The scaffolding adapter Gab1 is critical for signaling by a number of RTKs (10
) and possibly for some cytokine and antigen receptors (13
). Upon cell stimulation, Gab1 becomes tyrosyl phosphorylated on multiple sites and recruits several signal relay proteins (10
). The precise ways in which these proteins then transduce signals further downstream remain unclear. Previous studies implicated Gab1-Shp2 association in signaling to the Ras-Raf-Mek-Erk pathway, whereas interaction of Gab1 with p85 is implicated in PI3K activation. Our work has revealed further complexity in the interaction of Gab1 with p85 and Shp2. Specifically, we have found that Shp2 regulates the amount of p85 that is bound to Gab1 following EGF stimulation and thereby determines the kinetics and extent of activation of PI3K and its downstream targets Akt and GSK-3β. Surprisingly, this regulation appears to be RTK specific, such that PI3K activation following PDGF and IGF-1 stimulation is not increased—indeed, appears to be decreased—in the absence of Shp2. Thus, the Gab1-Shp2 interaction has distinct functions in cell signaling in response to different growth factors and may help to determine the specificity of signals delivered by RTKs.
Previous studies suggested that Gab1 and Gab2 might be Shp2 substrates (3
). Consistent with previous work (39
), however, we observed no difference in total Gab1 tyrosyl phosphorylation in response to EGF (Fig. ) or other growth factors (data not shown) in the absence of Shp2. Moreover, Shc association with Gab1 was similar in WT and Shp2−/−
cells (Fig. ). These findings left open the possibility that Shp2 might act on specific tyrosyl-phosphorylation sites on Gab1. Indeed, Gab1-associated p85 is increased dramatically in Shp2−/−
cells (Fig. ). Accordingly, Gab1-associated PI3K activity is enhanced (Fig. ) and PI3K-dependent downstream targets (Fig. ) exhibit increased activation in response to EGF stimulation of these cells. Analogous results were obtained by using a cell line that inducibly overexpresses catalytically inactive Shp2 (Fig. ). The truncated protein that is expressed in Shp2−/−
cells lacks its N-terminal SH2 domain (35
) and cannot bind to Gab1 (39
). Furthermore, a Gab1 mutant unable to bind Shp2 exhibits increased p85 binding compared to that for WT Gab1 following EGF stimulation.
Taken together, these results imply that Shp2 must be recruited to Gab1 and be catalytically active to regulate Gab1-p85 association. The most likely explanation for these findings is that, upon recruitment to Gab1, Shp2 is activated by engagement of its N-terminal SH2 domain (1
) and then dephosphorylates one or more of the PI3K binding sites on Gab1, thereby regulating Gab1-p85 interaction. Consistent with a direct effect of Shp2 deficiency on the tyrosyl phosphorylation of p85 sites on Gab1, far-Western analysis revealed a marked enhancement in the ability of p85 to bind to Gab1 isolated from Shp2−/−
cells (Fig. ), whereas binding of Shp2 was only minimally affected (Fig. ). Our results clearly exclude the possibility that enhanced p85 association with Gab1 in the absence of functional Shp2 reflects an indirect effect of Shp2 deficiency on p85 or another protein that affects p85 binding to Gab1. However, we cannot exclude the possibility that Shp2 deficiency indirectly affects the ability of Gab1 to bind p85, e.g., by leading to another type of modification on Gab1 that affects binding ability. Direct assessment of the stoichiometry of phosphorylation of the p85 binding sites on Gab1 will be required to demonstrate this unambiguously.
In contrast, Grb2 association with Gab1 appears to be decreased slightly but consistently in Shp2−/−
cells. The physiological significance of this observation remains to be determined, but notably, Shp2−/−
) (Fig. and ) and Gab1−/−
) fibroblasts have defective Ras→Erk pathway activation in response to EGF and other growth factors. Since Grb2 binds the Ras exchange factor Sos, it will be important to determine whether decreased Gab1-Grb2 association contributes to decreased Ras→Erk activation. The observed decrease in Gab1-Grb2 interaction has two other potentially interesting implications. First, since Gab1-Shc interaction and EGFR tyrosyl phosphorylation (and presumably kinase activity) are unaffected by Shp2 deficiency (Fig. ), these results suggest that different tyrosine kinases catalyze the phosphorylation of distinct sites on Gab1. Indeed, Gab2 is reportedly phosphorylated by both colony-stimulating factor 1 receptor and Src family kinases in macrophages (17
). More intriguingly, however, since Gab1-Grb2 binding is decreased in the absence of Shp2, it is possible that Shp2 regulates (directly or indirectly) the activity of the kinase that phosphorylates the Grb2 binding site(s) on Gab1. Further studies will be required to address these issues.
