From the Brookdale Center for Developmental and
Molecular Biology and the § Graduate Program in Molecular,
Cellular, Biochemical and Developmental Sciences, Mount Sinai School of
Medicine, New York, New York 10029
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ABSTRACT |
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Fibroblast growth factors (FGFs) stimulate tyrosine phosphorylation of a membrane-anchored adapter protein, FRS2/SNT-1, promoting its association with Shp-2 tyrosine phosphatase and upstream activators of Ras. Using the yeast two-hybrid protein-protein interaction assay, we show that FRS2/SNT-1 and a newly isolated SNT-2 protein directly bind to FGF receptor-1 (FGFR-1). A juxtamembrane segment of FGFR-1 and the phosphotyrosine-binding domain of SNTs are both necessary and sufficient for interaction in yeast and in vitro, and FGFR-mediated SNT tyrosine phosphorylation in vivo requires these segments of receptor and SNT. Our findings establish SNTs as direct protein links between FGFR-1 and multiple downstream pathways. The SNT binding motif of FGFR-1 is distinct from previously described phosphotyrosine-binding domain recognition motifs, lacking both tyrosine and asparagine residues.
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INTRODUCTION |
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Growth factors mediate biological responses through the
coordination of multiple signals emanating from cell surface receptors. Receptors with intrinsic tyrosine kinase activity
(RTKs)1 may initiate several
biochemical pathways by recruiting distinct cytosolic signaling
proteins to specific receptor docking sites following receptor
activation. RTK autophosphorylation can generate a set of receptor
phosphotyrosine motifs that interact with Src homology-2 (SH2) or
phosphotyrosine-binding (PTB) domains on target proteins, with the
sequence surrounding each phosphotyrosine providing specificity for
particular SH2 or PTB domains (1, 2). For example, distinct
phosphotyrosine motifs on epidermal growth factor receptor (EGFR)
recruit phospholipase C- (PLC
) and the adapter protein Grb-2 (3,
4). EGFR activates plasma membrane-associated Ras G proteins by
co-recruitment of the Grb-2-associated Ras guanine nucleotide exchange
factor Sos (5). The insulin receptor (IR) uses a variation on the above
theme to trigger multiple signaling cascades. Activated IR recruits and
tyrosine phosphorylates insulin receptor substrates (IRS-1, -2, -3, and
-4) (6), which, in turn, serve as multifunctional adapter proteins by
binding Grb-2/Sos, phosphatidylinositol-3'OH kinase, and Shp-2 tyrosine
phosphatase (7-9).
Fibroblast growth factors (FGFs) stimulate receptors (FGFRs) that, similar to IR, do not directly recruit Grb-2/Sos or Shp-2. Nevertheless, several FGF-induced biological responses require the activation of both Ras and Shp-2 (10-13). Grb-2/Sos and Shp-2 bind to membrane-associated adapter proteins termed SNTs (14) following FGF-induced SNT tyrosine phosphorylation (15-17). A previously characterized myristoylated SNT protein, termed FRS2 (16), bears an N-terminal PTB domain and multiple sites of FGF-induced tyrosine phosphorylation, which engage the SH2 domains of Grb-2 and Shp-2 (16, 17).2 Grb-2/Sos recruitment to SNT-1/FRS2 promotes Ras activation (16), whereas Shp-2 phosphatase may be activated by SNT engagement, analogous in mechanism to IRS-1-mediated Shp-2 activation (18). However, a direct interaction between activated FGFRs and SNT-1/FRS2 had not been established and seemed implausible because FGFRs lack NPx(pY) canonical PTB domain binding sites (19-21).
Using the yeast two-hybrid protein-protein interaction assay, we show here that SNT-1/FRS2 and a newly identified SNT-2 protein interact with FGFR-1. The PTB domain of SNTs and a juxtamembrane segment of FGFR-1 are both necessary and sufficient for this interaction, which is also seen between recombinant protein fragments in vitro, and these domains are also required for receptor-induced SNT tyrosine phosphorylation in vivo. These findings expand the repertoire of peptide motifs recognized by PTB domains and also establish SNT proteins as a family of FGF receptor signaling adapters that structurally and functionally resemble IRS proteins.
