c-Cb1 and Gab1 are multisubstrate signaling adapters that link activated growth factor and cytokine receptors to specific downstream signaling pathways (14
). Both proteins have been implicated in HGF/SF→c-Met receptor signaling pathways (14
). In particular, several studies have implicated Gab1 as an essential mediator of HGF/SF-induced morphogenesis (e.g., branching tubulogenesis of MDCK epithelial cells) (28
). Thus, overexpression of Gab1 could restore tubulogenesis mediated by a chimeric c-Met receptor defective in the ability to associate with Gab1 (28
). The Met→Gab1 tubulogenesis pathway required localization of Gab1 at sites of cell-cell contact, which, in turn, required the Gab1 PH domain and functional PI3K but did not require the PI3K-binding domain of Gab1. In the present study, overexpression of Gab1 but not of wt or mutant forms of Cb1 blocked HGF/SF protection against ADR-induced cytotoxicity and DNA damage. Interestingly, even a constitutively active mutant form of Cb1 (70Z) with transforming activity (43
) failed to modulate the ability of HGF/SF to protect MDCK epithelial cells. However, the Gab1 homolog Gab2 strongly inhibited HGF/SF protection of MDCK cells.
These findings implicate Gab1 as a regulator of an HGF/SF-mediated cell survival pathway(s). The overexpression of Gab1 did not induce cytotoxicity by itself, nor did it have a significant effect on sensitivity to ADR in the absence of HGF/SF, suggesting that effect of Gab1 was mainly restricted to the increased survival due to c-Met signaling. Studies of MDCK cell lines expressing mutant Gab1 proteins revealed that neither the PH nor the SHP2-binding domain was required for regulation of cell survival or DNA repair, but the PI3K-binding domain of Gab1 was essential for these activities. Further studies revealed that in parental MDCK cells, HGF/SF induced the sustained activation of c-Akt, which persisted at levels significantly greater than the unstimulated control after 24 and 48 h. In contrast, in cell lines overexpressing Gab1(wt) or the PH and SHP2 domain mutants, HGF/SF induced only transient activation (phosphorylation) of c-Akt, which returned to baseline by 2 h or less. However, cells expressing the PI3K-binding domain mutant Gab1 exhibited significant HGF/SF activation of c-Akt at the 48 h time point.
The forkhead family transcription factor FKHR, which induces the transcription of proapoptotic genes, is phosphorylated and functionally inactivated by Akt (41
). Utilizing a phosphospecific FKHR antibody, we found that the phosphorylation of FKHR in the different Gab1-expressing cell lines and the time course for HGF/SF-induced phosphorylation of FKHR correlated strongly with the HGF/SF-induced Akt activation. Coupled with the observation that inhibition of PI3K signaling by wortmannin reduced the degree of HGF/SF protection in untransfected MDCK cells, these findings suggest that Gab1 functions to regulate the PI3K→c-Akt cell survival pathway.
Consistent with this idea, the ectopic expression of p85(wt) overcame the inhibition of protection in Gab1-overexpressing cell lines and protected cells even in the absence of HGF/SF, while p85(DN) blocked the HGF/SF-mediated protection of parental MDCK cells. Somewhat surprisingly, the overexpression of wt or constitutive membrane-localized forms of p110, the catalytic subunit of PI3K, failed to overcome the Gab1 inhibition of HGF/SF protection or to significantly enhance the baseline MDCK cell survival, as did overexpression of p85, c-Akt, or Pak1. It should be recognized that c-Met, Gab1, and other signaling intermediaries such as c-Cb1 interact with the regulatory subunit of PI3K (p85) rather than the catalytic subunit (p110). Our findings suggest, although they do not formally prove, that for protection to occur, PI3K must be targeted to the c-Met receptor and that membrane targeting of the catalytic subunit of PI3K is not sufficient for protection.
The ability of HGF/SF to protect MDCK cells was abrogated by the overexpression of PTEN, a lipid 3′-phosphatase that converts phosphatidylinositol-(3,4,5)-triphosphate [PI(3,4,5)P3
] to PI(4,5)P2
. This finding suggests that the major lipid product of PI3K enzymatic activity [PI(3,4,5)P3
] is essential for the HGF/SF survival signal, although it is noted PI3K has protein kinase activity, which might also play a role in HGF/SF-mediated protection. Prior studies have suggested that PTEN blocks c-Akt-mediated signaling pathways for cell survival (39
). The Gab1 regulation of cell survival and DNA repair required the PI3K-binding but not the PH domain of Gab1. Thus, localization of Gab1 to cell-cell contacts, which is mediated by the PH domain (28
), is not required for cell survival. However, the failure of Gab1ΔPI3K to inhibit cell protection suggests that a physical interaction between Gab1 and PI3K is required to regulate cell survival. These findings are consistent with a model in which the recruitment of PI3K by Gab1 to a specifically constituted signaling complex determines which signaling pathway(s) downstream of PI3K is activated by HGF/SF.
