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INSERM, U-145 and IFR 50, 06107 Nice, France
Address all correspondence and requests for reprints to: Dr. Laurent Delahaye, INSERM U-145 and IFR 50, Faculté de Médecine, avenue de Valombrose, 06107 Nice Cedex 2, France. E-mail: delahaye{at}unice.fr
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Abstract |
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We were interested in finding a new molecule(s), tyrosine phosphorylated by the insulin receptor (IR), that could interact with Shp-2. To do so, we screened a human placenta complementary DNA (cDNA) library with the SH2 domain-containing part of Shp-2 using a modified yeast two-hybrid system. In this system we induce or repress the expression of a constitutive active IR ß-subunit. When expressed, IR phosphorylates proteins produced from the library that can then associate with Shp-2.
Using this approach, we isolated FRS2 as a potential target for tyrosine phosphorylation by the IR. After cloning the entire cDNA, we found that 1) in the yeast two-hybrid system, FRS2 interacts with Shp-2 in a fashion dependent on the presence of the IR; and 2) in the PC12/IR cell-line, insulin leads to an increase in FRS2 association with the phosphatase.
We next wanted to determine whether FRS2 could be a direct substrate for IR. In an in vitro kinase assay we found that wheat-germ agglutinin-purified IR phosphorylates glutathione-S-transferase-FRS2 fusion protein. Finally, in intact cells we show that insulin stimulates tyrosine phosphorylation of endogenous FRS2.
In summary, by screening a two-hybrid cDNA library, we have isolated FRS2 as a possible substrate for IR. We found that IR can directly phosphorylate FRS2. Moreover, in intact cells insulin stimulates tyrosine phosphorylation of FRS2 and its subsequent association with Shp-2. Taken together these results suggest that FRS2 could participate in insulin signaling by recruiting Shp-2 and, hence, could function as a docking molecule similar to insulin receptor substrate proteins.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunit
of its receptor (IR), the tyrosine kinase activity of the ß-subunit
becomes active, leading to IR autophosphorylation and subsequently
tyrosine phosphorylation of substrates. These tyrosine-phosphorylated
molecules then act as docking proteins, recruiting SH2
domain-containing effectors such as phosphatidylinositol 3-kinase (PI
3-kinase), the adaptor growth factor receptor-bound protein-2 (Grb-2),
and the phosphotyrosine phosphatase Shp-2 (1). The PI 3-kinase pathway triggers several cellular responses, such as 1) glucose transport, 2) glycogen synthesis, and 3) protein synthesis (2, 3). Grb-2 binding to tyrosine-phosphorylated substrates associates Sos, activates p21ras, and subsequently activates the mitogen-activated protein (MAP) kinase pathway. Shp-2 is a member of the phosphotyrosine phosphatase family. It was first described as the corkscrew gene product in Drosophila implicated in the torso tyrosine kinase receptor signaling pathway (4). Shp-2 is composed of two SH2 domains in its N-terminus and a catalytic domain at its C-terminus. The N-terminal SH2 domain of Shp-2 negatively regulates the phosphatase activity in resting conditions by binding the catalytic region. Upon binding to tyrosine-phosphorylated proteins such as insulin receptor substrate-1 (IRS-1) or Shc, the N-terminal SH2 domain no longer represses the catalytic domain, which allows subsequent activation of the enzyme (5). Shp-2 is phosphorylated in response to many growth factors, such as platelet-derived growth factor (PDGF) and epidermal growth factor (EGF), but not in response to insulin. Whether this phosphorylation influences the activation level of Shp-2 remains unclear. A Shp-2 molecule, deleted in 65 amino acids of the N-terminal SH2 domain, is lethal in utero at midgestation for mice homozygous for the mutant allele, suggesting a major role of Shp-2 in development (6).
