We generated two activated forms of SHP-2 by mutating N-SH2 domain residues (aspartate 61 [D61] or glutamate 76 [E76]) that form key contacts with the PTP domain (19
) (Fig. B). Our goal was to generate mutants that would target correctly within cells yet be activated by subthreshold levels of stimulation. We produced the two mutant proteins in E. coli
, purified them, and assayed their enzymological properties. Both were basally activated in vitro (Fig. C), although to different extents. Relative to wild-type SHP-2, D61A exhibited approximately 20-fold higher basal activity against the artificial substrate tyrosyl-phosphorylated RCML. Addition of a phosphotyrosyl peptide ligand for the N-SH2 domain led to further activation of D61A in a ligand concentration-dependent manner (Fig. C). In contrast, E76A was fully activated (~100-fold) in the absence of any added ligand. The increased activation of D61A upon ligand binding strongly suggested that this mutant retains a functional N-SH2 phosphotyrosyl binding pocket. However, to confirm that both of the mutant N-SH2 domains retain wild-type phosphotyrosyl protein-binding ability, we generated GST–N-SH2 fusion proteins for wild-type SHP-2, D61A, and E76A and compared their abilities to bind a known ligand for the N-SH2 of SHP-2, SHPS-1 (12
). All three N-SH2 domains bound to SHPS-1 with approximately equal affinities (Fig. D), indicating that neither the D61A nor the E76A mutation disrupts the N-SH2 phosphotyrosyl binding pocket. This conclusion is further supported by in vitro binding studies with phosphotyrosyl peptides (data not shown). Therefore, we conclude that mutation of D61 or E76 weakens the basal interaction between the N-SH2 and PTP domains, leading to enzymatic activation without affecting ligand binding.
We next assessed the biological activity of the activated SHP-2 mutants in animal cap assays. Untreated animal caps remain spherical. Treatment with an appropriate peptide growth factor results in elongation, characterized by distortion of the spherical shape of the animal cap (49
). As expected, animal caps stimulated with FGF elongated at stage 11-11.5 in a dose-dependent manner (49
) (Fig. A). Overexpression of wild-type SHP-2 in the absence of FGF does not evoke animal cap elongation (55
) (see Fig. A). In contrast, expression of either D61A or E76A induced substantial elongation in the absence of exogenous growth factors (Fig. A). The extent of elongation depended on SHP-2 protein levels: low levels of D61A induced detectable but slight elongation, whereas higher levels evoked elongation indistinguishable from that produced by FGF stimulation (Fig. A, lower panel). In contrast, E76A-expressing animal caps elongated at most doses tested (Fig. A), consistent with the higher catalytic activity of this mutant (Fig. C). Importantly, expression of a form of D61A in which the essential FLVRES arginines in both SH2 domains are mutated to lysines (KKDA) failed to evoke elongation (Fig. B). This result implies that phosphotyrosyl protein binding and, presumably, appropriate subcellular targeting of SHP-2 are required for activated mutant function.
FIG. 2 Expression of activated mutants induces elongation of Xenopus animal caps. (A) (Top panel) Stage 8 animal caps were stimulated with increasing concentrations of FGF, as indicated. Photographs were taken at stage 11.5 to assess elongation. (Second panel) (more ...)
FIG. 4 Analysis of differentiation in animal caps expressing activated mutants of SHP-2. (A and B) Histological sections from stage 39 animal caps that were unstimulated (C−) or stimulated with activin (5 ng/ml) (C+A) or FGF (100 ng/ml) (C+F) (more ...)
The requirement for a functional phosphotyrosyl binding pocket also implies that at least one ligand for the SH2 domains of SHP-2 must be phosphorylated in unstimulated animal caps. Previous studies have demonstrated that FGF is present and that low-level constitutive stimulation of the XFGFR pathway occurs in unstimulated animal caps (24
). In addition, our previous work established that SHP-2 function is required for XFGFR signaling (41
). Thus, it seemed possible, if not likely, that the activated mutants function to enhance low-level signaling through the XFGFR pathway. Such a model implies that XFGFR function should be required for activated mutant-induced elongation. To test this possibility, we inhibited endogenous XFGFR activity by expressing a dominant negative form of the XFGFR (XFD) that lacks the cytoplasmic domain (2
). The dose of XFD used in these experiments blocked FGF-induced animal cap elongation (data not shown), indicating that we had inhibited XFGFR activity. Moreover, elongation induced by either D61A or E76A also was inhibited by XFD coexpression (Fig. ), demonstrating that basal FGF signaling is required for the action of the activated SHP-2 mutants. These results further support the conclusion that D61A and E76A mimic the endogenous SHP-2 signaling pathway in animal caps.
