrate-trapping mutant binds to the protein in an
equilibrium mode, the observed enhanced population of com-
plex, compared with that in the presence of
WT
Shp2, suggests
that wild-type phosphatase is turning over pGrb2.
The above observations are important because they con-
rm the cycling of Grb2 between its FGFR2-phosphorylated
state and Shp2-dephosphorylated state. This cycling of Grb2
is fundamental to the maintenance of homeostasis of FGFR2
kinase activity. The rate of cycling maintains a steady-state level
of the phosphorylation of Grb2 and ensures that the phosphory-
lated receptor population is controlled and incapable of effect-
ing a downstream response. On exposure to an extracellular
ligand this cycling is presumably pushed toward increased con-
centrations of pGrb2 and hence expanding the population of
up-regulated FGFR2 molecules.
Y209 (Fig. 4 B). There are two phosphorylatable tyrosines on
C-SH3 (Y160 and Y209; Lin et al., 2012). To assess whether
Shp2 phosphatase activity was specic for either site we indi-
vidually mutated each of these residues to a nonphosphorylat-
able phenylalanine. Fig. 4 B shows that the amount of pGrb2
of the Y160F decreases in the presence of Shp2, but not in the
Y209F mutant. This result shows that Shp2 can dephosphory-
late the C-SH3 domain of Grb2 and is specic for pY209.
To conrm phosphatase activity of Shp2 toward pGrb2 in
a cellular context, HEK293T cells were cotransfected with
GFP-FGFR2 and strep-tagged Grb2 (strep-Grb2) and incubated
with either an FGFR2 kinase inhibitor (SU5402) or a Shp2
inhibitor (NSC87877), and the level of pGrb2 was determined
(Fig. 4 C). Under nonstimulated conditions, in the absence of
either inhibitor we observed the presence of a low level of
pGrb2. pGrb2 is not observed in the presence of the kinase in-
hibitor, conrming that the basal FGFR2 kinase activity is
required for Grb2 phosphorylation. Inhibition of the phospha-
tase resulted in increased concentrations of pGrb2 compared
with when the enzyme is uninhibited.
Having established that Grb2 is a substrate for Shp2, we
investigated the mechanism by which the phosphatase is re-
cruited. Because Shp2 phosphorylated on Y542 has been previ-
ously reported to bind Grb2 and in doing so affect auto-inhibition
of the phosphatase domain (Lu et al., 2001), we conrmed that
this was important for complex formation between these two
proteins in cells. FLIM data on A431 cells expressing N-terminal
CFP-tagged Grb2 (CFP-Grb2) and N-terminal RFP-tagged
Shp2 (RFP-Shp2) reveal that in the nonstimulated state a low
Figure 3. Shp2 phosphorylation by FGFR2 is inhibited by
Grb2. (A) Serum-starved HEK293T cells were incubated with
30 µM FGFR inhibitor (SU5402) for 2 h and then either stimu-
lated with 10 ng/ml FGF9 for 15 min or left untreated. Cell
lysates were prepared and analyzed by Western blotting
with the indicated antibody. Anti-pFGFR and anti-Y542 on
Shp2 antibodies were used to evaluate phosphorylation of
proteins. The immunoblot was stripped and reprobed with a
pan-antibody to determine total protein level. (B) Comparison
of ligand-stimulated Shp2 phosphorylation between A431-Ci
and A431-Grb2i cells in nonstimulated and on stimulation
by FGF2 or FGF9 for 1 h. Shp2 phosphorylation was de-
tected with anti-pY542 antibody (top). The immunoblot was
reprobed for total Shp2 as a loading control (middle) and
with Grb2 (bottom). (C) Densitometric quantification of basal
state Shp2 phosphorylation levels in A431 cells in control
shRNA (A431-Ci) and Grb2-shRNA (A431-Grb2i). Error bars
represent SD, n = 7.
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JCB • VOLUME 200 • NUMBER 4 • 2013 498
To demonstrate the cycling of Grb2 between the receptor-
bound/dephosphorylated state and the unbound/phosphory-
lated state we used an in vitro FRET-based spectroscopic assay
(Fig. 6 A). The cytoplasmic region of FGFR2 (FGFR2
cyto
resi-
dues 400–821) and full-length Grb2 N-terminally labeled with
GFP and RFP fusion proteins, respectively, were expressed and
puried from Escherichia coli. Initially the uorescence lifetime
of isolated GFP-FGFR2 was measured (2.64 ns; Fig. 6 B).