Shp-2 acts as a positive mitogenic transducer downstream of tyrosine kinase receptors, as shown by the fact that injection of either glutathione-S-transferase (GST)-SH2 of Shp-2 or antibodies to Shp-2 leads to inhibition of DNA synthesis in Rat1 cells (7). Furthermore, expression of a catalytically inactive mutant of Shp-2 in NIH-3T3 cells stably expressing insulin receptors blocks insulin-induced activation of MAP kinases (8). In CHO/IR cells, this catalytically inactive mutant or the N-terminal SH2 mutant fails to induce c-fos transcription in response to insulin (9). Considering these data, Shp-2 appears to be a key component in mitogenic signaling induced by various growth factors, such as PDGF, EGF, and insulin.
Although it has been demonstrated that upon PDGF stimulation the Grb-2/Sos complex is recruited to the PDGF receptor through binding to Shp-2 (10) and that this leads to stimulation of MAP kinases, it is not clear how Shp-2 enhances MAP kinase activity.
To identify tyrosine-phosphorylated substrates that recruit Shp-2 when phosphorylated by the IR or new proteins that interact with Shp-2, we screened a human placenta complementary DNA (cDNA) library using Shp-2 as bait. For this purpose we engineered a modified two-hybrid system in which we induce the expression of IR at the same time as two fusion proteins. The first is composed of the DNA-binding domain fused to Shp-2, and the second comprises the activation domain fused to a potential interacting protein. This system allows IR to phosphorylate proteins expressed from the library, revealing phosphotyrosine-dependent interaction(s) with the Shp-2 SH2 domain. Using this strategy, we identified a protein as a potential Shp-2-interacting molecule when phosphorylated by the IR tyrosine kinase. This protein has a region homologous to the phosphotyrosine-binding (PTB) domain of IRS-1, a consensus myristylation sequence in its amino-terminus, and putative binding sites for adaptors such as Grb-2 in its C-terminal part. After cloning, we found in the NCBI Blast Databank that this protein was similar to FRS2/SNT, a molecule shown to link the FGF receptor (FGFR) to the Ras-MAP kinase pathway (11, 12). Further, we examined whether FRS2 could bind Shp-2 in cells exposed to insulin. We found that Shp-2 associates with FRS2 after insulin treatment of a PC12 cell line stably expressing the IR. Further, FRS2 incubated with IR in vitro becomes tyrosine phosphorylated, indicating that FRS2 is likely to be a direct IR substrate. Consistent with this view, we observed in PC12/IR cells, that insulin leads to tyrosine phosphorylation of FRS2.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Oligonucleotides were purchased from Life Technologies, Inc. (Paisley, Scotland), restriction enzymes were obtained from New England Biolabs, Inc. (Beverly, MA), Pwo DNA polymerase was obtained from Roche Molecular Biochemicals (Strasbourg, France), and synthetic defined dropout yeast media lacking the appropriate amino acids were purchased from BIO 101 (La Jolla, CA). Cell culture media and geneticin were obtained from Life Technologies, Inc.. All chemical reagents used were purchased from Sigma (St. Louis, MO), except protein A-Sepharose, which was obtained from Pharmacia Biotech, Inc. (Uppsala, Sweden).
We produced precipitating rabbit antibodies to FRS2 against amino acids 325–508 fused to GST protein. Blotting antibodies to FRS2 were provided by J. Schlessinger (New York, NY). Antibodies to Shp-2 and Grb-2 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
The PC12/IR cell line was previously described (14).
Plasmid construction
The coding sequence of the IR cytoplasmic domain was inserted in
the plasmid pVJL9-IR downstream of the repressible promoter MET25 as
previously described (13). The SH2 domains of Shp-2 cDNA and p85 cDNA
were also subcloned in pVJL9-IR in-frame with the DNA-binding domain of
Lex A (15). The fusion proteins obtained (LexA-n/cSH2-Shp-2 and
LexA-p85) corresponded to the LexA DNA-binding domain 1–147 fused to
both proteins. Correct in-frame fusion between LexA and both Shp-2 and
p85 cDNA was verified by sequencing.