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EXPERIMENTAL PROCEDURES |
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Reagents--
The antibodies used were: monoclonal anti-EGFR for
extracellular domain (Oncogene Science), 4G10 anti-phosphotyrosine,
anti-PLC and anti-Shc (Upstate Biotechnology), anti-FGFR-1 C
terminus and anti-GST (Santa Cruz Biotech), anti-Eps8 (Tranduction
Labs), 9E10 anti-myc tag (courtesy of T. Moran),
peroxidase-conjugated secondary antibodies (Caltag).
p13suc1-agarose was from Upstate Biotechnology Inc.
Human SNT cDNA Clones-- Human SNT-1 cDNA (GenBankTM AF036717) encodes a 508-residue protein 96% identical to murine FRS2 (16), whereas human SNT-2 cDNA encodes a 492 residue protein bearing 49% sequence identity to SNT-1 (GenBankTM AF036718). Details of cloning procedures shall appear elsewhere.2
Expression of Epitope-tagged SNT Proteins in 3T3
Cells--
SNT-1, SNT-2, and Shc cDNA open reading frames modified
with a C-terminal triple myc epitope tag (22) were cloned in
a SR promoter-driven expression vector (23). Polymerase chain
reaction with Pfu DNA polymerase (Stratagene) was used to engineer
deleted forms of myc-tagged SNT-1:
PTB, residues
1-34/138-508;
M, 1-137/418-508;
C, 1-417. Vectors were
introduced into NIH 3T3 cells or derivative clones carrying chimeric
receptors by G418 or hygromycin selection of cotransfected plasmids.
Selected cell pools treated for 10 min with 100 ng/ml acidic FGF, EGF,
or mock were lysed in 1% Nonidet P-40, immunoprecipitated with 9E10,
electrophoresed and Western blotted with 9E10 or 4G10, using
peroxidase-conjugated secondary antibodies and ECL (Amersham Pharmacia
Biotech) detection. Immunoprecipitates from cells incubated for 8 h with 100 µCi/ml [3H]myristate (New England Nuclear)
were electrophoresed, and labeled proteins were detected by
fluorography.
Signaling by EGFR/FGFR Chimeras in 3T3 Cells--
cDNAs for
human EGFR (GenBankTM X00588) and murine FGFR-1
(GenBankTM U22324) were used to construct chimeric
receptors, all of which bear the full extracellular domain of
EGFR (residues 1-644): E/FR1,
EGFR(1-644)/FGFR(377-822);
E/FR1-8,
EGFR(1-695)/FGFR(434-822);
E/FR1-10,
EGFR(1-644)/FGFR(377-762);
E/FR1-12,
EGFR(1-644)/FGFR(377-406,434-822). pSR
vectors bearing chimeric receptor or native EGFR cDNA were cotransfected with pLTRneo into NIH 3T3 cells, and G418-resistant pools were lysed before or after 10 min of EGF treatment.
p13suc1-agarose captures and immunoprecipitates were
electrophoresed and Western blotted with 4G10.
Yeast Two-hybrid Assays--
Wild-type or mutant segments of
FGFR-1 cDNA were cloned into pAS2-1
(CLONTECH) for expression as GAL4 DNA
binding domain (BD) fusion proteins: BD-WT(401-822),
BD-K(401-822/K514R),
BD-
JM(401-406, 434-822),
BD-JM1(401-434), BDJM2(401-451), BD
(empty vector). Full-length or segments of SNT cDNAs were cloned
into pACT-2 (CLONTECH) for expression as GAL4
activation domain (AD) fusion proteins: SNT-1(2-508),
SNT-1(11-140), SNT-1(164-508),
SNT-2(2-492), SNT-2(11-141), AD (empty
vector). The N-terminal SH2 domain (SH2-N) of rat PLC
was also
cloned into pACT-2. BD and AD plasmids were cotransformed into
Saccharomyces cerevisiae CG1945 (leu2,trp1,his3,
GAL1UAS:HIS3, GAL417-mers:LACZ) and selected
for acquisition of plasmids on defined medium agar
Leu,
Trp, or for
fusion protein interaction on
Leu,
Trp,
His, +1 mM
3-aminotriazole. Co-activation of LACZ was confirmed by colony lift
X-gal staining. RIPA buffer cell lysates were immunoprecipitated with
FGFR-1 antibodies, and Western blotted with 4G10- and BD-specific
antibodies.
