INTRODUCTION

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Growth factor-initiated signal transduction is required in multiple developmental pathways. Mice with naturally occurring mutations in receptor tyrosine kinases (RTKs), such as c-kit (W) (reviewed in reference 65) and the epidermal growth factor receptor (EGFR) (42), have gross phenotypic abnormalities, as well as signaling defects in specific cell populations. The EGFR homolog let-23 is essential for vulval development in Caenorhabditis elegans (5), whereas multiple RTKs are required for normal Drosophila development (reviewed in references 18, 19, 59, 60, and 68). In Xenopus, overexpression of a dominant negative form of fibroblast growth factor (FGF) receptor 2 (FGFR2) results in severe gastrulation defects and abnormal mesoderm formation (3, 4). Likewise, mice lacking FGFR1 die prior to or during gastrulation (16, 86), demonstrating that FGF signaling pathways are critical for early development in vertebrates.

Biochemical and genetic studies have identified several downstream components of RTK signaling cascades. Following growth factor binding, tyrosyl phosphorylation sites on the activated receptor recruit secondary signaling molecules containing SH2 domains, which couple RTK activation to cytoskeletal and nuclear events (reviewed in reference 13). SH2-containing secondary signaling proteins include enzymes, such as phospholipase C (PLC) and SHP-2, and adapters, which contain other modules such as SH3, PTB, and PH domains. One central pathway in signaling from multiple RTKs is the Ras/Raf/MEK/mitogen-activated protein (MAP) kinase (MAPK) cascade (reviewed in references 44 and 67). This pathway involves the SH2/SH3-containing adapter, Grb2, which is bound via its SH3 domains to the Ras exchange protein, Sos. The Grb2 SH2 domains either bind directly to activated RTKs or, in some pathways, bind to adapters such as IRS-1, FRS-2 (35), and/or Shc (for a review, see reference 46). Genetic evidence for the role of Grb2 was provided by studies of its Drosophila homolog, drk (53), and its C. elegans homolog, sem-5 (12). The importance of the MAPK pathway in RTK-driven developmental pathways also has been demonstrated in invertebrates (reviewed in reference 83). However, the roles of most other secondary signaling molecules have not been validated by genetic analyses. There is virtually no information regarding the functions of specific subdomains in vivo.

SH2-containing protein-tyrosine phosphatases (SHPs) are key components of RTK signaling pathways and play important developmental roles (reviewed in references 48 and 78). SHPs share the same overall architecture, with two SH2 domains at their N termini, a protein-tyrosine phosphatase (PTP) domain, and a C terminus containing sites for tyrosyl and seryl/threonyl phosphorylation and, in some cases, a proline-rich domain (for reviews, see references 22 and 47). Two SHPs exist in vertebrates. SHP-1 is expressed primarily in hematopoietic and epithelial cells, where it acts predominantly as a negative regulator of RTK and cytokine receptor signaling pathways (for reviews, see references 10, 48, and 79). This is illustrated most vividly by the phenotype of mice with defective SHP-1 genes (motheaten and motheaten viable mice [reviewed in reference 10;]]). The ubiquitously expressed SHP-2, despite a high degree of sequence similarity to SHP-1 (approximately 60% overall identity), appears to have distinct functions. Experiments with tissue culture cells using dominant negative mutants (1, 8, 52, 87, 88) or antibody microinjection approaches (8, 28, 64, 85) established SHP-2 as a required positive component in several RTK pathways, acting upstream of MAPK. The Drosophila homolog of SHP-2, corkscrew (csw), is required in multiple developmental pathways (2, 57, 58). In Xenopus, SHP-2 is required for basic (bFGF)-induced MAPK activation, mesodermal marker induction, and completion of gastrulation; these functions cannot be subserved by SHP-1 (74). Most likely, SHP-2 has a similar role in early development of all vertebrates, since mice homozygous for targeted disruptions of SHP-2 die by embryonic day 10.5 (6, 66) and exhibit gastrulation defects and impaired bFGF and platelet-derived growth factor signaling (66).

Why the two vertebrate SHPs have distinct biological functions has remained unclear. They appear to participate in several of the same pathways; in some cases, they even bind to the same receptors (e.g., c-Kit, IL3-R, and EpoR) (reviewed in references 48 and 79). Their SH2 domains seem to recognize similar phosphotyrosyl peptides (see Discussion). Both also are enzymatically activated in vitro upon binding of phosphotyrosyl peptides to their respective SH2 domains (14, 15, 31, 38, 54, 56, 61, 63, 73, 80).