Full-length FRS2 was amplified by PCR using the following primers: sense, 5'-ccggaattcgaatgggtagctgttgtagctgtcc-3'; and antisense, 5'-cggctcgagccaggctcacatgggcagatcagtactattgtgtctag-3'. Then we subcloned it in-frame into pGex-4T2 and pActII polylinker using EcoRI (5'-side) and XhoI (3'-side). Correct in-frame fusion between GST and FRS2 cDNA was verified by sequencing. Correct in-frame fusion between the activation domain of Gal4 and FRS2 was verified by sequencing.
Yeast transformation and reporter gene activity
L40 yeast were transformed with the pVJL-IR-n/c-SH2-Shp-2 used
as a bait and a human placenta cDNA library expressed in pGAD10 by the
improved lithium acetate method of Gietz et al. (16).
Selection of positive clones was realized according to the
manufacturer’s recommendation (CLONTECH Laboratories, Inc.).
To study protein-protein interactions, L40 yeast were cotransformed with pBTM116 (or pVJL-IR) and pActII plasmids expressing hybrid proteins of interest, using the lithium acetate method (16). L40 were grown for 48 h on plates containing Trp-, Leu- complete supplemented medium to select clones containing both plasmids (pVJL-IR and pACTII carry the Trp+ and Leu+ selection markers, respectively). Suppression of IRß gene expression carried on the pVJL-IR vector was accomplished by addition of L-methionine (Sigma) at 2 mM to the medium.
The histidine reporter gene was tested by replicating the clones expressing the different sets of plasmids on plates containing complete supplemented medium without tryptophan, leucine, and histidine and by growing them at 30 C for 48 h. Double transformants were also assayed for ß-galactosidase activity, using a color filter assay as previously described (17).
Selection of positive clones using the yeast two-hybrid
system
After yeast transformation with both the human placenta cDNA
library containing vector and the vector carrying the bait, we isolated
clones growing on selective media. We then tested the specificity of
interactions to discriminate false positives. To do so we first tested
their ability to induce transcription of the ß-galactosidase reporter
gene by color filter assay (17). To discriminate between yeast
containing the vector with the bait and yeast with the vector carrying
the cDNA from the library, we grew the yeast and isolated the cDNA.
Thereafter, we transformed HB101 bacteria auxotropic for leucine,
growing only in presence of the vector from the library. We isolated
the cDNA from those cells and retransformed yeast to confirm the
interaction in the presence, but not in absence, of IRß-subunit. We
also tested the interaction of the clone with unrelated proteins such
as Ras or lamin to confirm the specificity.
cDNA amplification of the partial clones obtained by screening the
human placenta library
To isolate the full-length sequence of the partial cDNA clone
obtained by using a two-hybrid library, we screened a human liver
Marathon-ready cDNA library with the Marathon-ready cDNA amplification
kit (CLONTECH Laboratories, Inc.) and the advantage cDNA
polymerase mix (CLONTECH Laboratories, Inc.) according to
the conditions recommended by the manufacturer. The PCR products were
subcloned in the pCR 2.1 vector using Original TA Cloning Kit
(Invitrogen).
Northern blot analysis
The FRS2 cDNA (reading frame) was amplified by PCR and purified.
[-32P]Deoxy-CTP radiolabeled FRS2 cDNA probe
was prepared using a random priming kit (Amersham Pharmacia Biotech, Piscataway, NJ), and Northern blot analysis was
performed using a rat Multiple Tissue Northern (CLONTECH Laboratories, Inc.). The hybridization was realized according to
the manufacturer’s recommendations. The membrane was washed with
0.2 x SSC (standard saline citrate) and 0.1% (wt/vol) SDS and
visualized by autoradiography.
Cell culture and transfection
PC12/IR cells were cultured in RPMI 1640 medium containing 10%
(vol/vol) FCS, 10% (vol/vol) horse serum, and 500 µg/ml geneticin.
Cells were starved for 16 h before the experiment in 0.2%
(wt/vol) BSA and 0.2% Biomedia serum (Foster City, CA).
