The FGFR is responsible for mediating a diverse array of effects in different biological systems including cell proliferation, angiogenesis, mesoderm induction, patterning, and neurite outgrowth. In skeletal muscle, ligand activation of the FGFR stimulates Erk activation, myoblast proliferation, and repression of differentiation (22
). In this study, we have examined the role of SHP-2 in FGFR signaling in myoblasts. We have found that SHP-2 is recruited to a complex containing the multisubstrate adaptor protein FRS-2α following FGFR activation. Furthermore, the catalytic activity of SHP-2 is required for activation of the Erks and, subsequently, for the transactivation of Elk-1 in response to FGF-2 in myoblasts. Using a constitutively active mutant of SHP-2, we have also found that SHP-2 mediates the suppressive effects of the FGFR on myogenesis. Interestingly, the suppressive effect of the activated mutant of SHP-2 on myogenesis appears to occur via an Erk-independent pathway. Finally, our data suggest that the ability of the activated mutant of SHP-2 to recapitulate some of the FGFR signaling pathways is due to its ability to induce hyper-tyrosyl phosphorylation of FRS-2α in response to FGF-2. Thus, SHP-2 may participate in the early regulation of signaling pathways downstream of the FGFR that are responsible for myogenic regulation.
Activation of the FGFR leads to tyrosyl phosphorylation of a 90-kDa protein termed FRS-2α, a lipid-anchored, multisubstrate adaptor protein that contains a PTB domain (25
). Using C2C12 myoblasts, we show that FRS-2α is expressed, becomes tyrosyl phosphorylated, and complexes with SHP-2 following FGFR activation (Fig. ). Both FRS-2α and IRS-1 belong to the multisubstrate adaptor protein family. However, unlike the FGFs, which suppress myogenesis, the IGFs promote myogenesis via IRS-1 tyrosyl phosphorylation. Thus, it was formally possible that the IGFs could induce tyrosyl phosphorylation of FRS-2α, especially since ligands other than the FGFs, such as nerve growth factor, have been shown to induce FRS-2α tyrosyl phosphorylation (34
). Also, there is the potential for the PTB domain of FRS-2α to compete for the same Shc binding site on the insulin receptor, allowing FRS-2α to bind and become tyrosyl phosphorylated by insulin (34
). A recent report has suggested that insulin can induce FRS-2α tyrosyl phosphorylation in PC12 cells overexpressing the insulin receptor (10
). This issue is of particular importance because the ability of the IGFs to initiate signaling via FRS-2α could have significant implications for our understanding of how these ligands antagonistically regulate skeletal muscle function. Our experiments indicate that under conditions where either insulin or IGF-I induces tyrosyl phosphorylation of IRS-1, FRS-2α does not become tyrosyl phosphorylated (Fig. ). Conversely, FGF-2 stimulation of myoblasts does not result in tyrosyl phosphorylation of IRS-1 (Fig. ). These data demonstrate that the signaling pathways for the IGFs and FGFs in skeletal muscle are indeed distinct.
The PTP domain of SHP-2 has been shown to be required for signaling to the Erks following FGF stimulation of fibroblasts (53
), PC12 (17
), and Xenopus
ectodermal explants (59
). However, a role for SHP-2 in FGF-2 signaling in myoblasts has yet to be demonstrated. We now show that the PTP activity of SHP-2 is required for FGF-2 stimulation of MAPK-mediated Elk-1 transactivation (Fig. ). Following FGF-2 stimulation of myoblasts, we noted that SHP-2 becomes tyrosyl phosphorylated (Fig. ), prompting us to test the requirement for the C-terminal Grb2 binding-tyrosyl phosphorylation sites in this pathway. We observed a modest but consistent inhibition of FGF-2-induced Elk-1 transactivation when the two C-terminal tyrosyl residues (Y542 and Y580) were mutated (Fig. ). When Erk2 kinase activity was assessed following expression of the Grb2 binding-tyrosyl phosphorylation site mutant, maximal potentiation of Erk2 activity was not achieved following FGF-2 stimulation. Thus, as is the case in other systems, our data demonstrate that in myoblasts, the catalytic activity of SHP-2 is required for transmission of the predominant signal to the Erks and subsequently to Elk-1 following FGF-2 stimulation.