PTB Domain in Vitro Capture Assay-- His6-tagged FGFR-1(401-451), FGFR-1(763-822), or a random express sequence tag-derived 30-kDa protein fragment were expressed from pET3D vector in BL21DE3 and purified on nickel agarose. GST-SNT-1(11-140) (PTB domain) and native GST were expressed from pGEX2T in Escherichia coli BL21DE3, purified on glutathione-agarose, and eluted with free glutathione. Nickel agarose conjugates (5 µl) were used to capture aliquots of GST proteins in Nonidet P-40 lysis buffer, which were eluted, electrophoresed, and Western blotted with anti-GST. To detect His-tagged proteins, aliquots of proteins on nickel agarose conjugates were electrophoresed and stained with Coomassie Blue.
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RESULTS AND DISCUSSION |
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Sequence Similarities between SNT and IRS Protein Families-- SNTs were originally characterized as 85,000-90,000 apparent molecular weight proteins that undergo rapid NGF- and FGF-induced tyrosine phosphorylation in neuronal cells and that bind to immobilized yeast cell cycle protein p13suc1 (14). Tyrosine-phosphorylated p13suc1-binding proteins were later characterized in several non-neuronal cell lines stimulated with FGF (15). We have cloned and characterized two human SNT genes encoding structurally related proteins. SNT-1 is 508 amino acids in length and is the human homolog of murine FRS2 (16), whereas SNT-2 is 492 residues in length and bears overall 49% amino acid sequence identity to SNT-1. SNT-1(FRS2) likely corresponds to previously described p13suc1-binding proteins (14-16), whereas SNT-2 protein displays faster gel electrophoretic mobility (see below) and does not bind to p13suc1 (data not shown). Both SNTs are myristoylated by virtue of the conserved N-terminal modification consensus sequence MGSCCS (Fig. 1, A and B). Both proteins also bear highly related PTB domains (Fig. 1A) and share several tyrosine-containing motifs in the C-terminal two-thirds of each protein that govern Grb-2 and Shp-2 recruitment following SNT tyrosine phosphorylation (16).2
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The Juxtamembrane Segment of FGFR-1 Is Required for Ligand-induced
Tyrosine Phosphorylation of SNTs--
SNT proteins are tyrosine
phosphorylated in response to FGF and neurotrophin receptor
stimulation, but not following stimulation of other receptors,
including IR and EGFR (14, 15). To localize FGFR domains required for
SNT tyrosine phosphorylation, chimeric receptors were constructed
bearing different segments of FGFR and EGFR transmembrane and
cytoplasmic regions. All chimeras contained the EGFR extracellular
domain, so that chimeric receptor signaling could be assayed in NIH 3T3
cells lacking endogenous EGFR (15). SNT was detected by capture with
p13suc1-agarose followed by anti-phosphotyrosine Western blot
(14). As shown in Fig. 2A, EGF
stimulation of E/FR1, which bears the entire transmembrane and
cytoplasmic regions of FGFR1, induced strong SNT tyrosine
phosphorylation, whereas stimulation of EGFR or chimeras E/FR1-12 and
E/FR1-8, in which juxtamembrane residues 407-433 of FGFR1 were either
deleted or replaced with EGFR sequence, respectively, induced very weak
SNT phosphorylation. E/FR1-8 and E/FR1-12 still could undergo
autophosphorylation and induce tyrosine phosphorylation of PLC (27),
Shc (24), and Eps8 (26) (Fig. 2A). By contrast, the
C-terminally truncated E/FR1-10 chimera, which lacks FGFR-1 residues
763-822, could induce SNT tyrosine phosphorylation (Fig.
2A) but not PLC
phosphorylation as expected (27).
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The PTB Domain Is Required for SNT-1 Tyrosine Phosphorylation
Following FGFR Stimulation--
Myristoylation is required for
efficient SNT-1/FRS2 tyrosine phosphorylation by anchoring the protein
to the plasma membrane (16). To determine other SNT motifs required for
FGF-induced tyrosine phosphorylation, myc-tagged SNT-1
proteins bearing deletions of residues 35-137 (PTB), 138-417
(
M), or 418-508 (
C) were expressed in 3T3 cells and assayed for
FGF-induced tyrosine phosphorylation. Although deletions of the middle
or C-terminal portions of SNT-1 did not prevent FGF-induced tyrosine
phosphorylation, deletion within the PTB domain completely abolished
tyrosine phosphorylation (Fig. 3),
despite retention of
PTB myristoylation (Fig. 1B) and association with the post-nuclear particulate fraction (data not shown), consistent with membrane localization. The requirement of the
PTB domain for SNT tyrosine phosphorylation suggested that this domain
interacts with FGFR or an intermediary protein.