The molecular details by which SHP-2 participates in RTK signaling in vivo also have remained unclear. SHP-2 binds, via its SH2 domains, to phosphotyrosine motifs in several activated RTKs and adapters or accessory molecules, including IRS-1 (36), Gab1 (30), Dos (29), and SHPS1/SIRP1 (25, 33). Nevertheless, the importance of the SH2 domains in signaling in vivo has not been demonstrated explicitly. In response to stimulation by some growth factors, SHP-2 is phosphorylated on one or both of the tyrosyl residues in its C-terminal tail, Y542 and Y580 (9, 82). These sites can bind to Grb2 (9, 40, 75, 76, 84), which led to the suggestion that SHP-2 might act as an adapter, coupling RTKs to the Ras pathway (9, 40, 47). However, the biological significance of this interaction has not been demonstrated. Recently, tyrosyl-phosphorylated SHP-2 also has been found to bind to the SH2 domain of the inositol monophosphatase SHIP in cytokine-stimulated hematopoietic cells (41); the function of this complex and whether it occurs in nonhematopoietic cells remain unclear. Between the two C-terminal tyrosines is a proline-rich region, which potentially could bind SH3 and/or WW domain-containing proteins. SHP-1 lacks this proline-rich region, raising the possibility that it could contribute to SHP signaling and/or specificity. However, no proteins have been shown to bind to the SHP-2 proline-rich region, nor has a function for this domain been demonstrated. Experiments using PTP-inactive mutants of SHP-2 strongly suggest that the PTP domain is required for normal signaling (see above), but whether the PTP domain also contributes significantly to SHP specificity has remained undetermined.

We chose to address these unresolved questions by using bFGF-induced mesoderm induction as an experimental model. Signals from vegetal pole cells of Xenopus embryos direct the presumptive ectodermal cells of the animal cap to adopt a mesodermal cell fate (50). Mesoderm induction can be evoked, to various degrees, by purified growth factors, including bFGF, serving as the basis for the animal cap assay (reviewed in reference 34). Animal cap explants cultured in low-salt media undergo ectodermal differentiation. Addition of bFGF results in dramatic elongation of the explants, MAPK activation, and induction of mesodermal markers.

It has been shown previously that expression of an SHP-2 mutant lacking 31 amino acids of the PTP domain (P) (see Fig. 1A) in animal caps blocks normal bFGF responses, demonstrating that SHP-2 and, in particular, its PTP domain are required downstream of the Xenopus FGFR (XFGFR) (74). We now have determined which other domains of SHP-2 are necessary for transmission of the XFGFR signal. Both SH2 domains of SHP-2 are required, although the N-terminal SH2 domain (N-SH2) is more critical than the C-terminal SH2 domain (C-SH2). The C-terminal tyrosines and the proline-rich region are dispensable for rescue of the dominant negative effects of P, suggesting that the adapter model is not the major mechanism for bFGF signal transmission. Finally, by analyzing the effects of SHP-2/SHP-1 chimeras, we have found that the PTP domain of SHP-2 accounts for much of the specificity between SHP-1 and SHP-2 in bFGF-induced mesoderm induction. Our results establish an absolute requirement for the SH2 and PTP domains of SHP-2 in this pathway, provide the first demonstration that biological specificity resides within the phosphatase domain of a PTP in vivo, and suggest that PTP specificity is determined by combinatorial mechanisms.

	MATERIALS AND METHODS

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Mutant construction. Constructs containing RK mutations in the essential arginines within the FLVRES sequences of N-SH2 (R32), C-SH2 (R138), and both SH2 domains (R32,138) of human SHP-2, cloned into pET vectors, were described previously (73). An upstream EcoRI site was introduced into the 5' ends of the pET constructs by PCR using the oligonucleotides 5'-CTGACTGAATTCATGACATCGCGGAG-3' and 5'-ATCTCTGGTCTCAGCTAA-3', with pET FLVRES mutants as templates. The 0.7-kb products were digested with EcoRI and BglII and cloned via three-way ligations with either a BglII-EcoRI 3' fragment of wild-type (WT) SHP-2 or a BglII-EcoRI 3' fragment of P into pXT7SR1 vectors (17) or pSP64RI (74), each linearized with EcoRI. pSP64RI-R32K, pXT7SRI-R138K, and pXT7SRI-R32,138K contain point mutations of the essential arginines in the FLVRES sequence in the context of the full-length protein. pXT7SRI-R32K/P, pSP64RI-R138K/P, and pXT7SRI-R32,138K/P contain point mutations of the essential arginines in the FLVRES sequence in the context of P; this deletion includes the essential VHCSAG motif (74).

Plasmid constructs and in vitro transcription. For in vitro transcription, constructs were subcloned into the pSP64RI (74) or pXT7SRI (17) vectors, which contain a polylinker flanked by Xenopus -globin 5' and 3' untranslated sequences. In vitro transcription of linearized plasmids was carried out by using SP6 polymerase for constructs in pSP64RI and T7 polymerase for constructs in pXT7SRI and a MEGAscript kit (Ambion).

Embryo manipulations. Fertilization and embryo culture in 0.1× MMR were performed as described previously (49). Embryos were transferred to 0.5× MMR-3% Ficoll (Pharmacia) and injected with 10 nl of RNA in the animal pole of both cells of two-cell-stage embryos. The concentration of P mRNA injected was determined by preliminary experiments with each batch of RNA in which the minimal level required to block bFGF-dependent elongation was determined. The RNA concentrations for all other mutants were determined by the amount required to produce protein levels comparable to those of P. Animal pole explants were excised at stage 7.5 to 8.5 and analyzed as described previously (74).

