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. 2001 Aug;21(15):4968-84.
doi: 10.1128/MCB.21.15.4968-4984.2001.

The multisubstrate adapter Gab1 regulates hepatocyte growth factor (scatter factor)-c-Met signaling for cell survival and DNA repair

S Fan  1 , Y X Ma, M Gao, R Q Yuan, Q Meng, I D Goldberg, E M Rosen
Affiliations

The multisubstrate adapter Gab1 regulates hepatocyte growth factor (scatter factor)-c-Met signaling for cell survival and DNA repair

S Fan et al. Mol Cell Biol. 2001 Aug.

Abstract

Hepatocyte growth factor (scatter factor) (HGF/SF) is a pleiotrophic mediator of epithelial cell motility, morphogenesis, angiogenesis, and tumorigenesis. HGF/SF protects cells against DNA damage by a pathway from its receptor c-Met to phosphatidylinositol 3-kinase (PI3K) to c-Akt, resulting in enhanced DNA repair and decreased apoptosis. We now show that protection against the DNA-damaging agent adriamycin (ADR; topoisomerase IIalpha inhibitor) requires the Grb2-binding site of c-Met, and overexpression of the Grb2-associated binder Gab1 (a multisubstrate adapter required for epithelial morphogenesis) inhibits the ability of HGF/SF to protect MDCK epithelial cells against ADR. In contrast to Gab1 and its homolog Gab2, overexpression of c-Cb1, another multisubstrate adapter that associates with c-Met, did not affect protection. Gab1 blocked the ability of HGF/SF to cause the sustained activation of c-Akt and c-Akt signaling (FKHR phosphorylation). The Gab1 inhibition of sustained c-Akt activation and of cell protection did not require the Gab1 pleckstrin homology or SHP2 phosphatase-binding domain but did require the PI3K-binding domain. HGF/SF protection of parental MDCK cells was blocked by wortmannin, expression of PTEN, and dominant negative mutants of p85 (regulatory subunit of PI3K), Akt, and Pak1; the protection of cells overexpressing Gab1 was restored by wild-type or activated mutants of p85, Akt, and Pak1. These findings suggest that the adapter Gab1 may redirect c-Met signaling through PI3K away from a c-Akt/Pak1 cell survival pathway.

