Friend Virus induced acute erythroleukemia provides an experimental system to study the multi-stage events involved in the pathogenesis of hematologic neoplasias. Friend erythroleukemia progresses in a biphasic manner: the initial phase is characterized by the polyclonal expansion of infected erythroid precursor cells, which is promoted by the activation of a naturally-occuring truncated form of the Stk receptor called short-form stk (Sf-Stk); and the later phase of the disease is marked by the acquisition of fully transformed erythroid leukemic cells in the spleen, blood, bone marrow and liver, leading to the development of erythroleukemia. Studies from our lab have demonstrated the importance of the kinase activity of Sf-Stk, its Grb2 binding site and recruitment of a Grb2/Gab2 complex in the development of Friend Virus induced erythroleukemia. In addition, we have shown that fusion proteins of Sf-Stk and Gab2 (Sf-Stk/Gab2), in which the C-terminal docking site tyrosines of Sf-Stk were replaced with the coding sequence of Gab2, are able to support Epo-independent colony formation. Here we demonstrate that Gab2−/− mice are partially resistant to Friend erythroleukemia in vivo and Gab2−/− erythroblasts fail to form Epo-independent colonies in response to Friend Virus infection. This defect can be rescued by exogenous expression of wild-type Gab2, but not Gab1. Moreover, we find that the Sf-Stk/Gab2 fusion protein, but not the Sf-Stk/Gab1 fusion protein, which fails to support hematopoietic transformation in this system, results in enhanced tyrosine phosphorylation of and interaction with signal transducer and activator of transcription 3 (Stat3). Our in vivo studies suggest that Stat3 is tyrosine phosphorylated and interacts with Gab2 in splenocytes following injection of sensitive mice with Friend Virus. Exogenous expression of dominant negative Stat3 in sensitive erythroblasts significantly inhibits BFU-e and CFU-e colony formation induced by Friend Virus. Furthermore, bone marrow cells from floxed-Stat3 mice fail to form Epo-independent colonies in response to Friend Virus infection following the retroviral introduction of Cre-recombinase into primary erythroblasts. In order to map the domains of Gab2 which are critical for the activation of Stat3 and the Epo-independent growth of Friend Virus infected cells, we utilized a series of Gab1/Gab2 chimeric molecules. Our data suggest that the region between Q120 and P358 of Gab2 is required for the tyrosine phosphorylation of Stat3. By scanning this region, we identified a Y194LHQ site, a potential Stat3 binding motif, in Gab2, but not in Gab1. The ability of Sf-Stk /Gab2 to recruit and activate Stat3, and support Epo-independent colony formation of primary erythroblasts in response to Friend Virus infection in vitro was abolished when Y194 motif was mutated to phenylalanine. In addition, mutation of this tyrosine in the context of wide type Gab2 abrogated its ability to rescue the defective response of Gab2−/− bone marrow to Friend Virus. In conclusion, these results suggest that a Sf-Stk/Grb2/Gab2/Stat3 signaling cascade serves as a key mediator in the Friend Virus induced transformation of erythroid cells and that the Y194LHQ motif of Gab2 is required for the activation of Stat3 during the transformation of primary erythroid cells in this system.

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