In this report we demonstrate that overexpression of wild-type SHP-2 potentiates both serum- and FGF-2-induced inhibition of myogenesis (Fig. ). However, overexpression of a catalytically inactive mutant of SHP-2 abrogates both serum- and FGF-2-induced inhibition of muscle-specific gene expression. Therefore, these data reveal that the catalytic activity of SHP-2 participates in a pathway that contributes to the inhibitory actions of both serum and FGF-2 on myogenesis. When SHP-2 was overexpressed, its inhibitory effects on muscle-specific gene expression were significantly greater in the presence of FGF-2 than in the presence of serum. Previously, we have shown that SHP-2 complexes with both Gab-1 and SHPS-1 in C2C12 myoblasts in the presence of serum (24
). However, in the presence of FGF-2, SHPS-1, Gab-1, and FRS-2α all complex with SHP-2 (Fig. ). It is conceivable that the ability of SHP-2 to further suppress muscle-specific gene expression in the presence of FGF-2, compared to serum, is reflected by its ability to form an additional complex with tyrosyl-phosphorylated FRS-2α. Given that SHP-2 has been shown to bind tyrosyl-phosphorylated FRS-2α directly through phosphotyrosyl residues pY436
), we hypothesize that the interaction of SHP-2 with these residues plays a critical role in mediating the inhibitory myogenic effects by the FGFR. FRS-2α also interacts directly with Grb2 (17
), which could also participate in transmitting the inhibitory myogenic signals from the FGFR. Further work is required to determine the significance of the FRS-2α tyrosyl phosphorylation sites in FGF-2-mediated inhibition of myogenesis.
We have utilized a constitutively active mutant of SHP-2 to assess the sufficiency of the PTP activity of SHP-2 to inhibit myogenesis. These data demonstrate that the catalytic activity of SHP-2 is sufficient to promote myogenic inhibition prior to the initiation of differentiation. However, the activated mutant of SHP-2 no longer suppresses differentiation once the myogenic process has been initiated (Fig. and ). This is consistent with the notion that once myogenesis has been initiated following growth factor removal, myoblasts become irrevocably committed to terminal differentiation (8
). These observations indicate that the suppressive effects of the activated SHP-2 mutant are likely restricted to myoblasts that have not yet begun the process of myogenesis. The inhibitory effects of the activated SHP-2 mutant on myogenesis provide a potential mechanism whereby the mitogenic and the inhibitory myogenic effects of the FGFR can be separated (22
). SHP-2 may represent the bifurcation in the signaling pathway downstream of the FGFR that mediates both the Erk-dependent proliferative and the Erk-independent suppressive myogenic effects of FGF-2.
Our data do not support the interpretation that activation of Erk is necessary to mediate the suppressive effects of FGFR signaling on myogenesis. Although overexpression of an activated mutant of SHP-2 suppresses myogenesis, no appreciable increase in either Erk activity or Elk-1 transactivation is observed (Fig. and ). Importantly, the suppressive effects of the activated SHP-2 mutant are not likely due to its mislocalization, since this mutant interacts appropriately with both FRS-2α and the p120 complex (containing Gab-1 and SHPS-1) (Fig. ). These data raise two possibilities for how SHP-2 is involved in mediating the repressive effects of FGF-2 on myogenesis. SHP-2 may either signal in a completely distinct pathway, independent of the Erks, to repress myogenesis, or it may activate the Erks to levels just sufficient to suppress myogenesis. We favor the former explanation because direct Erk2 kinase assays of lysates from SHP-2 E76A-transduced myoblasts showed that SHP-2 did not alter the level of basal Erk2 activity (Fig. ). Although there is evidence to indicate that Erk is sufficient to repress myogenesis, there is also evidence for Erk-independent pathways that participate in suppressing myogenesis (22
). In MM14 cells, a skeletal muscle satellite cell line that is strictly dependent on the FGFs, proliferation and repression of differentiation are separate events (26
). Moreover, Erk activity is dispensable for myogenic repression by FGF-2 in these MM14 cells (22
). It is conceivable that our data implicate SHP-2 as a component of the Erk-independent signaling pathway that represses differentiation.