FIG. 3 A functional XFGFR is required for the effects of the activated SHP-2 mutants. (A) Animal caps were injected with 5 ng of either D61A or E76A RNA (left) or coinjected with the indicated activated SHP-2 mutant and XFD (2.5 ng) (right). (B) Protein levels (more ...)
In addition to evoking elongation, FGF stimulation promotes expression of mesodermal markers and differentiation of animal cap ectoderm to ventrolateral mesoderm (see the introduction). We therefore expected that like FGF treatment (51
), expression of the activated SHP-2 mutants would evoke tissue differentiation concomitant with animal cap elongation and, indeed, that the induced mesodermal derivatives might drive elongation movements. We looked for evidence of such tissues in animal caps expressing the activated mutants. E76A expression induced low levels of mesodermal tissue, including a mesothelial layer and mesenchyme (Fig. A and B). Thus, high levels of SHP-2 (e.g., as generated by E76A) can evoke some (low) level of mesodermal tissue differentiation, consistent with weak activation of the full range of FGF signaling events by E76A. Surprisingly, however, no mesodermally derived tissues were detected in D61A-expressing animal caps (Fig. A and B). D61A-expressing animal caps displayed numerous vacuoles, but these structures were clearly distinct from those associated with notochord (Fig. B); their origin remains unknown. As expected, FGF-stimulated caps contained muscle and other ventrolateral mesodermal derivatives, including mesothelium and mesenchyme (Fig. A and B), whereas activin-stimulated animal caps developed dorsal structures, including notochord and neural tissue (Fig. B). Thus, D61A-expressing animal caps elongate fully (i.e., comparably to FGF-stimulated animal caps) yet fail to exhibit significant (or even detectable) evidence of mesodermal cytodifferentiation.
In addition to mesodermal derivatives, such as notochord and muscle, neural tissue and endoderm also can contribute to elongation movements in animal caps (17
). D61A-expressing animal caps exhibited a yolky appearance, characteristic of either endoderm or lack of tissue differentiation. However, molecular marker analysis (see below and Fig. F) strongly suggests that no endodermal differentiation occurs in D61A (or E76A)-injected animal caps. Furthermore, in contrast to activin-stimulated animal caps, there was no histological evidence of neural differentiation in response to activated mutant expression (Fig. B).
We further analyzed differentiation in animal caps expressing the activated mutants by surveying the expression of molecular markers. The late mesodermal marker muscle actin was detected on Northern blots following FGF stimulation and, to a markedly lower degree, upon E76A expression. However, in agreement with the histological sections (Fig. A and B), no muscle actin mRNA was detected in D61A-expressing animal caps (Fig. C). We also analyzed early marker expression by RT-PCR. As expected, FGF stimulated Xbra expression in a dose-dependent manner. Notably, doses of FGF (11 ng/ml) that did not induce elongation stimulated significant Xbra expression (Fig. D). Also as expected, expression of an activated form of MEK (MEK*) (32
), a downstream effector in the FGF pathway, induced Xbra expression (Fig. D). In contrast, at low doses of E76A, no Xbra mRNA was detected (Fig. D), although such doses were capable of inducing elongation comparably to FGF (Fig. A). At higher levels of expression, E76A did evoke significant Xbra expression. However, Xbra was induced barely, if at all, by even the highest levels of D61A expression (Fig. D). Again, it should be emphasized that these animal caps elongated dramatically (Fig. A and data not shown). Interestingly, one FGF-regulated gene, Xwnt-8, was induced significantly by both activated mutants (Fig. D), raising the possibility that Xwnt-8 participates in a pathway selectively induced by SHP-2 action. Thus, expression of activated forms of SHP-2 in animal caps leads to induction of some but not all FGF-mediated downstream events.