On adding RFP-Grb2 the uorescence lifetime in solution
was measured as they formed the complex in which two recep-
tor molecules were recruited to the Grb2 dimer (Fig. 6 A and
Fig. S3; Lin et al., 2012). The formation of the complex results
in FRET between the GFP and RFP uorophores, and hence, a
decrease in the uorescence lifetime. ATP/Mg
2+
was added to
activate the FGFR2 kinase and the uorescence lifetimes were
measured at given time points. The lifetime was seen to in-
crease as a consequence of the reduction in direct interaction
between receptor and adaptor protein as Grb2 is phosphory-
lated by the activated RTK (Fig. 6 B). The lifetime reaches a
steady state as the concentration of pGrb2 maximizes (2.64 ns).
Shp2 was then added to the solution of FGFR2
cyto
and Grb2
(Fig. 6 A) and the lifetime was recorded. The presence of the
phosphatase results in the decrease in the uorescence lifetime
of GFP-FGFR2
cyto
due to FRET as the concentration of pGrb2
is reduced and the resulting dephosphorylated Grb2 binds to the
receptor (Fig. 6, A and B). These in vitro data mirror the observa-
tions in cells described in Fig. 4 by reproducing the interactions
and turnover of Grb2 phosphorylation in the presence of kinase
and phosphatase.
By way of additional conrmation that Grb2 was a sub-
strate for Shp2 we used the substrate-trapping C459S mutant
of Shp2 in the uorescence spectroscopic assay. This mutant
has no inherent catalytic activity, and hence Grb2 should not be
dephosphorylated in its presence. The addition of this mutant
results in no reduction in the uorescence lifetime, consistent with
no recovery of the Grb2–FGFR2 complex (Fig. 6 B). Further-
more, to conrm the requirement of pY542 to promote phos-
phatase activity against Grb2 we used the nonphosphorylatable
Y542F mutant Shp2 in place of
WT
Shp2. In this case the recov-
ery of the Grb2–FGFR2 complex was abrogated (the uores-
cence lifetime remains at 2.64 ns; Fig. 6 C), reecting the
reduction in the binding of the mutant Shp2 to Grb2 and hence
reduced dephosphorylation of Grb2.
We conrmed these observations using the independent
approach of time-correlated single-photon counting (TCSPC)
to measure the GFP-FGFR2
cyto
lifetime. This experiment mea-
sures FRET between the RFP-Grb2 and FGFR2-GFP uor-
ophore fusion tags in a similar way to the previous experiment;
however, rather than measuring one lifetime at a specic wave-
length, this method uses a dicrotic lter to detect uorescence
emission over a range of wavelengths. The data are measured
and presented as a percentage of the total population of inter-
actions (Fig. 6 C and Fig. S3). The FLIM data for the interaction
between FGFR2
cyto
and Grb2 in the presence of
WT
Shp2 and the
C459S and Y542F mutants was entirely consistent with the
spectroscopic data. Interestingly, after an extended period (18 h)
in the presence of the Y542F Shp2 mutant, the percentage of
Figure 4. Shp2 dephosphorylates Grb2. (A) Wild-type or FGFR2 stably
transfected HEK293T were starved overnight, then stimulated using 10 ng/ml
FGF9 for either 15 or 60 min. Cells were lysed in the presence of prote-
ase and phosphatase inhibitors. 50 µg of total cell lysate were used for
immunoblotting studies. Phosphorylation of FGFR2 was examined using
anti-pFGFR2 (first panel). To examine the Grb2 phosphorylation states in
the absence or presence of FGFR2 expression, 1 mg of total cell lysates
were used for immunoprecipitation using an anti-Grb2 antibody, and
probed with an anti-Grb2 antibody. The immunoprecipitated Grb2 from
FGFR2-overexpressing cells show multiple bands (both serum starved and
FGF9 stimulated), suggesting the high molecular weight species is tyrosine-
phosphorylated Grb2, which is only phosphorylated in the presence of
FGFR2. (B) Recombinant Grb2 C-SH3 mutants (Y160F, left; Y209F, right)
were expressed and purified from E. coli and incubated with pure FGFR2
cytoplasmic domain in a 1:1 molar ratio in the presence of ATP and MgCl
2
at room temperature for 1 h. Recombinant GST-fused pShp2 was obtained
via the same protocol. A general anti-pY antibody was used to examine the
phosphorylation state of FGFR2-phosphorylated Shp2 (lanes 4, 6, 10, and
12; panel 1) and Grb2 C-SH3 domains (lanes 2 and 8; panel 4). A specific
anti-pY542 Shp2 antibody was also used to confirm that Y542 of Shp2 is
phosphorylated. A pool of both proteins was dephosphorylated by mixing
phosphatase (either pShp2 or Shp2) with phosphorylated protein substrates
(either pGrb2 C-SH3 Y160F or phospho-Grb2 C-SH3 Y209F) at 4°C over-
night. The anti-pY blot shows only the pGrb2 C-SH3 Y160F can be dephos-
phorylated by both pShp2 and Shp2 (lanes 5 and 6; panel 4). However,
the phosphorylation state of pGrb2 C-SH3 Y209F is not affected by Shp2,
suggesting that the Y209 is the target of Shp2. A total Shp2 antibody (panel 3)
and total Grb2 antibody (panel 5) were used to confirm equal protein
loading. (C) HEK293T cells were cotransfected with FGFR2-GFP and Grb2-
strep-tag. After 48 h cells were starved for 4 h and incubated with either
FGFR-specific inhibitor (50 µM SU5402) or Shp2-specific inhibitor (100 µM
NSC87877) for 1 h. Cell lysates were subjected to affinity purification
using strep-tactin agarose beads and immunoblotted with anti-pY antibody
(top) followed by anti-Grb2 antibody (bottom).