Recently, it was reported that the recruitment of Gab1 to the EGFR initiates a positive feedback loop in which initial tyrosyl phosphorylation of Gab1 leads to p85 recruitment, PI3K activation, and PIP3 production, which then leads to further recruitment (and phosphorylation) of Gab1 via PIP3 binding to the Gab1 PH domain (31
). Our data suggest that, by dephosphorylating p85 binding sites on Gab1, Shp2 helps to regulate the Gab1-PI3K positive feedback loop, thereby controlling the extent, kinetics, and location of PI3K activation in response to EGF (Fig. ). Indeed, in the absence of Shp2, EGF induces sustained translocation of Gab1 to the plasma membrane (Fig. ). Since Gab1 activation of the Erk pathway also requires membrane recruitment (and Gab1 signaling to Erk requires Shp2), Shp2 may contribute to both Erk activation and inactivation as well. However, we do not exclude the possibility that other mechanisms, such as the recruitment and activation of the 5′-inositol phosphatase Ship-2 and/or serine/threonine phosphorylation of Gab1 and/or PI3K, also contribute to negative regulation of the Gab1-PI3K positive feedback loop. A recent report also indicated that phosphorylation of Gab1 by Erk enhances p85 association with Gab1 (45
). Importantly, the increased interaction of p85 with Gab1 in EGF-stimulated Shp2−/−
cells cannot be explained by differential Erk activity, since EGF-evoked Erk activation is diminished in the absence of functional Shp2 (e.g., Fig. ). However, it is possible that decreased Erk activation may explain or help explain the decreased association of p85 with Gab1 in PDGF- and/or IGF-1-stimulated Shp2−/−
cells (compared to WT cells).
FIG. 8. Model for regulation of kinetics of EGF-induced PI3K activation by Gab1-Shp2 interaction. Following EGF stimulation, the docking protein Gab1 is recruited to the plasma membrane through binding of its Met binding domain to the EGFR directly and through (more ...)
Although Gab1-Shp2 interaction is critical for regulation of EGF-evoked PI3K activation, Shp2 is dispensable for, and may potentiate, PDGF and IGF-1 stimulation of PI3K-dependent pathways. Notably, unlike the EGFR, the PDGF receptor (PDGFR) has direct binding sites for p85, and p85 binding to these sites is believed to provide the major route to PI3K activation from the PDGFR (15
). Previous studies suggest that there is no preferential dephosphorylation of these sites by Shp2 (16
), which could explain why there is no increase in Akt activation in response to PDGF in Shp2−/−
cells. Less clear is why PDGF-evoked Akt activation appears to be decreased in these cells. Conceivably, as discussed above, a tyrosine kinase regulated by Shp2 could contribute to phosphorylation of the p85 binding sites on the PDGFR. Alternatively, and perhaps more likely, decreased Ras activation may lead to lower PI3K activation in response to PDGF, since Ras binds to and activates the catalytic subunit (p110) of PI3K (32
). Like the EGFR, the IGF-1 receptor lacks direct binding sites for PI3K. In the IGF pathway, however, insulin receptor substrate (IRS) proteins, particularly IRS-1, are more important for PI3K activation than is Gab1. Previous studies of the role of Shp2 in regulating insulin or IGF-1 activation of PI3K yielded conflicting results. One group, studying the effects of IRS-1 mutants that cannot bind Shp2, reported that Shp2 negatively regulates p85 binding to IRS-1, presumably by dephosphorylating its p85 binding sites (23
). Another group demonstrated that expression of dominant-negative Shp2 attenuates IRS-1-associated PI3K activity (42
). Our results are consistent with the latter report and suggest that Shp2 is a positive regulator of PI3K activation by IGF-1 or insulin, at least in fibroblast cell lines. How and why Shp2 can act as a negative regulator of EGF-induced PI3K activation and a positive regulator of PI3K activation by other RTKs remain topics for future work.
It is increasingly clear that signal thresholds and the temporal aspects of signaling are important for determining the cellular response. One way that cells can utilize the same (or similar) signaling pathways to control a wide range of cellular processes is to vary the amplitude and duration of pathway activation and to convert this into qualitatively different biological responses. In perhaps the best studied example, the EGFR induces transient Erk activation and stimulates proliferation of PC12 cells, whereas the NGF receptor and fibroblast growth factor receptor evoke a sustained Erk response, culminating in neuronal differentiation (21
). By regulating the extent and magnitude of p85 association with Gab1 and thereby helping to regulate the extent and kinetics of PI3K activation in response to some, but not all, RTKs, Shp2 may play an important role in directing the varied effects of different growth factors.