RNA and protein analysis. RNA extraction and Northern analysis of late mesodermal markers were performed as described elsewhere (32). For MAPK shift assays, animals caps were isolated at stage 7.5 to 8 and dissociated in calcium-free, magnesium-free normal amphibian media (27). Dissociated cells were collected and stimulated for 5 min at 25°C with 100 ng of Xenopus bFGF per ml and then pelleted for 10 s at 14,000 rpm. The pellets were lysed immediately in Nonidet P-40 (NP-40) lysis buffer (1% NP-40, 150 mM NaCl, 50 mM Tris [pH 7.4]) containing protease inhibitors (10 µg of leupeptin per ml, 1 µg of aprotinin per ml, 1 µg of pepstatin A per ml, 1 µg of antipain per ml, and 20 µg of phenylmethylsulfonyl fluoride per ml) and phosphatase inhibitors (1 mM sodium vanadate) and incubated for 10 min on ice. The lysates were clarified for 10 min at 14,000 rpm at 4°C, electrophoresed, and transferred onto Immobilon P membranes (Millipore). Immunoblots were probed with rabbit polyclonal anti-Xenopus MAPK C-terminal antibodies (a generous gift of James Maller). Levels of SHP-2 and chimeric proteins containing the SHP-2 N terminus were quantified by probing total NP-40 lysates from animal caps at stage 8 or 9 with mouse monoclonal antibodies against the N-SH2 of SHP-2 (Transduction Laboratories). For experiments comparing expression of chimera 12 with that of SHP-2, polyclonal antibodies against the C terminus of SHP-2 (Santa Cruz Biotechnology, Inc.) were used instead.

Glutathione S-transferase (GST) fusion proteins and PTP assays. SHP-2, SHP-1, and chimeras 21, 212, and 12 were subcloned into the EcoRI site of pGEX4T (Pharmacia). The constructs were transformed into DH5. Log-phase bacterial cultures (100 ml) were induced with 0.1 µM isopropyl--D-thiogalactopyranoside (IPTG) (Gibco-BRL) for 4 h at 37°C. Bacterial pellets were lysed in 1× STE (10 mM Tris-Cl [pH 7.5], 10 mM NaCl, 1 mM EDTA) containing 100 µg of lysozyme per ml for 10 min on ice and then pelleted at 4°C and 12,500 rpm for 15 min. Triton X-100 was added to a final concentration of 1.9%. The lysates were incubated with 250 µl of a 50% slurry of glutathione agarose (Sigma) for 1 h. The beads were washed four times in ice-cold phosphate-buffered saline, and proteins were eluted with 15 mM glutathione in 50 mM Tris-150 mM NaCl-5 mM dithiothreitol at pH 7.4 and then dialyzed for 4 h in 50 mM HEPES-150 mM NaCl. Protein concentrations were determined by Coomassie staining and bicinchoninic acid assay (Pierce).

	RESULTS

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To determine which domains of SHP-2 are required for signaling downstream of the XFGFR, we generated a series of mutants with alterations in the SHP-2 SH2 domains and the C-terminal tail (Fig. 1B and C). For subsequent studies to assess which domains contribute to specificity in XFGFR signal transduction, we generated several SHP-2/SHP-1 chimeras (Fig. 1D). RNA was prepared from each of these constructs by in vitro transcription (see Materials and Methods) and injected into both animal poles of two-cell embryos. Animal caps from injected and control embryos were monitored for their ability to elongate, activate MAPK, and induce expression of the late mesodermal marker, muscle actin, in response to bFGF stimulation. In one series of experiments, we examined whether mutations in various SHP-2 domains, when introduced into the P mutant, retained the ability to block these bFGF-stimulated events (Fig. 1B and C). Mutants (in the context of P) that fail to block bFGF signaling indicate a requirement for that domain to mediate the P block and, by inference, a requirement for that domain to transmit the SHP-2 signal. Due to the nature of dominant negative experiments, proteins that block signaling must be expressed at levels substantially higher than that of endogenous SHP-2, creating the possibility that subtle effects of functional-domain mutations on the ability of P to block signaling might not be detected. For this reason, we also utilized a complementary, more sensitive approach in which the functional-domain mutants were introduced into WT SHP-2 (Fig. 1B and C) and then were assessed for their ability, when coexpressed with P, to rescue the P-induced block of FGF signaling. For these experiments, we used the minimal amount of P required to block animal cap elongation (as determined by preliminary titration experiments). Low levels of the mutant proteins were coexpressed with this minimal amount of P. The abilities of mutant and WT SHP-2 to rescue were compared. Again, we interpreted mutant SHP-2 proteins that failed to rescue the P-induced block as indicating a requirement for that domain of SHP-2 in bFGF signaling.