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Page 6
499Grb2 controls FGFR2 and Shp2 activity • Ahmed et al.
MAP kinase pathway once cells are stimulated). However,
Grb2-depleted HEK293T and A431 cells do display lower levels
of growth factor–stimulated MAP kinase activity as reected
in levels of phosphorylated ERK (pERK; Fig. 7, A, C, and D;
Fig. S4, A and C). One explanation for this could be that the
binding of Grb2 to FGFR2 stabilizes receptor dimers, increas-
ing the probability of active signaling on growth factor stimula-
tion. This obviously would not occur in the absence of Grb2.
Another possibility is that in the absence of Grb2, ligand-
activated receptors are rapidly and concomitantly deactivated
by Shp2, rendering them signaling incompetent. This would
result in reduced FGF-stimulated MAP kinase activation. We
investigated this latter hypothesis using the Shp2-specic inhib-
itor to demonstrate that, if Shp2 is responsible for deactivating
FGFR2 in Grb2i cells, then its inhibition would allow MAP
interactions increases. This is presumably the result of phospha-
tase activity that still occurs, but the rate is signicantly reduced
in the absence of the amplifying effect of the pY542 interaction
with Grb2.
Shp2 inhibition restores MAP kinase
activity in Grb2 knockdown cells
The focus of this work is on the cycling of kinase and phospha-
tase activity under the control of Grb2 in the nonstimulated
state. However, this control could ultimately have an effect on
signal transduction once the cells are exposed to growth factor.
The general viability of Grb2 knockdown cells indicates some
of the primary functions generally associated with the presence
of Grb2 in these cells are still intact (e.g., the reduction in over-
all Grb2 concentration does not abrogate signaling through the
Figure 5. Interaction between Grb2 and
Shp2. CFP-Grb2 and RFP-Shp2 colocalization
and direct interaction measurement using FLIM
in A431 cells. (A) Control lifetime measurement
for CFP alone. (B) Interaction of CFP-Grb2 with
RFP-tagged wild-type Shp2 (RFP-
WT
Shp2) at
basal and after 20 ng/ml FGF9 stimulation.
(C) Co-localization and direct interaction of
Y542F mutant Shp2 with CFP-Grb2 at basal
and after FGF9 stimulation. (D) Constitutive
interaction of the C459S substrate-trapping
Shp2 mutant with Grb2. A left-shifted peak rel-
ative to the line drawn along 2.2 ns indicates
a binding. A peak centered on the 2.2 ns line
indicates nonbinding. Bar, 20 µM.
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Page 7
JCB • VOLUME 200 • NUMBER 4 • 2013 500
and MAP kinase activation, as manifested by phosphorylation
ERK (Fig. 2, A and C), were monitored under basal conditions
and after FGF9 stimulation. The results of these experiments
show that inhibition of Shp2 in Grb2 knockdown cells does
indeed restore FGF9-stimulated pERK (Fig. 7, A and C). Further-
more, decreased
WT
FGFR2 phosphorylation in Grb2i cells can
be rescued by Shp2 inhibition (Fig. 7, A and B). Shp2 inhibition
in Grb2-depleted A431 cells also leads to increased receptor
phosphorylation and restoration of the FGF-stimulated MAP
kinase response (Fig. 7, D and E). However, Shp2 inhibition
elicits little effect in EGF-stimulated MAP kinase, which re-
mains unchanged (Fig. 7 F and Fig. S4 B). This therefore pro-
vides further evidence that Grb2 is an inhibitor of Shp2 in
FGFR2 signaling, and in the absence of Grb2, Shp2 inhibition
limits receptor activation and downstream signaling. Importantly,
the Shp2 inhibitor pretreatment increases basal receptor phos-
phorylation, eliciting a signicant downstream MAP kinase
response. This increase in receptor phosphorylation appears
signicantly higher than the specic FGF-induced receptor
phosphorylation that restored the downstream MAP kinase
response (Fig. 7). This observation is important because it em-
phasizes that receptor phosphorylation alone is not sufcient for
signal transduction.