In vitro kinase assay
Wheat-germ agglutinin (WGA)-purified IR (150 fmol) was incubated
for 25 min in HNT buffer (50 mM HEPES, 150 mM
NaCl, and 0.1% Triton X-100, pH 7.4) containing 4 mM
MnCl2, 8 mM
MgCl2, 30 µM ATP, and 1
mM vanadate with or without 10-7
M insulin. GST protein or various amounts (1, 10, or 20
µg) of GST-FRS2 were preincubated with glutathione-coupled Sepharose
beads for 1 h at 4 C. Then WGA-IR, activated or not, was added to
the GST-FRS2 pellets in addition to
[
-32P]ATP. After 30 min of shaking at room
temperature, the supernatant was removed, and the pellets were washed
three times with HNT buffer containing 50 mM HEPES, 150
mM NaCl, and 0.5% Triton X-100, pH 7.4. The pellets were
dried, and the proteins were resuspended in Laemmli buffer before being
subjected to SDS-PAGE under reducing conditions. The gel was incubated
with Coomassie Blue in trichloroacetic acid to visualize the GST-FRS2
and then autoradiographed.
Immunoprecipitation and immunoblotting
Cells were pretreated with 2 mM vanadate
(Na3VO4) for 40 min,
stimulated or not with 10-6 M
insulin for 5 min, and washed in PBS. Then they were incubated in lysis
buffer containing 20 mM HEPES, 150 mM NaCl, 1.5
mM MgCl2, 50 mM NaF, 1
mM EDTA, 1 mM EGTA, 10 mM
Na4P2O7,
10% glycerol, 2 mM vanadate, 1 mM
PhMeS02F, 100 UI/ml aprotinin, 20
µM leupeptin, 2 µM pepstatin, 4
mM benzamidine, and 1% (vol/vol) Triton X-100. The lysates
were centrifuged at 15,000 x g at 4 C for 15 min, and
the samples were immunoprecipitated at 4 C with the appropriate
antibodies preadsorbed on protein A coupled to Sepharose beads. After
3 h, the pellets were washed with lysis buffer and 2
mM vanadate, and the proteins were analyzed by
SDS-PAGE under reducing conditions. Then the proteins were transferred
to an Immobilon membrane (Immobilon polyvinylidene difluoride,
Millipore Corp., Milford, MA). After blocking the membrane
with TBS buffer (10 mM Tris-HCl and 140
mM NaCl, pH 7.4) containing 0.5% (wt/vol) BSA
and cutting it, we immunoblotted with antibodies to FRS2, Shp-2, or
phosphotyrosine followed by [125I]protein A and
autoradiography or used the Enhanced Chemiluminescent Light System
(Pierce Chemical Co., Rockford, IL).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). L40 cells
were transformed with two plasmids. One plasmid contains the nSH2- and
cSH2-containing part of Shp-2 fused to the DNA-binding domain of LexA.
The other contains cDNAs from a library fused to the activation domain
of Gal4. In addition, we repressed or induced expression of the active
IR ß-subunit (repression of expression is induced by
L-methionine in the culture medium). Hence, the IR
ß-subunit is able to phosphorylate the protein expressed from the
library, which allows for a phosphotyrosine-dependent interaction with
the nSH2 and/or cSH2 domain(s) of Shp-2. Yeast containing both plasmids
grow on Trp-, Leu-
medium. In clones interacting with the bait, transcription of the HIS
reporter gene is induced, permitting growth on
His- medium. Our system is schematized in Fig. 1A.