View larger version (14K): [in this window] [in a new window]   FIG. 1.   Microinjection constructs: schematic representations of human SHP-2 and chimeric cDNA clones showing functional domains that might participate in bFGF signaling, including the two SH2 domains, the SH2-PTP linker, the PTP domain, the C-terminal tyrosine phosphorylation sites, and the proline-rich region. Amino acid numbers corresponding to human SHP-2 (24) are indicated above the diagram. (A) Full-length SHP-2 and P, a mutant with a 31-amino-acid deletion in the PTP domain, which acts as a dominant negative mutant. (B) SH2 domain mutants with point mutations in the essential b5 arginine of both SH2 domains (R32,138K) or individual N-SH2 (R32K) or C-SH2 (R138K) domains in the context of WT SHP-2 or P, as indicated. (C) C-terminal tail mutants with tyrosine (Y)-to-phenylalanine (F) mutations at position 542 and/or 580 in the context of WT SHP-2 or P, as indicated. pro, 10-amino-acid deletion of the proline-rich region between the two tyrosines. (D) Chimeras between SHP-2 and SHP-1. SHP-1 domains (white boxes) and SHP-2 domains (black boxes) are indicated. Shown are SHP-1; the 21 chimera, containing the SH2 domains and linker region of SHP-2 fused to the PTP and C-terminal tail of SHP-1; the 212 chimera, containing the SH2 domains and linker of SHP-2 fused to the PTP domain of SHP-1 and the C-terminal tail of SHP-2; and the 12 chimera, containing the SH2 domains of SHP-1 fused to the linker, PTP domain, and C-terminal tail of SHP-2.

The SH2 domains of SHP-2 are required for bFGF-induced signaling in animal caps. To assess the role of the SH2 domains of SHP-2, we generated mutants in which the essential FLVRES motifs of N-SH2, C-SH2, or both SH2s (N+CSH2) were converted to FLVKES in the context of WT SHP-2 or P (Fig. 1B). Previous studies revealed that such mutants display markedly reduced (approximately 50-fold), although not absent, binding to phosphotyrosyl peptides in vitro (73). Normally, animal caps undergo dramatic elongation in response to stimulation with bFGF (Fig. 2A, C+) which is accompanied by induction of mesodermal markers, including muscle actin (Fig. 3C), and activation of MAPK (Fig. 3D). As was shown previously (74), P blocks elongation (Fig. 2A and others), the activation-dependent shift of MAPK (Fig. 2C and others), and muscle actin mRNA induction (Fig. 3C and others), most likely by competing with endogenous, full-length SHP-2 for its target(s). Mutation of the essential arginines of both SH2 domains (R32,138K/P) abolished the ability of P to act as a dominant negative mutant in the bFGF pathway: animal caps from embryos injected with R32,138K/P RNA elongated at all doses tested (Fig. 2A). The failure to block was not due to an inability of the mutant protein to be expressed stably, since levels of R32,138K/P protein ranging from equal to the minimal amount of P required to block elongation (Fig. 2B) to up to 10-fold-higher levels (data not shown) failed to block elongation. R32,138K/P also failed to block activation of MAPK (Fig. 2C). This result demonstrates that at least one functional SH2 domain is required for the dominant negative effect of P on bFGF signaling and supports a model in which P acts as a dominant negative mutant by binding, via its SH2 domain(s), to one or more endogenous phosphotyrosyl proteins, thus preventing proper localization and/or activation of endogenous SHP-2.

View larger version (48K): [in this window] [in a new window]   FIG. 2.   SH2 domains are required for P to act as a dominant negative mutant. Representative experimental results show animal cap elongation, protein levels, and MAPK activation following injection of the indicated SHP-2 mutants. (A) bFGF-stimulated animal caps at stage 10.5. The injected mRNA is indicated beneath each panel. C+, uninjected animal caps stimulated for 2 h with bFGF (100 ng/ml). These caps are elongated. In contrast, P-injected caps (P) show no elongation and are scored as blocked. 1× and 5×, concentrations used (see Materials and Methods). (B) Immunoblot analysis of injected animal caps at stage 8. Total lysates of animal caps were probed with anti-PTP1D/SHP-2 monoclonal antibodies (Transduction Laboratories). P proteins are indicated (lower band). This antibody cross-reacts with endogenous Xenopus SHP-2 (XSHP2), which serves as a loading control. The injected mRNA is indicated beneath each lane. (C) MAPK activation in animal caps stimulated with bFGF (100 ng/ml) at 25°C for 5 min. Total lysates of animal caps were probed with anti-Xenopus MAPK (anti-XMAPK antibodies) (see Materials and Methods). The injected mRNA is indicated beneath each lane. The amount of R138/P injected here is equivalent to 5× in panel A. (D) Quantitation of animal cap elongation. Mean percentages of elongated caps are shown. Error bars show the standard error of the mean for each injected mRNA. Injected mRNA is indicated beneath each lane. Percentages are based on a minimum of 45 total animal caps in three or more separate experiments.