Discussion
The constitutive turnover of RTK phosphorylation requires
control mechanisms to abrogate the possibility of aberrant sig-
nal transduction. This can be achieved by controlling kinase
activity, blocking the attainment of the fully active state, and
inhibiting recruitment of downstream effector proteins to the
RTK. The data reported herein establish a role for Grb2 in which
it applies a controlling inuence over the basal kinase activity
of FGFR2 and inhibits Shp2-mediated receptor dephosphory-
lation. This central control exerted by Grb2 can be represented
by the cycle of contributing catalytic activity shown in Fig. 8
and summarized below.
Our recent ndings revealed that under nonstimulated
conditions, dimeric Grb2 can bind to two receptor molecules
resulting in partial phosphorylation of FGFR2 including activa-
tion loop tyrosine residues (Lin et al., 2012; Fig. 8 A). In forming
this heterotetrameric complex Grb2 inhibits both the dephos-
phorylation of FGFR2 by Shp2 and the phosphorylation of
Shp2 by FGFR2 (Fig. 8, B and C, respectively). The inhibition
of the dephosphorylation of partially phosphorylated FGFR2
(Fig. 8 B) can be demonstrated in cells in which Grb2 expres-
sion has been knocked down which show negligible basal
receptor phosphorylation (Fig. 1). In the absence of Grb2, Shp2
is able to interact with FGFR2. This results in receptor dephos-
phorylation. As a result, inhibition of Shp2 under these con-
ditions results in the recovery of the phosphorylated receptor
(Fig. 1 D and Fig. 7). Not only does this conrm an inhibitory
role for Grb2 on phosphatase activity, but it also suggests that
an inherent background phosphorylation of FGFR2 occurs even
in the absence of Grb2 in nonstimulated cells. We show that
inhibition of Shp2 activity is likely to result from the inability of
Shp2 to bind to the receptor in the presence of the Grb2–FGFR2
Figure 6. In vitro demonstration of catalytic cycling of FGFR2 and Shp2
in the presence of Grb2. (A) Schematic of interactions performed in vitro
to demonstrate catalytic activity of FGFR2 and Shp2 on Grb2. Mixing
FGFR2
cyto
(blue) with Grb2 (red) promotes the formation of a heterotetra-
meric complex (Lin et al., 2012). Addition of ATP and MgCl
2
to this results
in phosphorylation of FGFR2 and Grb2 (green circle). Addition of Shp2
(orange) results in dephosphorylation of FGFR2 and Grb2 (blue line). The
heterotetrameric complex is recovered under these conditions. (B) Fluor-
escence lifetime measurement between GFP-FGFR2
cyto
and RFP-Grb2 as
a function of time. The first point corresponds to the fluorescence lifetime
for isolated GFP-FGFR2 (black arrow). On addition of Grb2 (red arrow)
a heterotetrameric complex between Grb2 and FGFR2 forms. This results
in FRET between the GFP and RFP and the concomitant reduction in fluor-
escence lifetime. On addition of ATP/Mg
2+
(purple arrow) up-regulation
of the RTK ensues and Y209 on Grb2 becomes phosphorylated and the
FGFR2–Grb2 complex dissociates. The lifetime increases, reflecting reduc-
tion in complex concentration and the accumulation of pGrb2. After 80 min
Shp2 was added (orange arrow). At this point clear reassociation of
Grb2 and FGFR2 is observed as Grb2 is dephosphorylated in the presence
of Shp2 and consequently the fluorescence lifetime decreases (blue line on
graph). Replacing
WT
Shp2 with the Y542F (red line) or C459S (green line)
mutant results in no immediate reduction in lifetime, confirming that the
FGFR2–Grb2 complex is not rescued by adding these compromised phos-
phatases. (C) Measurement of FRET between GFP-FGFR2
cyto
(Cyto) and
RFP-Grb2 in solution using FLIM. Cyto alone is GFP-FGFR2
cyto
and repre-
sents the background false-positive percentage FRET readout. Cyto+Grb2
is the population of molecules undergoing FRET when RFP-Grb2 is present.
Cyto+Grb2+ATP is the population of GFP-FGFR2
cyto
undergoing FRET with
RFP-Grb2 when the FGFR2 kinase was