Since Friend disease was first reported in 1957, the acute erythroleukemia induced by the various strains of Friend virus has provided an excellent model to study multistage carcinogenesis. In the first stage, the virus infects erythroid progenitor cells, resulting in the Epo-independent (Epo^ind ) expansion of erythroid progenitor cells. In the late stage, integration of the virus into the Spi-1 locus leads to inappropriate expression of Pu.1 and subsequent inactivation of the p53 locus in erythroid cells, causing a block in their differentiation and eventually acute erythroleukemia.

Friend virus induced erythroleukemia in susceptible mice is initiated by the interaction of the Friend virus encoded glycoprotein, gp55, with the erythropoietin receptor and the product of the host Fv2 gene, a naturally occurring truncated form of the Stk receptor tyrosine kinase (Sf-Stk). We have previously demonstrated that the activation of Sf-Stk and recruitment of a Grb2/Gab2/Stat3 signaling complex is required for the ability of Friend virus to induce Epo independent colony formation of erythroid progenitor cells. Here we demonstrate that gp55 interacts with Sf-Stk through the cysteine residues Cys³⁰⁶, Cys³⁰⁹, Cys³³⁷ and Cys ³³⁸ in the ecotropic domain of gp55 and the cysteine residues Cys ⁸, Cys¹⁹, Cys³⁷ and Cys⁴² in the extracellular domain of Sf-Stk. This interaction does not promote the dimerization of Sf-Stk, but results in the enhanced phosphorylation of pre-formed Sf-Stk dimers. Point mutation of these cysteine residues or deletion of these domains abrogates the interaction between gp55 and Sf-Stk, resulting in the inability of these mutants to promote the Epo independent growth of erythroid progenitor cells.

We further demonstrate that cell surface localization of Sf-Stk is critical for receptor phosphorylation and activation of downstream signaling. We show that the interaction between gp55 and Sf-Stk induces the cell surface translocation of Sf-Stk. We also show that mutation of the cysteines in the extracellular domain of Sf-Stk results in enhanced cell surface localization in the absence of gp55, but not enhanced phosphorylation of Sf-Stk. Alternatively, while a constitutively activating mutation in the kinase domain of Sf-Stk is not sufficient to induce receptor phosphorylation and downstream signaling, mutation of the cysteines in the extracellular domain of Sf-Stk in this context restores full activity of Sf-Stk in the absence of gp55. These results indicate that both kinase activation and cell surface localization play an essential role in the activation of Sf-Stk signaling by gp55.

Finally, we demonstrate that gp55 activation of Sf-Stk results in the recruitment and activation of Gab1 through the C terminal docking sites Tyr429 and Tyr436, and that Gab1 activation by Sf-Stk correlates with its ability to promote the phosphorylation of Erk and the activation of AP1. Further, we show that Src interacts with Sf-Stk in the presence of gp55 and that Src activity is essential for the phosphorylation of Gab1 and Gab2 and the ability of gp55 to promote Sf-Stk activation of the MAPK signaling pathway. We also demonstrate that Src kinase activity is essential for the Epo independent growth of primary erythroblasts in response to Friend virus infection.