View larger version (61K): [in this window] [in a new window]   FIG. 3.   Intact SH2 domains are required for SHP-2 to rescue the P-induced block of bFGF signaling. Representative experimental results show elongation, protein levels, muscle actin induction, and MAPK activation in response to bFGF stimulation of animal caps from embryos injected with the indicated RNAs. (A) bFGF-stimulated animal caps at stage 10.5 were analyzed as for Fig. 2A. Shown are caps from embryos injected with P alone (P), with P plus WT SHP-2 (P + FL), or with P plus the indicated increasing amounts of the R32,138K mutant (1×, 5×, and 10×). (B) Immunoblot analysis of the injected animal caps from panel A. (C) Northern blot analysis of induction of the mesoderm-specific marker muscle actin. Caps collected at stage 21 were analyzed for expression of muscle actin (lowest band). The two upper bands represent cytoplasmic actin, which cross-reacts with the probe and acts as a loading control for the experiment. The injected mRNA is indicated beneath each lane. (D) MAPK activation in animal caps injected with the indicated constructs, monitored as described for Fig. 2D. XMAPK, Xenopus MAPK. (E) Quantitation of animal cap elongation, as described for Fig. 2D. The injected mRNA is indicated beneath each lane. Percentages are based on a minimum of 30 total animal caps in three separate experiments.

The C-terminal tyrosines and proline-rich domain are dispensable for rescue of the P block of bFGF signaling. We next examined the role of domains within the C-terminal tail of SHP-2. Phosphorylated Y542 and Y580 both can bind Grb2, which led to the proposal that by binding Grb2, SHP-2 links RTKs to Ras activation (see the introduction). Tyrosyl phosphorylation of WT SHP-2 in response to bFGF treatment of animal caps is weak and inconsistent, although catalytically inactive forms of SHP-2, such as P, are constitutively and strongly phosphorylated (data not shown). This suggests that SHP-2 probably is phosphorylated in this system but then rapidly autodephosphorylates. We were concerned that instead of sending a positive signal via Grb2, the increased tyrosyl phosphorylation of the SHP-2 C terminus in catalytically impaired mutants such as P might actually sequester Grb2. If this were the case, the block in bFGF signaling caused by mutants such as P might be artifactual, rather than reflecting a specific role for the SHP-2 catalytic domain in bFGF signaling; this concern has been raised also by others (71). To rule out this possibility, we generated a triple mutant (Y542,580F/P) containing the P deletion and both Y542 and Y580 converted to phenylalanine (Fig. 1C). Animal caps expressing levels of the triple-mutant protein equal to the level of P required to block bFGF signaling (Fig. 4B) failed to elongate (Fig. 4A) or activate MAPK (Fig. 4C) in response to bFGF stimulation. The effects of this mutant were rescued by small amounts of WT SHP-2 (Fig. 4A), demonstrating that the triple-mutant blocks mesoderm induction by a mechanism similar to that of P. In addition to confirming that the PTP domain itself is required for bFGF signaling, this result demonstrates that the C-terminal tyrosines are not required for the dominant negative effects of the P mutant.

View larger version (67K): [in this window] [in a new window]   View larger version (18K): [in this window] [in a new window]   FIG. 4.   Tyrosine-to-phenylalanine mutations do not affect the ability of P to block bFGF signaling. Representative experimental results show animal cap elongation, protein levels, and MAPK activation. (A) bFGF-stimulated animal caps at stage 10.5, prepared from embryos injected with the indicated mRNA. (B) Immunoblot analysis, carried out as described for Fig. 2B. Positions of P proteins and WT full-length SHP-2 (FL) are indicated. In this exposure, endogenous Xenopus SHP-2 levels are too low to be detected. The injected mRNA is indicated beneath each lane. (C) MAPK activation in animal caps assayed as described for Fig. 2C. The injected mRNA is indicated beneath each lane. XMAPK, Xenopus MAPK. (D) Quantitation of animal cap elongation, as for Fig. 2D. The injected mRNA is indicated beneath each lane. Percentages are based on a minimum of 45 total animal caps in three or more separate experiments.

View larger version (67K): [in this window] [in a new window]   FIG. 5.   Mutation of tyrosines to phenylalanines does not affect the ability of full-length SHP-2 to rescue the P block. (A) bFGF-stimulated animal caps at stage 10.5 from embryos injected with the indicated mRNA. Shown are caps injected with P alone (P), P plus WT full-length SHP-2 (P + FL), and P plus the indicated single or double tyrosyl phosphorylation site mutants. (B) Immunoblot analysis of SHP-2 expression, as described for Fig. 2B. (C) Quantitation of animal cap elongation, as for Fig. 2D. Percentages are based on a minimum of 70 total animal caps in three or more separate experiments.

View larger version (52K): [in this window] [in a new window]   FIG. 6.   Deletion of the C-terminal prolines has no effect on the ability of SHP-2 to rescue the P block. Representative experimental results show animal cap elongation and protein levels. (A) bFGF-stimulated animal caps from embryos injected with the indicated mRNAs. (B) Immunoblot analysis, as described for Fig. 2B. (C) Quantitation of animal cap elongation, as for Fig. 2D. Percentages are based on a minimum of 37 total animal caps in two or more separate experiments.