Expression of DN mutants of either c-Akt or Pak1 strongly inhibited the ability of HGF/SF to protect parental MDCK cells, while the expression of wt or constitutively active mutant forms of c-Akt and Pak1 partially restored the HGF/SF-mediated protection in Gab1-overexpressing cell lines. These findings implicate c-Akt and Pak1 as signaling intermediaries for cell survival that function downstream of Gab1. In some cellular contexts, Pak1 may act downstream of c-Akt to cause the phosphorylation and inactivation of Bad, a cell death agonist that binds to and inhibits the cell survival mediator Bcl-XL
). Although Pak1 activity is required for HGF/SF protection, and two Rho family GTPases that can function as activators of Pak1 (cdc42 and Rac) (45
) are activated by HGF/SF in MDCK cells (36
), preliminary studies indicate that expression of DN inhibitors of these GTPases (cdc42-N17 and Rac-N17) do not block HGF/SF protection of MDCK or MtLn3 rat mammary carcinoma cells (unpublished findings). Thus, the HGF/SF-mediated c-Akt/Pak1 survival pathway may be independent of cdc42 and Rac.9
We previously reported that HGF/SF blocks apoptosis induced by DNA-damaging agents such as ADR in various epithelial cell types, including MDCK cells (12
). HGF/SF inhibition of apoptosis was demonstrated by a significant reduction in the extent of DNA fragmentation caused by ADR. Consistent with this finding, a caspase-3 inhibitor and, to a much less extent, a caspase-6 inhibitor enhanced cell survival in ADR-treated MDCK cells in the absence of HGF/SF. Although the increase in survival attributable to the caspase-3 inhibitor in Gab1(wt) cell clones was relatively modest (20 to 25%), this increase was observed in the absence or presence of HGF/SF, suggesting that Gab1 functions to inhibit an antiapoptosis pathway that ultimately prevents the activation of caspase-3. The inability of the caspase-3 inhibitor to fully restore cell viability, especially in the Gab1(wt)-transfected cell lines, could have several explanations: (i) the inhibitor only partially blocked caspase-3 activity; (ii) additional caspases can independently mediate ADR-induced cell death; and (iii) ADR also induces a component of nonapoptotic cell death.
Several studies suggest that Gab1 associates with the c-Met receptor by at least two distinct mechanisms: one involving recruitment to the canonical Grb2-binding site (1356
YVNV) of c-Met by interaction with Grb2, and the second governed by a non-Grb2-dependent mechanism (27
). The overexpression of Gab1 restored c-Met-mediated tubulogenesis even in cells with chimeric receptors mutated at the Grb2-binding site (i.e., 1356
Y→F and 1358
), suggesting that a Grb2-independent pathway may contribute to tubulogenesis. Our studies using the chimeric CSF-Met-transfected MDCK cell lines suggest that the Grb2-binding site is particularly important for c-Met-mediated cell survival signaling, although other are also involved. Thus, Gab1 may compete with or sequester other substrates that associate with c-Met through the Grb2 site, possibly including the p85 regulatory subunit of PI3K. This hypothesis is consistent with the finding that the N1358H mutant receptor showed significantly reduced association with Gab1 and with p85 compared with the wt CSF-Met receptor.
Previous studies have established that PI3K can be activated through an interaction with Ras (34
) and that Ras can be recruited to tyrosine kinase receptors through interaction with Sos (Son of sevenless), a protein that functions as a guanine nucleotide exchange factor for Ras and interacts with tyrosine kinase receptors (23
). Sos, which interacts with the multifunctional docking site of the Met receptor (1349
), also binds to the Grb2 adapter. Interestingly, a recent study suggests that the multifunctional docking site of Met is not required for receptor-mediated Ras signaling or cell scattering (42
). Thus, residual Ras signaling could explain why there is some protection by mutant receptors lacking either or both tyrosines (1349
Y and 1356
Y). Thus, expression of a DN Ras (Ras-N17) abrogated the protection mediated by the Y(1349+1356)F mutant receptor and partially but significantly inhibited protection by the wt chimeric CSF-Met receptor. Alternatively, the small degree of protection observed by some of the mutant chimeric Met receptors, but not observed for the N1358H mutant, could be due to clonal variability.
Figure shows one possible model for Gab1-dependent signaling pathways in MDCK cells. In this scheme, PI3K is an effector for both the morphogenesis and antiapoptosis pathways, and Gab1, through its PI3K-binding domain, directs the signal away from the antiapoptosis pathway. Several features of this model are noteworthy: (i) PI3K functions both to activate Gab1 morphogenesis signaling (28
) and, in a different context, to activate the c-Akt/Pak1 survival pathway; and (ii) Gab1 acts upstream of c-Akt and Pak1 to regulate cell survival signaling.
Model for Gab1 signaling pathways in MDCK cells. See text for discussion.
This model may also have implications for understanding the role of HGF/SF in embryonic morphogenesis. Since regulated apoptosis is essential during embryonic development, Gab1 may function to render cells undergoing morphogenesis more susceptible to cell death signals required for the next embryonic transition. In our studies, overexpression of Gab1 had a much greater effect on the HGF/SF-stimulated cell survival than on the baseline survival in the absence of exogenous HGF/SF. Although the hypothesis is speculative, our findings support a significant role for Gab1 in the regulation of cell survival pathways in epithelial cells.