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-32P]deoxy-CTP. This was used as a probe on
the rat Multiple Tissue Northern membrane (CLONTECH Laboratories, Inc.). As shown in Fig. 1B
, we observed that the corresponding
messenger RNA is about 6.5 kb. This messenger RNA is present in every
tissue tested, but at a higher level in brain, lung, liver, kidney, and
testis. To obtain the full-length coding region of this protein, we used the Marathon-ready cDNA amplification kit (CLONTECH Laboratories, Inc.). We screened a human liver cDNA library and obtained a cDNA corresponding to the complete coding region with approximately 1.5 kb. This cDNA was 99.5% identical to one cloned by Rabin et al. (18) coding for SNT (suc-associated neurotropic factor-induced tyrosine phosphorylated target) and more recently identified by Kouhara et al. as FRS2 (FGF receptor substrate-2) (11). Our cDNA displays 7 different nucleotides of 1524, and in 2 cases it leads to amino acid changes, L175M and S447F. SNT is involved in the Ras-independent nerve growth factor signaling pathway, promoting neuronal differentiation (19). FRS2 has been characterized in fibroblast growth factor (FGF) signaling, where it links FGFR tyrosine kinase activity to the Ras/MAP kinase pathway, promoting FGF-induced neurite outgrowth (11). However, the fact that we cloned this protein using IR indicates that this protein may also be involved in insulin signaling and act as a substrate for insulin receptors.
FRS2 interacts with Shp-2 in the yeast two-hybrid system
As IRS proteins interact with SH2-containing proteins such as
Shp-2 and p85, we tested in our two-hybrid system whether FRS2 would
bind to Shp-2 and p85, the regulatory subunit of PI 3-kinase. We
subcloned full-length cDNA of FRS2 in pActII vector and coexpressed
FRS2 in yeast with p85 or different forms of Shp-2 subcloned in pVJL-IR
vector. Next we repressed or induced expression of IR by growth in the
presence or absence of L-methionine. Figure 2 represents the results obtained with a
color filter assay. When FRS2 is coexpressed with wild-type (WT) Shp-2,
no interaction is observed. This could be explained if Shp-2 catalytic
activity can dephosphorylate the necessary tyrosine(s) on FRS2 that
functions as a docking site for the SH2 domain of the phosphotyrosine
phosphatase. Hence, we used a catalytically inactive form of Shp-2, in
which cysteine 459 of the catalytic domain is replaced by a serine,
resulting in an inactive enzyme (8). This Shp-2 mutant can interact
with FRS2, but only when IRß is expressed (Fig. 2). We also tested
the n/cSH2-containing part of Shp-2 that does not have the catalytic
domain of the phosphatase. Yeast cotransformed with this construct
shows blue coloration within 1 h, reflecting interaction with
FRS2. This occurs only in presence of IRß, as repression of the IRß
expression prevents interaction. We did not detect any association
between FRS2 and p85 in the presence of IR as might be expected, as
FRS2 does not possess YXXM motifs that are docking sites for PI
3-kinase regulatory subunits. Taken as a whole these results suggest
that 1) FRS2 can bind to both a catalytically inactive form of Shp-2
and the n/cSH2 domain of Shp-2 dependent on the presence of IR; and 2)
FRS2 does not interact with p85, even in presence of IRß.
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). In unstimulated conditions, Shp-2 is
poorly associated with FRS2, but after insulin treatment, an increased
amount (
35%) of Shp-2 is coimmunoprecipitated with FRS2 (Fig. 3B).
We conclude that in intact PC12/IR cells, Shp-2 associates with FRS2 in
an insulin-dependent fashion.
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We first tested whether purified IR phosphorylates FRS2 in an in
vitro kinase assay. We incubated WGA-IR with or without insulin
(10-7 M) for 25 min in the
presence of 30 µM ATP. Then WGA-IR was added to
increasing amounts of GST or GST-FRS2 in the presence of
[-32P]ATP. After 30 min, we washed the
pellets and analyzed the proteins by SDS-PAGE. The experiment presented
in the lower panel of Fig. 4
illustrates the increasing amounts of GST-FRS2 on the Coomassie
blue-stained gel. The upper panel is an autoradiogram
showing that WGA-IR previously activated with insulin phosphorylates
GST-FRS2, whereas nonactivated WGA-IR does not. The level of
phosphorylation increases in parallel with the amount of fusion protein
incubated. GST alone was not phosphorylated by the WGA-IR, even after
insulin incubation. To conclude, in an in vitro kinase
assay, WGA-purified IR phosphorylates FRS2 upon insulin activation,
suggesting that FRS2 is probably a direct substrate of IR.