Determinants of SHP specificity map to the PTP domain. The above results (combined with our previous studies) show that the SH2 domains of SHP-2, and N-SH2 in particular, as well as the PTP domain are required for bFGF-stimulated animal cap elongation, MAPK activation and mesodermal marker induction. SHP-1 cannot substitute for SHP-2 in this system (reference 74 and data not shown), so we next addressed the question of which domain(s) is responsible for this difference. All known nontransmembrane PTPs contain noncatalytic domains capable of directing interactions with other proteins and/or lipids. Since isolated PTP catalytic domains generally display relatively low levels of substrate specificity, it has been suggested that targeting PTP catalytic domains to the correct location in the cell is a (the) major determinant of PTP specificity (45).

View larger version (69K): [in this window] [in a new window]   View larger version (43K): [in this window] [in a new window]   FIG. 7.   Chimeras containing the PTP domain of SHP-1 act as dominant negative mutants in the animal cap assay. Representative experimental results show animal cap elongation, protein levels, muscle actin induction, and MAPK activation. (A) bFGF-stimulated animal caps at stage 10.5 from embryos injected with either P or the 21 chimera. Note that the two constructs block elongation equivalently. (B) Immunoblot analysis as described for Fig. 2B and MAPK activation in animal caps, assessed as for Fig. 2C. The injected mRNA is indicated beneath each lane. XMAPK, Xenopus MAPK. (C) bFGF-stimulated animal caps at stage 10.5. Shown are caps from embryos injected with P alone (P), P plus WT full-length SHP-2 (P + FL), the 212 chimera alone (212), and the 212 chimera plus WT full-length SHP-2 (212 + FL). (D) Immunoblot analysis as in Fig. 2B. (E) Northern blot analysis of muscle actin mRNA induction, carried out as for Fig. 3C. No muscle actin band is seen in either the P or the 212 lane, even upon longer exposure. (F) MAPK activation in animal caps, as in Fig. 2C. The injected mRNA is indicated beneath each lane. (G) Quantitation of animal cap elongation, as in Fig. 2D. Percentages are based on a minimum of 45 total animal caps in three or more separate experiments.

View larger version (43K): [in this window] [in a new window]   FIG. 8.   The P block is partially rescued by coexpression of a chimera containing the PTP domain of SHP-2. Representative experimental results show animal cap elongation, protein levels, muscle actin induction, and MAPK activation. (A) bFGF-stimulated animal caps at stage 10.5 from embryos injected with the indicated mRNAs. Note that P is fully rescued by coexpression of WT full-length SHP-2 (P + FL) and partially rescued upon coexpression of the 12 chimera (P + 12). (B) Immunoblot analysis carried out as for Fig. 2B, except that an antibody directed against the C terminus of SHP-2 (Santa Cruz Biotechnology) was used to allow comparison of the levels of full-length SHP-2, P, and the 12 chimera protein, which share the C terminus of SHP-2 (see Materials and Methods for details). (C) Northern blot analysis of induction of muscle actin mRNA, as for Fig. 3C. (D) MAPK activation in animal caps injected with the indicated constructs, analyzed as for Fig. 2C. XMAPK, Xenopus MAPK. (E) Quantitation of animal cap elongation, as in Fig. 2D. Percentages are based on a minimum of 88 total animal caps in three or more separate experiments.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

Using mesoderm induction in animal cap explants as a model system, we have elucidated the structural requirements for SHP-2 function in bFGF signaling. We found that both SH2 domains are important for normal signaling, although N-SH2 plays the more critical role. Surprisingly, we can find no role for the C-terminal tyrosines in this system, arguing strongly that the Grb2 adapter model for positive signaling by SHP-2 (9, 40, 47) does not apply to the bFGF pathway. Likewise, the proline-rich domain appears to be dispensable for bFGF signaling. The PTP domain is essential and, presumably via its ability to dephosphorylate only certain phosphotyrosyl proteins, is a, if not the, major determinant of biological specificity in vivo. The SH2 domains also appear to contribute to SHP specificity. Our results identify for the first time key structural determinants of biological specificity for a member of the PTP family.

Previous work suggests that the SH2 domains of SHPs may serve at least two types of function. First, several groups have shown that SHP-1 and SHP-2 are targeted to various plasma membrane proteins upon stimulation with growth factors or cytokines. In some cases, the SHPs bind directly to RTKs themselves (21, 37, 81); in others, SHPs may bind to transmembrane proteins, such as SHPS-1/SIRP family members (25, 33, 51, 52, 89), or to adapter or accessory molecules, such as IRS-1 (36), Gab1 (30), and FRS-2 (35). SH2 domain-directed binding serves to relocate SHPs to new intracellular locations, where they may gain access to potential substrates. Additionally, however, in vitro studies using recombinant SHPs and known phosphotyrosyl peptide ligands for their SH2 domains have suggested that SH2 domain engagement increases catalytic activity (14, 15, 31, 38, 54, 56, 61, 63, 73, 80). Basally, SHP-2 and SHP-1 appear to exist in a closed conformation wherein the SH2 domains bind to and inhibit the PTP domain; SH2 engagement relieves inhibition, presumably by promoting conversion to an open conformation. For SHP-1, N-SH2 alone can repress basal activity; C-SH2 cannot subserve this function (54). However, both SH2 domains can bind to phosphotyrosyl peptide targets. We cannot determine from our data which of these functions accounts for the critical requirement for the SHP-2 SH2 domains for proper bFGF-induced signaling. However, if, as in SHP-1, only N-SH2 of SHP-2 participates in basal repression, whereas both SH2 domains can direct binding to the appropriate intracellular target(s), the absolute requirement for N-SH2, as well as the contribution of C-SH2, may be explained (Fig. 2 and 3).