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Yeast Two-hybrid Assay Detects Direct Interaction between SNT PTB Domains and Receptor Juxtamembrane Motif-- FGFR-1 and SNTs do not readily coimmunoprecipitate from mammalian cell lysates, suggesting the absence of stable FGFR/SNT complexes (15).3 We chose to use the yeast two-hybrid system to detect and characterize weaker FGFR/SNT interactions, as this assay has facilitated analysis of weak interactions between IR and IRS (28).
The cytoplasmic portion (residues 401-822) of wild-type (wt) or kinase-negative (K
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In Vitro Binding of Recombinant FGFR-1 and SNT-1 Protein Fragments-- As an additional assay for confirming direct SNT/FGFR interaction, prokaryotically expressed fragments of FGFR-1 and SNT-1 were tested for their ability to associate in vitro. FGFR-1 juxtamembrane residues 401-451, and control peptides were expressed as polyhistidine-tagged proteins and purified on nickel-agarose beads, which were then used for in vitro capture of soluble GST-SNT1(11-140) PTB fusion protein or native GST. Whereas agaroses bearing FGFR-1(763-822) or an unrelated 30-kDa polyhistidine-tagged protein nonspecifically captured a small fraction of the input GST-SNT1(11-140) protein, agarose bearing a juxtamembrane fragment of FGFR-1(401-451) quantitatively captured GST-SNT1(11-140) protein from solution but did not react with native GST (Fig. 5).
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Conclusions-- We have shown that a direct interaction between FGFR-1 and SNTs is required for receptor-induced SNT tyrosine phosphorylation. These findings establish SNTs as adapters that directly link activated FGF receptors to multiple downstream signaling pathways (Grb2/Sos and Shp-2), similar to the role of IRS proteins in governing insulin receptor signaling. The juxtamembrane sequences of FGFRs are not significantly related to cytoplasmic sequences in other RTKs, probably accounting for the failure of most RTKs to activate SNTs (14, 15).
PTB domains generally recognize peptide motifs containing the sequence NPx(pY) (19-21), although variant motifs for some PTB domains lacking phosphotyrosine or asparagine have been described (29-31). The SNT PTB domains bind to a region of FGF receptor lacking both tyrosine and asparagine residues, thereby further expanding the repertoire of motifs recognized by PTB domains. The phosphotyrosine independence of SNT/FGFR-1 interaction suggests that SNTs may stably or transiently contact FGFR-1 prior to receptor activation. A potentially important feature of SNT PTB domains is their retention of signature arginine residues at SNT positions 63 and 78 (Fig. 1A). The corresponding PTB domain arginines at positions 212 and 227 of IRS-1 interact with phosphotyrosine on insulin and interleukin-4 receptors (20, 21). We speculate that an alternative phosphotyrosine-dependent mode of epitope recognition may govern association of SNTs with other binding partners, such as the neurotrophin receptors. ![]() |
ACKNOWLEDGEMENTS |
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We thank Jon Schoorlemmer and Ron Kohanski for many helpful discussions and Ming-Ming Zhou and Hank Sadowski for critical evaluation of the manuscript.
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FOOTNOTES |
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* This work was supported by Research Grant R21-GM55666 (to M. G.) and by Predoctoral Training Grant T32-GM08553 (to K. W. L.).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.
¶ These authors contributed equally to this work.
To whom correspondence should be addressed: Brookdale Center
for Developmental and Molecular Biology, Box 1126, Mount Sinai School
of Medicine, 1 Gustave Levy Place, New York, NY 10029. Tel.:
212-241-3394; Fax: 212-860-9279; E-mail:
mgoldfa{at}smtplink.mssm.edu.
1
The abbreviations used are: RTK, receptor with
intrinsic tyrosine kinase activity; SH2, Src homology-2; PTB,
phosphotyrosine-binding; EGF, epidermal growth factor; EGFR, EGF
receptor; PLC, phospholipase C-
; IR, insulin receptor; IRS,
insulin receptor substrate; FGF, fibroblast growth factor; FGFR, FGF
receptor; BD, binding domain; AD, activation domain; GST, glutathione
S-transferase; WT, wild type; IP, immunoprecipitation; PY,
phosphotyrosine.
2 H. Xu and M. Goldfarb, manuscript in preparation.
3 H. Xu and M. Goldfarb, unpublished data.
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REFERENCES |
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