Role of N‐terminal sequences of the tyrosine kinase sf‐Stk in transformation of rodent fibroblasts by variants of Friend spleen focus‐forming virus

Abstract

Infection of erythroid cells by Friend spleen focus-forming virus (SFFV) leads to acute erythroid hyperplasia in mice, due to expression of its unique envelope glycoprotein, gp55. Erythroid cells expressing SFFV gp55 proliferate in the absence of their normal regulator, erythropoietin, because of the interaction among the viral envelope protein, the erythropoietin receptor, and a short form of the receptor tyrosine kinase Stk (sf-Stk). This leads to constitutive activation of several signal transduction pathways. Our previous studies showed that sf-Stk interacts with SFFV gp55, forming disulfide-linked complexes. This covalent interaction, along with other noncovalent interactions with SFFV-gp55, results in constitutive tyrosine phosphorylation of sf-Stk and rodent fibroblast transformation. Here, we determined the precise amino acid region within sf-Stk that contributes to fibroblast transformation by the polycythemia-inducing (SFFV-P) and the anemia-inducing (SFFV-A) strains of SFFV. Sf-Stk deletion mutants showed different transforming abilities in fibroblasts infected with SFFV-P and SFFV-A, although the N-terminal extracellular domain of sf-Stk was essential for fibroblast transformation by both viruses. Point mutations of sf-Stk indicated that cysteine 19 was critical for fibroblast transformation by SFFV-P, although all four cysteines (8, 19, 37 and 42) appeared to be important for fibroblast transformation by both SFFV-P and SFFV-A. Mutation of sf-Stk cysteine 19 abolished its ability to form dimers with SFFV-P and SFFV-A gp55. These results suggest that the interaction between sf-Stk and the envelope proteins of the polycythemia-and anemia-inducing variants of SFFV is architecturally different.The Friend spleen focus-forming virus (SFFV) is a highly pathogenic retrovirus that induces rapid erythroblastosis in susceptible strains of mice.1 Friend SFFV is a replication-defective virus with deletions in its env gene that give rise to a unique glycoprotein, SFFV gp55. This unique glycoprotein confers pathogenicity on the virus; a vector encoding SFFV gp55 alone is sufficient to induce erythroblastosis in susceptible strains of mice.2 The Fv-2 gene encodes one of the key susceptibility factors for SFFV-induced erythroid disease 3,4 : the receptor tyrosine kinase Stk/RON, a member of the Met family of receptor tyrosine kinases. 5,6 Susceptibility to SFFVinduced disease is associated with expression of a short form of Stk, termed sf-Stk, which is transcribed from an internal promoter within the Stk gene of susceptible mice (Fv-2 ss ) but not in resistant mice (Fv-2 rr ), 4 and is abundantly expressed in erythroid cells.5 Infection of Fv-2 ss erythroid cells with the polycythemia-inducing strain of SFFV (SFFV-P) induces erythropoietin (Epo)-independent proliferation and differentiation, whereas erythroid cells infected with the anemiainducing strain of SFFV (SFFV-A) proliferate in the absence of Epo but still require Epo for differentiation.7 A number of signaling pathways, normally activated in erythroid cells...