Homozygous deletion of 64 amino acids in the N-SH2 domain of murine SHP-2 results in mice with severe developmental abnormalities and impaired bFGF signaling (66). Although these results are consistent with a requirement for N-SH2 in bFGF signaling, another explanation is possible. The deletion mutant retains the essential FLVRES motif of N-SH2, but this mutant most likely is nonfunctional for either appropriate targeting or basal repression. Therefore, it is unclear whether the phenotype of these mice results solely from loss of normal SHP-2 function (hypomorphism) or a combination of hypomorphism and abnormal function (neomorphism) resulting from the presence of an activated PTP in an abnormal cellular location. Our data indicate that the SH2 domains of SHP-2 are indeed essential contributors to SHP-2 function in bFGF signaling in vivo.

The SH2 domains of SHP-2 also appear to account for some of the specificity between SHP-1 and SHP-2. The 12 chimera rescues the P-induced block only partially (Fig. 8). If the SH2 domains of SHP-1 and SHP-2 were interchangeable, this chimera would be equivalent to WT SHP-2 in the rescue assay. Most likely, the SH2 domains of SHP-1 have an affinity which is lower than those of SHP-2 (but not absent) for the target of the P SH2 domains. When expressed at high enough levels, the SH2 domains of the 12 chimera can cross over and bind what are normally SHP-2 binding targets. Consistent with the idea of some specificity resident within the SH2 domains, WT SHP-1 does not act as a dominant negative mutant for bFGF-induced signaling in animal caps (53a). Again, if the SH2 domains of the two SHPs were fully interchangeable, the results of our experiments with the 21 and 212 chimeras would predict that targeting of an incorrect PTP domain (i.e., SHP-1) to the normal location of SHP-2 would lead to a block in signaling.

Further study is needed to clarify the molecular basis by which the SH2 domains of the two SHPs confer specificity. Optimal binding sequences, determined by peptide library screening, for the N-SH2 domains of both SHPs have been determined (references 11, 69, and 70 and Table 1). The similarity between these consensus sequences renders it easy to see why, particularly at high concentrations, the two SHPs may be able to bind to the same intracellular target (see above). The precise determinants within phosphotyrosyl peptide ligands for the SHPs that permit discrimination between them remain unclear. Comparison of peptide sequences of binding targets for SHP-2 shows a general trend in which no basic residues are present between amino acids 2 and +5 (with position 0 being pY), an observation that is compatible with the crystal structures of the SHP-2 SH2 domains (20, 39). In contrast, many SHP-1 binding sites that have been mapped have basic residues within the region from 2 to +5 (Table 1). However, some proposed binding sites for SHP-2 (e.g., CTLA-4 [43]) deviate from these simple consensus sequences. It is possible that sequences distant from the phosphotyrosyl peptide ligand within proteins that bind to the SH2 domains of SHP-2 may influence binding; such influences would not be reflected in attempts to define a linear consensus sequence. Similarly, additional structural determinants within the SH2 domains may participate in recognition of specific peptides. Determination of the crystal structure of the SH2 domains of SHP-1 bound to an appropriate ligand and comparison with the available structures of SHP-2 should provide key insights into the resolution of these questions.

The C terminus of SHP-2 has structural motifs that could play important roles in signal transduction, including two tyrosyl phosphorylation sites, a proline-rich stretch, and potential serine phosphorylation sites. The proline-rich region and at least one of the two C-terminal tyrosines are conserved from Drosophila Csw to vertebrate SHP-2 (24, 58, 74), suggesting that this region contributes to signaling in at least some pathways. Studies of SHP-2 function in mammalian cells and in Xenopus have relied heavily on catalytically inactive mutants of SHP-2, which exhibit abnormally high levels of tyrosyl phosphorylation in response to some growth factors, including insulin and Xenopus bFGF (53a, 71). Since tyrosyl-phosphorylated SHP-2 can recruit Grb2 via SH2 domain interactions, we were concerned that aberrantly phosphorylated SHP-2 might sequester Grb2 away from its normal role in promoting Ras activation. Aberrant phosphorylation might also promote excessive SHP-2/SHIP association, with unclear functional consequences.