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Figures

FIG. 1
FIG. 1
Structural requirements for c-Met signaling for cell survival in MDCK cells. (A) c-Met protein levels in cell lines expressing different chimeric Met receptors. Subconfluent proliferating cells were harvested, and equal aliquots of total cell protein (50 μg per lane) were Western blotted, using antibodies directed against c-Met (N-Term), c-Met (C-Term), the multisubstrate adapter Gab1, and α-actin (control for loading and transfer). (B) MTT assays. Parental MDCK cells or clones expressing wt or mutant chimeric CSF-Met receptors were tested for protection against ADR by the chimeric receptor. Subconfluent proliferating cells in 96-well dishes were sham treated (negative control) or preincubated with recombinant human HGF/SF (100 ng/ml; positive control) or recombinant human CSF-1 (50 ng/ml) for 48 h, exposed to ADR (15 μM for 2 h), washed, postincubated in fresh drug-free culture medium for 72 h, and assayed for cell viability using the MTT assay. Cell viability values (relative to sham-treated controls) are means ± standard errors of 10 replicate wells. The survival of untransfected parental cells was not altered by CSF-1, while in wt CSF-Met cells, the protection by CSF-1 was similar to that of HGF/SF. For cells with mutant CSF-Met but not for wt CSF-Met cells, the viability of CSF-1–ADR-treated cells was less than that for HGF/SF-ADR-treated cells (P < 0.001, two-tailed t tests). (C) Decreased association of Gab1 and p85 with N1358H mutant Met receptor. Subconfluent proliferating cultures of MDCK cells expressing wt CSF-Met or N1358H mutant Met receptors were treated with or without CSF-1 (50 ng/ml for 48 h) and subjected to IP using antibodies against c-Met (C-Term), Gab1, or normal mouse IgG as a control. IPs were Western blotted for c-Met (C-Term), Gab1 (C-20), or p85 (the catalytic subunit of PI3 kinase). (D) Inhibition of protection by mATP and Y(1349+1356)F receptors by DN Ras. Subconfluent proliferating cells in 100-mm-diameter dishes were transfected overnight without or with 10 μg of a DN Ras expression vector (Ras-N17), washed, subcultured into 96-well dishes, preincubated with or without CSF-1 (50 ng/ml for 48 h), exposed to ADR (15 μM for 2 h), postincubated for 72 h, and assayed for MTT dye conversion. Ras-N17 caused a significant reduction in the survival of CSF-1-treated cells for all three cell types (P < 0.001) but did not affect the cell survival in the absence of CSF-1.
FIG. 2
FIG. 2
Overexpression of Gab1 but not c-Cb1 blocks HGF/SF protection of MDCK. (A) Anti-HA, Gab1 (C-20), and c-Met (C-Term) Western blots of MDCK cell lines stably expressing different forms of HA-Gab1. Subconfluent proliferating cells were harvested, and equal aliquots of total cell protein (50 μg per lane) were blotted as described in Materials and Methods. (B) Anti-HA Western blots of MDCK cell lines stably expressing HA-Cb1 cDNAs. Assays were performed as described for panel A. (C) Gab1 and its homolog Gab2 block protection against ADR-induced toxicity. (Top) Subconfluent proliferating cells in 96-well dishes were preincubated with or without HGF/SF (100 ng/ml for 48 h), exposed to ADR (15 μM for 2 h), washed, postincubated in fresh drug-free culture medium for 72 h, and assayed for MTT dye conversion. Cell viability values are means ± standard errors for cell lines expressing wt Cb1 (HA-c-Cb1), mutant Cb1 (HA-70Z-Cbl), Gab1 (wt) (Bottom) and parental cells. Values plotted were calculated from 10 replicate wells. (Bottom) Parental MDCK cells were transfected overnight with Gab2 or empty vector (10 μg of plasmid DNA per 100-mm-diameter dish) plus Lipofectamine, inoculated in 96-well dishes, and assayed as described above. For each experimental condition, two separate dishes were transfected; 10 replicate wells were tested per dish. Values are means ± ranges of the two dishes. For each ADR dose, cell survival based on pooled data for the two dishes was significantly increased by HGF/SF treatment of empty vector (P < 0.001) but not Gab2-transfected (P > 0.05) cells. (D) Gab1 blocks HGF/SF-stimulated repair of DNA strand breaks. Subconfluent proliferating cells in 100 mm-diameter dishes were preincubated with or without HGF/SF (100 ng/ml for 48 h), exposed to ADR (20 μM for 2 h), washed, and postincubated for 24 h in fresh drug-free culture medium. The number of SSBs or DSBs was determined by DNA filter elution assays and expressed as the fraction of DNA eluted minus that for sham-treated control cells. Results are shown for the same cell types as in panel C. Values are means ± ranges or standard errors for two or three clones of each type.