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, lower panel). We then
stripped the membrane and reblotted it with antiphosphotyrosine
antibodies. As shown in the upper panel of Fig. 5, insulin
strongly induced tyrosine phosphorylation of FRS2.
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Discussion |
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We tested whether in the absence of IR expression the interactions detected in the yeast two-hybrid system between FRS2 and other proteins occur. We found that FRS2 interacts with Shp-2 in a phosphotyrosine-dependent manner, as the repression of IR by L-methionine abolishes all interactions observed when IR is expressed. Those experiments in the yeast two-hybrid system show that IR should phosphorylate FRS2, allowing the interaction with Shp-2.
Using Northern blotting we found that FRS2 appears to be ubiquitously expressed, but is most abundant in brain, lung, liver, kidney, and testis. As Shp-2 is involved in insulin-induced Ras activation (24) and has been described as a positive effector in insulin signaling (9), we wanted to determine whether FRS2 associates Shp-2 in insulin-treated cells. We found that in unstimulated PC12/IR cells, a small fraction of FRS2 is bound to Shp-2. Upon insulin stimulation, an increased amount of Shp-2 associates with FRS2. FRS2 contains a consensus myristylation sequence in its amino-terminus, which lies in a MGXXXS/T motif (25). This myristylated head targets FRS2 to the membrane and is essential for membrane localization, tyrosine phosphorylation, Grb2/Sos recruitment, and MAP kinase activation in response to FGF (11). In the light of our findings, Shp-2 could also be recruited to the membrane through its interaction with FRS2 upon insulin treatment of cells.
In PDGF signaling, the PDGF receptor binds and phosphorylates Shp-2, which then acts as an adaptor linking Grb2/Sos complex to activation of the Ras/MAP kinase pathway. In contrast, Shp-2 is not phosphorylated in response to insulin, and thus, the mechanism by which Shp-2 exerts an effect in insulin signaling remains unclear. We have attempted to detect alterations in Grb2 association with FRS2 in insulin-treated cells. However, we found no change in the Grb2 protein content associated in FRS2 immunoprecipitates from insulin-treated cells (data not shown). FRS2 contains docking sites for Grb2 at Tyr196, Tyr306, Tyr349, and Tyr392 (11), and Shp-2 contains also potential binding sites at Tyr279, Tyr304, and Tyr356 for the Grb2 SH2 domain (26). Considering these data, we conclude that in insulin-treated cells Grb2 is not recruited by the FRS2/Shp-2 complex, as it does not associate with either FRS2 or Shp-2 under these conditions.
FRS2 contains a region that is 29% homologous to the IRS-1 PTB domain. In contrast to genuine IRS molecules, FRS2 does not contain a pleckstrin homology (PH) domain. This domain is implicated in targeting IRSs to the membrane, probably by binding phospholipids. IRS-1 lacking its PH domain is no longer able to undergo in vivo tyrosine phosphorylation in response to insulin stimulation (27). FRS2 does not possess a PH domain, but its myristylated tail may target it to the membrane in a manner analogous to the PH domains of other IRSs. In addition, FRS2 could interact through its PTB domain with the IR before phosphorylation.
We examined whether FRS2 could be a direct substrate for the tyrosine kinase of the insulin receptor. We found in an in vitro kinase assay that GST-FRS2 could be phosphorylated by WGA-purified IR. Furthermore, we observed that in intact cells FRS2 is tyrosine phosphorylated after treatment with insulin. It should be mentioned that we cannot exclude the possibility that upon insulin treatment of cells, another kinase is activated and phosphorylates FRS2. However, based on our in vitro association and phosphorylation data we favor the idea that FRS2 is a direct substrate of IR.