Consistent with the lack of histological evidence of neural differentiation, there was no detectable expression of NCAM in activated mutant-expressing animal caps (Fig. E). Furthermore, there was no expression of the endodermal marker mixer, sox-17α, or sox17β (Fig. F), arguing that the activated mutants do not induce endoderm. Elongation induced by the activated mutants probably is not due to gratuitous activation of the activin signaling pathway, since we did not detect expression of several activin-induced genes, including Gsc, pintallavis (Fig. E), and the early endodermal markers discussed above (Fig. F).
We next looked at earlier signaling events. FGF stimulation results in the rapid activation of MAPK. To assess the effects of the activated mutants on the MAPK pathway, we took advantage of the fact that D61A- and E76A-expressing animal caps exhibited cold sensitivity: mutant-expressing animal caps failed to elongate at 13°C, whereas incubation at higher temperatures resulted in elongation (Fig. A and data not shown). Injected embryos were incubated at 13°C until stage 8. Cells from dissociated animal caps were then stimulated by acutely raising the temperature to 24°C in the presence or absence of FGF. Under these conditions, E76A activated MAPK (as indicated by reduction in electrophoretic mobility) in a dose-dependent manner, whereas activation of D61A resulted in minimal, often undetectable MAPK activation (Fig. B). When animal caps expressing either activated mutant were maintained at 13°C, no MAPK activation was observed, and expression of wild-type SHP-2 failed to activate MAPK at any temperature tested (data not shown).
FIG. 5 Activated mutants induce minimal MAPK activation. (A) Cold sensitivity of D61A mutant. Embryos were injected with wild-type (WT) or D61A RNA (5 ng) at the two-cell stage and cultured at 13°C until stage 8. Animal caps were excised and then incubated (more ...)
These data indicate that elongation can occur with minimal activation of MAPK. Moreover, activation of MAPK by FGF was not sufficient to induce maximal elongation. Stimulation of uninjected animal caps with either 11 or 100 ng of FGF per ml significantly activated MAPK (Fig. B), yet only animal caps stimulated with the higher dose elongated fully (Fig. ). In contrast, animal caps expressing D61A elongated dramatically, but MAPK activation was minimal (Fig. and B). These results suggest that FGF-stimulated animal cap elongation may require activation of a parallel pathway that is regulated preferentially by SHP-2.
Overexpression of Xbra in animal caps induces mesoderm (9
). However, the minimal activation of MAPK and Xbra transcription by activated SHP-2 mutants, which evoke strong elongation, raised the possibility that Xbra expression per se might be dispensable for FGF-induced elongation movements. Xbra function can be inhibited effectively by expression of a fusion of the DNA binding domain of Xbra with the repressor domain from Drosophila engrailed
). Coexpression of Xbra-EnR and E76A led to complete inhibition of Xbra transcription (data not shown) and prevented E76A-induced elongation (Fig. ).
FIG. 6 Inhibition of Xbra activity prevents activated mutant-induced elongation. (A) Animal caps injected with E76A (5 ng), E76A (5 ng) and Xbra-EnR (200 pg), or E76A (5 ng), Xbra-EnR (200 pg), and eFGF (5 pg) were left unstimulated and allowed to develop until (more ...)