In many regards, the results presented here are similar to those reported by O'Reilly et al., who found that the activated mutant of SHP-2 was sufficient for mesoderm induction to levels similar to those with FGF stimulation (46
). However, unlike FGF, which induces a robust activation of Erk in Xenopus
explants, the activated mutant of SHP-2 fails to significantly induce Erk activity (46
). The activated SHP-2 mutant may retain the capacity to engage the Ras/Raf/Erk pathway weakly but may signal more robustly to other pathways, presumably those that lead to the control of differentiation and/or other morphogenic processes. SHP-2 has been shown to be upstream of Ras (42
) and has recently been implicated as a regulator of other small GTP-binding proteins such as Rho (23
). Interestingly, activated mutants of RhoG, Rac1, and cdc42Hs have been shown to prevent myogenesis (35
). These observations raise the possibility that the activated mutant of SHP-2 could potentially inhibit myogenesis by engaging a Rho GTP-binding protein.
Our data have revealed an intriguing and unexpected finding; they indicate that the activated mutant of SHP-2 promotes and subsequently potentiates FGF-2-induced FRS-2α tyrosyl phosphorylation (Fig. ). These observations may explain why the activated mutant of SHP-2 recapitulates the actions of FGF-2 to inhibit myogenesis. It is conceivable that under low concentrations of FGF-2, FRS-2α is not stoichiometrically tyrosyl phosphorylated and FGF-2-mediated signaling is silent. However, expression of the activated mutant of SHP-2 in the absence of exogenous FGF-2 may promote tyrosyl phosphorylation of FRS-2α to levels that are sufficient to propagate the FGFR signal. In fact, the activated mutant of SHP-2 can synergize with subthreshold levels of exogenous FGF-2, resulting in the full restoration of animal cap elongation (46
). In the absence of growth factors, autocrine production of FGF-2 in myoblasts is insufficient to repress myogenesis. The activated mutant of SHP-2 may cooperate, either additively or synergistically, with these low levels of FGF-2 to induce an inhibitory myogenic signal by increasing FRS-2α tyrosyl phosphorylation. In agreement with this hypothesis, we did observe a slightly higher level of FRS-2α tyrosyl phosphorylation (data not shown), and SHP-2 is basally associated with FRS-2α in the absence of growth factors (Fig. ). Recent evidence suggests that Ras is responsible for the autocrine production of FGF-2 in the MM14 skeletal muscle satellite cell line (12
). One possibility is that the activated mutant of SHP-2 may promote FGF-2 autocrine production via a Ras, or another small GTP-binding protein-dependent pathway, in addition to regulating tyrosyl phosphorylation of FRS-2α. In combination, these two mechanisms could culminate in an FGFR/FRS-2α signal that would be sufficient to suppress myogenesis.
Although the activated mutant of SHP-2 did not cause activation of the Erks, it did induce hyper-tyrosyl phosphorylation of FRS-2α (Fig. ). One might have anticipated that hyper-tyrosyl phosphorylation of FRS-2α would be sufficient to induce Erk activation. One interpretation of these observations is that there are tyrosyl phosphorylation sites that reside on FRS-2α which do not couple to the Erk pathway but rather couple the FGFR to the myogenic pathway. These data are supported by the longstanding observation that FGFR-mediated proliferation and inhibition of myogenesis are separable events (22
). The precise mechanisms by which the activated mutant of SHP-2 causes hyper- and sustained tyrosyl phosphorylation of FRS-2α in response to FGF-2 remain to be elucidated. However, one could speculate that this mutant of SHP-2 leads to the activation of a tyrosine kinase that subsequently phosphorylates FRS-2α. One obvious candidate is the FGFR itself; however, we find no difference in the levels of FGFR auto-tyrosyl phosphorylation when the activated mutant of SHP-2 is expressed (Fig. ). An alternative mechanism could involve inhibition of a PTP by the activated SHP-2 mutant that negatively regulates FRS-2α tyrosyl phosphorylation. Clearly, much work is needed to resolve the precise mechanistic basis of these effects of the activated SHP-2 mutant on FRS-2α tyrosyl phosphorylation. In summary, our results reveal a pathway in which SHP-2 functions to mediate both the proliferative Erk signaling pathway and the inhibitory myogenic signals. Further investigation into the targets of SHP-2 in myogenic signaling is required to elucidate the details of this apparent dual signaling role for SHP-2 in myoblasts.