Taking these results as a whole, we suggest that FRS2 may act as an adaptor molecule in insulin-treated cells. After phosphorylation by IR, FRS2 would recruit the SH2-containing effector(s), such as the phosphotyrosine phosphatase Shp-2. IRS-1 is phosphorylated upon insulin treatment by interacting with the IR ß-subunit. The IRS-1 PTB domain binds to the juxtamembrane tyrosine 960, whereas the IRS-1 PH domain links to membrane phospholipids (28). If FRS2 undergoes tyrosine phosphorylation by IR in intact cells, we hypothesize that its myristyl tail may target it to the membrane while its PTB domain interacts with the NPXY motif comprising tyrosine 960.
It was recently proposed that FRS2 could compete with Shc for binding to the TrkA receptor, as both proteins bind to the same phosphotyrosine of the receptor (29). Hence, FRS2 could also compete with other IRS molecules or adaptor proteins such as Shc or Gab-1.
It has been suggested that Shp-2 could play dual roles in cells. Kim et al. (30) demonstrated that the gp130 subunit of the IL-6 receptor can bind Shp-2, which then undergoes tyrosine phosphorylation. When the gp130-binding sites on Shp-2 are mutated, phosphorylation of the receptor, the associated JAK activity, and the DNA-binding activity of STAT1 and STAT3 are maintained at elevated levels for a prolonged time compared with WT gp130. This suggests that Shp-2 could play a negative role by dephosphorylating substrates and attenuating cytokine signaling. Myers and al. (31) also showed that insulin-induced tyrosine phosphorylation of IRS-1 on residues 1172 and 1222 leads to binding and subsequent activation of Shp-2. When they mutated both tyrosines, IRS-1 became more heavily phosphorylated on tyrosine, and more PI 3-kinase was associated with IRS-1, leading to an increase in protein synthesis. However, they did not find changes in the proliferative behavior of cells expressing the IRS-1 mutant compared with WT-IRS-1. Those data demonstrate that Shp-2 could down-regulate, at least in this cellular context, the metabolic effect of insulin transduced through PI 3-kinase. In contrast to these examples of negative regulation of signaling by Shp-2, numerous studies using deficient Shp-2 cell lines or injection of inhibitory antibodies have demonstrated the positive role of Shp-2 in growth factor signaling. Recently, Takada et al. showed that Shp-2 is recruited to the membrane by the SHPS (SHP substrate) in response to insulin (32). Shp-2 binds the Y449 and Y473 of SHPS, is activated, and potentiates insulin-induced MAP kinase activation, suggesting a positive role for Shp-2 in insulin signaling.
Whether FRS2 is playing overall a positive or a negative role in insulin signaling remains to be determined. After FRS2 is tyrosine phosphorylated by the IR, Shp-2 moves to the membrane and binds FRS2. This is thought to activate the phosphatase activity, possibly leading to MAP kinase activation and enhancement of gene expression. Alternatively, Shp-2 could dephosphorylate FRS2 or the receptor, and by doing so, down-regulate signaling by the tyrosine kinase receptor.
Finally, we can envision that cross-talk between signaling by insulin and FGF could impact on FRS2. The skin disorder acanthosis nigricans is often found in disease states associated with extreme insulin resistance. It is intriguing to note that acanthosis nigricans is also observed in Crouzon syndrome, which is known to be due to mutations of FGFR3 (33, 34). Therefore, the issue of whether for this skin disorder FRS2 could be the point on which insulin receptor and FGF receptor signals converge represents a potentially promising area of investigation.
To summarize we have shown that FRS2 may act as a direct substrate of the activated IR. Furthermore, after its phosphorylation, FRS2 is able to recruit Shp-2 in intact cells in response to insulin, probably through interaction with the SH2 domains of Shp-2. Therefore we propose a role for FRS2 as a newly identified IRS.
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Acknowledgments |
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Footnotes |
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Received July 13, 1999.
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