The Activation of Stat3 by the Stk Receptor Tyrosine Kinase Requires Src Kinases and Gab2, and Is Essential for Friend Virus Induced, Epo-Independent Growth of Primary Erythroid Cells.

Friend virus-induced erythroleukemia is a model for the study of the multi-stage nature of carcinogenesis. The early stage of the disease is characterized by a polyclonal expansion of infected erythroid precursor cells, and the later stage is marked by the emergence of fully transformed cells in the spleen, blood, bone marrow and liver, leading to the development of erythroleukemia. Previous findings have demonstrated that a naturally occurring, N-terminally truncated form of the stk receptor tyrosine kinase (Sf-stk) provides signals necessary for the polycolonal expansion of infected cells. Signal transducer and activator of transcription (Stat) proteins play an important role in normal and malignant hematopoiesis. Moreover it has been shown that growth factors can induce Stat3 in a Src family kinase (SFK)-dependent manner. Here we show that dominant negative Stat3 and c-Src significantly inhibit the BFU-e and CFU-e colony number induced by friend virus. In addition, the non-specific SFK inhibitor PP1 also abrogated Epo-independent colony formation. Moreover we found that the inhibition of SFKs by PP1 suppresses Stat3 tyrosine phosphorylation activated by Sf-stk. To elucidate the signaling mechanism by which Sf-stk activates Stat3, we mutated the docking site tyrosines to phenylalanine. Our results indicated that the mutation of Y436F eliminates Sf-stk-induced Stat3 tyrosine phosphorylation. Studies from our lab have further demonstrated that tyrosine 436 of Sf-stk is essential for the cytokine-independent growth of primary erythroblasts induced by Friend virus, by providing a docking site for Grb2, which recruits Gab2 to induce erythroleukemia. To further determine the relationship between Gab2 and Stat3, we generated fusion proteins, in which Sf-stk lacking the docking site tyrosines is fused to Gab2 or Gab1. The Sf-stk/Gab2, but not Sf-stk/Gab1, fusion protein supported Epo-independent colony formation and induced Stat3 tyrosine phosphorylation. In conclusion, these results suggest that SFKs, Gab2 and Stat3 are all important in Friend virus induced erythroleukemia mediated by Sf-stk, and that Gab2 and SFKs lie upstream of Stat3 in this signaling pathway.