At first glance, this result appears to imply that at least some Xbra function is required for both mesoderm induction and elongation. However, in animal caps, the relationship between XFGFR signaling and Xbra is complex. XFGFR activation leads to Xbra transcription (24
). Xbra in turn induces transcription of the gene for the XFGFR ligand eFGF (47
). This results in a positive feedback loop that maintains FGF signaling throughout early mesoderm induction and gastrulation (24
) (see Discussion). XFD blocks mesoderm induction evoked by overexpression of Xbra, demonstrating the importance of this feedback loop (47
). Because the activated mutants require an N-SH2 ligand for activity, and generation of this ligand requires XFGFR function (see above), the inhibitory effect of Xbra-EnR could be due to failure to generate eFGF. To test this possibility, we coexpressed E76A and Xbra-EnR and provided exogenous FGF to the media in order to bypass the FGF feedback loop. Addition of FGF to the media did not rescue induction of elongation by E76A (data not shown), suggesting either that Xbra is directly required for elongation to occur or that exogenous FGF is not stable enough to provide a continuous signal throughout the assay. To allow continuous production of ligand, we next coinjected RNAs encoding E76A, Xbra-EnR, and the XFGFR ligand, eFGF (23
). Although the dose of eFGF used was sufficient to induce MAPK activation (Fig. B) and Xbra expression (data not shown), eFGF did not rescue E76A-induced animal cap elongation (Fig. A). These results clearly demonstrate that Xbra function is required for elongation in response to activated mutants of SHP-2. However, since D61A and low doses of E76A do not induce Xbra to a significant extent, these results indicate that the amount of Xbra required for elongation is much lower than that required for mesodermal tissue differentiation (see Discussion).
We previously showed that SHP-2 activity is required for FGF-induced MAPK activation, Xbra induction, and elongation (55
). However, activated mutants of SHP-2 are, at best, weak inducers of MAPK activation and Xbra expression. These results imply that XFGFR activation sends additional signals to yield full MAPK activation. To determine if such signals combine with SHP-2 to enhance MAPK activation, we asked whether subthreshold levels of FGF and D61A or E76A would synergize. To test this prediction, we established doses of FGF and D61A or E76A RNA that lead to minimal induction of elongation, MAPK activation, and Xbra induction (Fig. ). When animal caps expressing low-dose D61A or E76A were stimulated with low-dose FGF, markedly enhanced elongation (Fig. A), MAPK activation (Fig. B), and Xbra induction (Fig. C and data not shown) were observed.
FIG. 7 Activated SHP-2 mutants synergize with FGF. (A) Animal caps were either uninjected (top panel) or injected with 0.2 ng of D61A (left side, middle panel) or 0.2 ng E76A (right side, middle panel). Uninjected animal caps (top panel) or activated mutant-injected (more ...)
Finally, we sought to identify downstream components of FGF- or activated SHP-2-mediated signaling leading to elongation. Previous reports have demonstrated a requirement for Ras downstream of the XFGFR. Consistent with these data, expression of dominant negative Ras (63
) (DNRas) blocked FGF-dependent elongation (Fig. A), as well as MAPK activation (Fig. B) and Xbra induction (Fig. C). Similarly, DNRas completely abolished elongation movements induced by E76A (Fig. A). This result is consistent with the notion that Xbra induction by signaling through the Ras/Raf/MAPK cascade is required for E76A-dependent elongation. The weak E76A-dependent induction of MAPK (data not shown) and Xbra (Fig. C) also was completely inhibited by coexpression with DNRas.
FIG. 8 Pathways required for signaling by activated SHP-2 mutants. (A) Animal caps injected with RNA encoding DNRas (64) (5 ng), C-trunc (33) (20 pg), or Rho19N (100 pg), as indicated, were analyzed for elongation after either stimulation with FGF (100 ng/ml) (more ...)
Activin-stimulated morphogenetic movements are dependent on regulation of C-cadherin-mediated cell-cell adhesion (5
). We examined whether C-cadherin also plays a role in FGF- and E76A-induced elongation. Interestingly, expression of a dominant negative form of C-cadherin that lacks its cytoplasmic domain (C-trunc) (33
) did not prevent either FGF- or E76A-induced elongation (Fig. A). The same dose of C-trunc dramatically reduced activin-stimulated morphogenetic movements (data not shown), as reported previously (33
Recently, SHP-2 has been implicated in regulation of the actin cytoskeleton in tissue culture cells (40
). The small G protein Rho regulates actin cytoskeleton reorganization (56
) and is required for completion of gastrulation in Drosophila
). Expression of dominant negative Rho (Rho19N) (27
) completely abolished FGF- and E76A-induced elongation (Fig. A). In contrast to the results seen upon expression of DNRas, Rho19N expression had no effect on MAPK activation or induction of Xbra by either FGF or E76A (Fig. B and C). These findings indicate that Rho acts downstream of SHP-2 in the pathway leading to elongation.