FIG. 3
FIG. 3
Ability of Gab1 to abrogate HGF/SF-mediated protection requires the PI3K-binding domain of Gab1. (A) MTT assays of cell viability. Assays were carried out as described for Fig. 2C. Cell viability values (relative to sham-treated control cells) are means ± standard errors for three separate Gab1(wt) cell clones and means ± ranges for two separate clones each of Gab1ΔPH, Gab1ΔSP, and Gab1ΔPI3K. For each cell line, 10 replicate wells were tested. Cell viability values were significantly higher in HGF/SF-ADR-treated parental or Gab1ΔPI3K cells than in the corresponding ADR-treated cells (P < 0.001). (B) DNA filter elution assays of DNA strand breaks. Assays were performed as described for Fig. 2D, using the same clonal types as in panel A. Values are means ± standard errors or ranges for two or three clones of each type.
FIG. 4
FIG. 4
Gab1 overexpression inhibits HGF/SF-induced sustained activation of c-Akt. (A) HGF/SF induces prolonged c-Akt activation and FKHR phosphorylation in parental but not Gab1(wt) cells. Subconfluent proliferating cultures of two clones of each type in 100-mm-diameter dishes were preincubated with or without HGF/SF (100 ng/ml for 48 h), harvested, and Western blotted to detect total Akt, Akt phosphorylated on Ser-473 (phospho-Akt), total FKHR, or FKHR phosphorylated on Ser-256 (phospho-FKHR). Each lane corresponds to an aliquot of 50 μg of total cell protein. The bar graphs show the ratios of phospho-Akt and of phospho-FKHR in cells treated with (+) HGF/SF relative to cells treated without (−) HGF/SF, as determined by densitometry. Values are means ± ranges for two clones of each clonal type. (B) HGF/SF causes only transient c-Akt activation and FKHR phosphorylation in Gab1(wt) cells. Assays were performed as for panel A except that shorter incubations with HGF/SF (10 min to 24 h) were used.
FIG. 4
FIG. 4
Gab1 overexpression inhibits HGF/SF-induced sustained activation of c-Akt. (A) HGF/SF induces prolonged c-Akt activation and FKHR phosphorylation in parental but not Gab1(wt) cells. Subconfluent proliferating cultures of two clones of each type in 100-mm-diameter dishes were preincubated with or without HGF/SF (100 ng/ml for 48 h), harvested, and Western blotted to detect total Akt, Akt phosphorylated on Ser-473 (phospho-Akt), total FKHR, or FKHR phosphorylated on Ser-256 (phospho-FKHR). Each lane corresponds to an aliquot of 50 μg of total cell protein. The bar graphs show the ratios of phospho-Akt and of phospho-FKHR in cells treated with (+) HGF/SF relative to cells treated without (−) HGF/SF, as determined by densitometry. Values are means ± ranges for two clones of each clonal type. (B) HGF/SF causes only transient c-Akt activation and FKHR phosphorylation in Gab1(wt) cells. Assays were performed as for panel A except that shorter incubations with HGF/SF (10 min to 24 h) were used.
FIG. 5
FIG. 5
Effects of wt and mutant p85 (PI3K) and of wt PTEN expression vectors on HGF/SF-mediated protection of MDCK cells. (A) The regulatory subunit of PI3K (p85) modulates MDCK survival. Subconfluent proliferating cells in 100-mm-diameter dishes were transiently transfected overnight, using Lipofectamine and 10 μg of each vector per dish: empty vector (control), p85(wt), or p85(DN). Cells were washed, subcultured into 96-well dishes, preincubated with or without HGF/SF (100 ng/ml for 48 h), exposed to ADR (15 μM for 2 h), washed three times to remove ADR, postincubated for 72 h in fresh drug-free medium, and assayed for MTT dye conversion. Values of cell viability are based on 10 replicate wells. p85(wt) protected parental and Gab1 (wt) cells against ADR in the absence of HGF/SF (P < 0.001); p85(DN) blocked the HGF/SF-mediated protection of parental cells against ADR (P < 0.001). (B) PTEN blocks HGF/SF-mediated protection of parental MDCK cells. Transient transfection assays were performed as for panel A. For the pooled data from the two dishes for each assay condition, cells treated with empty vector plus HGF/SF and ADR showed a significantly reduced cell viability compared with cells treated with (PTEN, HGF/SF, and ADR) (P < 0.001); there was little or no increase in survival of cells treated with PTEN, HGF/SF, and ADR as compared with PTEN plus ADR.