Our finding that the triple mutant P/Y542,580F acts as a potent dominant negative mutant (Fig. 4) argues against the possibility that the dominant negative effects of P (and other catalytically inactive mutants) might be the result of inappropriately sustained tyrosyl phosphorylation and subsequent Grb2 binding. Furthermore, our results argue that the C-terminal tyrosines of SHP-2 are not absolutely required for positive signaling in the bFGF pathway, since tyrosine-to-phenylalanine mutants rescue the dominant negative effects of P in a manner indistinguishable from that of WT SHP-2 (Fig. 5). Deletion of the proline-rich region also has no effect on the ability of SHP-2 to rescue the P-induced block (Fig. 6). We attempted to study the signaling properties of an SHP-2 deletion mutant completely lacking its C terminus but were unable to detect accumulation of the mutant protein in Xenopus embryos. Therefore, we cannot exclude the possibility that other motifs within the C terminus (e.g., seryl phosphorylation sites) play some role in the positive signaling function of SHP-2 or that this region plays a modulatory role in bFGF-induced mesoderm induction that is undetectable in our assays.

Our results clearly establish that the PTP domain, together with the SH2 domains of SHP-2, is primarily responsible for its positive signaling functions. Since SHP-1 cannot replace SHP-2 in early Xenopus development (74) (Fig. 7 and data not shown), we questioned which regions of the two PTPs are critical for specificity in vivo by studying a series of SHP-2/SHP-1 chimeras. Both the 21 and the 212 chimeras act as potent dominant negative mutants, providing strong evidence that in vivo specificity resides within the PTP domain: even when targeted to the correct location in the cell by the SH2 domains of SHP, the PTP domain of SHP-1 cannot substitute for the PTP domain of SHP-2. Most likely, residues within the PTP domains determine substrate specificity. However, we cannot exclude the possibility that specificity is dependent solely upon the magnitude of the catalytic activity resident within the PTP domains of the two SHPs. Since proteins containing the PTP domain of SHP-1 have 30-fold-higher activity against artificial substrates than those containing the PTP domain of SHP-2, it is possible that the dominant negative effects are a result of too much PTP activity. Determination of residues responsible for substrate binding and for setting the level of activity for each PTP should resolve this issue.

Compared to the amount of P required, somewhat larger amounts of chimeric protein are needed to block animal cap elongation (Fig. 7 and data not shown). The N-SH2 domains of both SHPs are known to bind to and inhibit the PTP domain (see above). The endpoints of the deleted region within P fall within important secondary structural elements, as predicted from the crystal structure of PTP-1B (7), so the PTP domain of P probably is unfolded and thus unable to bind to the N-SH2. Therefore, P likely exists basally in an open conformation. Conversely, the chimeras are likely to be basally repressed through binding of their N-SH2 domains to the PTP domain. Energy would be required to open this closed conformation. Accordingly, larger amounts of the chimera proteins may be required to exert the same degree of inhibition of bFGF signaling.

Perhaps the most compelling evidence that the PTP domain determines specificity in vivo is provided by the ability of the 12 chimera to partially rescue the P-induced block. Although the 21 and 212 chimeras retain PTP activity in vitro, it remained formally possible that other parts of these molecules were improperly folded, leading to an artifactual block of signaling activity. However, the positive finding that the 12 chimera can to a large extent rescue the effects of a dominant negative SHP-2 mutant clearly shows that the PTP domain of SHP-2 provides critical information for proper signaling in vivo.

Our results are consistent with several recent studies using tissue culture systems. Using substrate-trapping approaches, p130Cas was identified as a likely substrate of PTP-PEST, arguing for PTP domain specificity for this nontransmembrane PTP as well (26). Chimeras similar to those used here were used in transient-transfection experiments with 293 cells (77). The researchers of that study concluded that the PTP domain of SHP-1 confers specificity for dephosphorylation of the EGFR. The biological significance of these observations is unclear, since the EGFR has not been identified as a bona fide substrate for SHP-1 in vivo. Moreover, the enzymatic activities of these chimeras were not reported, nor was it clear that the chimeras were expressed at comparable levels following transient transfection. Although these studies, together with our results, clearly indicate that the PTP domain can confer considerable specificity for several nontransmembrane PTPs, for others targeting seems to play a more dominant role. For example, although full-length PTP-1B appears to have highly restricted substrate specificity in transient-transfection studies (23), truncated PTP-1B lacking its C-terminal targeting sequence is relatively nonselective (26).

Most likely, all nontransmembrane PTPs use a combination of specificity conferred by targeting sequences and intrinsic PTP domain specificity to select the correct substrate(s) in vivo. By combining specificities resident in noncatalytic and catalytic domains, rapid evolution of a large number of selective PTPs may have been facilitated. For the SHPs, combinatorial specificity probably is of particular importance, since in several signaling pathways SHP-2 and SHP-1 appear to be targeted to similar, if not identical, sites, yet have drastically different downstream effects. The existence of specificity within the PTP domains of the SHPs would allow each molecule to select different targets from the same local milieu, potentially explaining the distinct effects of SHP-2 and SHP-1. Moreover, it may be possible to exploit the specificity resident within PTP catalytic domains to develop highly selective PTP inhibitors. Future studies should be directed to understanding the structural details underlying the specificities of the PTP domains of the SHPs.