Gab1 Is Involved in Erythropoietin Receptor-Mediated Survival Signal through Activation of the Erk Pathway.

Erythropoietin (EPO) is required for the survival, proliferation and differentiation of erythroid progenitor cells. The scaffolding adaptor protein Grb2-associated binder-1 (Gab1) is tyrosine phosphorylated upon stimulation of EPO in several cell lines and erythroid progenitor cells, and interacts with signaling molecules such as SHP2 phosphatase and the p85 subunit of phosphatidylinositol 3-kinase (PI3K). However, biological functions of Gab1 in EPO receptor (EPOR)-mediated signaling has not yet been established. In this study, to explore the biological functions of Gab1 in vivo, Gab1-deficient F-36P human erythroleukemia cells were generated by means of transfection of the expression vector of siRNA against Gab1. WST-1 assay revealed that growth of Gab1-deficient F-36P cells was reduced to 61% and 77%, respectively, 5 days after incubation with lower concentrations of EPO (0.001 and 0.01 ng/ml), compared with that of mock-transfected F-36P (F-36P-mock) cells. In contrast, growth of Gab1-deficient F-36P cells at sufficient concentration of EPO (10 ng/ml) was similar to that of F-36P-mock cells. Analysis of apoptosis by flow cytometry using FITC-labeled annexin-V showed that the percentage of annexin-V-positive apoptotic cells in Gab1-deficient F-36P and F-36P-mock cells was increased to 19% and 34%, and 8% and 17%, respectively, 72 h after incubation with 0.01 and 0.001 ng/ml of EPO. These results indicate that Gab1 plays a crucial role in transducing EPOR-mediated survival signals. Next, we examined the molecular mechanism of EPOR-mediated signaling involved in survival of erythroid cells through Gab1. Western blot analysis showed that EPO-induced phosphorylation of threonine 202/ tyrosine 204 on Erk-1 and Erk-2 in Gab1-deficient F-36P but not in F-36P-mock cells was significantly suppressed. Interestingly, phosphorylation of serine 473 on Akt in Gab1-deficient F-36P cells in response to EPO was slightly suppressed in comparison with that in F-36P-mock cells. Therefore, Gab1-mediated survival signals appear to be mainly transmitted to downstream through activation of the Erk pathway, although the PI3K/Akt pathway may be involved in EPO-initiated survival signal transduction mediated by Gab1. Furthermore, EPO induced the association of SHP2 with EPOR in Gab1-deficient F-36P cells. Gab1 was associated with SHP2 in EPO-treated F-36P cells. In addition, Gab1 was constitutively associated with Grb2 in F-36P cells. Taken together, EPO induces the recruitment of Gab1 to EPOR through binding of Gab1 to SHP2, which is associated with EPOR. Because the guanine nucleotide exchange factor SOS1 is known to bind to the SH3 domain of Grb2, SOS1-Grb2 complex is recruited to vicinity of Ras at the plasma membrane to activate this GTP-binding protein through the interaction of Grb2 with Gab1, leading to activation of Erk.