FIG. 6
FIG. 6
Effect of mutant Akt constructs on HGF/SF-mediated cytoprotection in MDCK cells expressing wt or mutant forms of Gab1. (A) DN Akt strongly inhibits HGF/SF-mediated protection. Transient transfection assays were performed analogously to those shown in Fig. 5A. Briefly, cells were transiently transfected overnight, washed, subcultured into 96-well dishes, preincubated with or without HGF/SF, exposed to ADR (15 μM for 2 h), postincubated for 72 h, and assayed for MTT dye conversion. For the parental and Gab1ΔPI3K cell types, DN Akt caused a large and significant reduction in the survival of HGF/SF-ADR-treated cells (P < 0.001). (B) wt Akt and Akt-myr enhance cell survival without HGF/SF and overcome Gab1 inhibition of HGF/SF protection. Assays were performed as described for panel A. Compared with the empty vector controls, wt Akt and Akt-myr enhanced the survival of ADR-treated parental MDCK cells and of Gab1(wt) cells in the absence of HGF/SF. The survival of wt Akt and Akt-myr cells but not empty vector-transfected Gab1(wt) cells, was significantly increased in cells treated with HGF/SF plus ADR compared to ADR alone (P < 0.001). (Inset) Parental cells and one clone each of Gab1(wt) and Gab1ΔPI3K cells were cotransfected with wt Akt and with plasmid pSV-β-gal, under conditions similar to those described above, to assess transfection efficiency. At 48 h after transfection, cultures were stained using X-Gal to detect β-Gal staining. (C) Pak1 positively modulates HGF/SF protection. Transient transfection assays were performed as above, using wt and DN Pak1 expression vectors. wt Pak1 enhanced the survival of parental or Gab1(wt) cells in the absence or presence of HGF/SF (P < 0.001); DN Pak1 abolished the ability of HGF/SF to protect the parental cells.
FIG. 7
FIG. 7
Failure of Gab1ΔPI3K to associate with the PI3K regulatory subunit (p85). Subconfluent proliferating parental, HA-Gab1(wt), or HA-Gab1ΔPI3K cell cultures were treated without (−) or with (+) 100 ng of HGF/SF per ml for 10 min and then harvested for IP. Cells were subjected to IP using an anti-HA (A) or anti-human Gab1 (B) antibody. As a control, parental MDCK cells were subjected to IP using an equivalent quantity of normal mouse IgG. The precipitates were subjected to Western blotting using anti-HA antibody, anti-Gab1 antibody C-20 (different from the Gab1 IP antibody), and an anti-p85 antibody. Protein densities (+HGF/SF/−HGF/SF ratio) were determined by densitometry (C).
FIG. 8
FIG. 8
Contribution of apoptosis to ADR cytotoxicity and Gab1 inhibition of protection. (A) Inhibition of caspase-3 partially reverses ADR toxicity in parental and Gab1(wt) cells. Subconfluent proliferating parental MDCK cells and two clones of Gab1 (wt) cells in 96-well dishes were preincubated with or without HGF/SF for 48 h, treated with ADR (15 μM for 2 h), washed, and postincubated for 72 h in the presence of caspase-3 inhibitor (10 μM), caspase-6 inhibitor (10 μM), or neither (see Materials and Methods). Cells were then assayed for MTT dye conversion. For each individual cell line, 10 replicate wells were assayed, and the mean cell viability values were calculated. For parental cells, values plotted are means ± standard errors of the 10 replicate wells. For the Gab1(wt) cells, the values are means ± ranges of the two separate clones. For both parental and Gab1(wt) cells treated with HGF/SF plus ADR or with ADR alone, the survival was significantly greater for cells treated with caspase-3 inhibitor compared with no inhibitor (P < 0.001 for all comparisons). (B) Gab1 blocks HGF/SF-mediated protection against ADR-induced apoptosis. Parental cells and two Gab1(wt) cell clones were treated with or without HGF/SF (100 ng/ml) and/or ADR (15 μM for 2 h), postincubated for 72 h in drug-free medium, and harvested for assays of DNA laddering as described in Materials and Methods. Pretreatment with HGF/SF blocked the formation of DNA ladders in the parental cells but not in the Gab1(wt) cell clones.
FIG. 9
FIG. 9
Model for Gab1 signaling pathways in MDCK cells. See text for discussion.
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