The Tyrosine Kinase sf-Stk and Its Downstream Signals Are Required for Maintenance of Friend Spleen Focus-Forming Virus-Induced Fibroblast Transformation

Abstract

Infection of erythroid progenitor cells by Friend spleen focus-forming virus (SFFV) leads to acute erythroid hyperplasia and eventually to erythroleukemia in susceptible strains of mice. The viral envelope protein, SFFV gp55, forms a complex with the erythropoietin receptor (EpoR) and a short form of the receptor tyrosine kinase Stk (sf-Stk), activating both and inducing Epo-independent proliferation. Recently, we discovered that coexpression of SFFV gp55 and sf-Stk is sufficient to transform NIH 3T3 and primary fibroblasts. In the current study, we demonstrate that sf-Stk and its downstream effectors are critical to this transformation. Unlike SFFV-derived erythroleukemia cells, which depend on PU.1 expression for maintenance of the transformed phenotype, SFFV gp55-sf-Stk-transformed fibroblasts are negative for PU.1. Underscoring the importance of sf-Stk to fibroblast transformation, knockdown of sf-Stk abolished the ability of these cells to form anchorageindependent colonies. Like SFFV-infected erythroid cells, SFFV gp55-sf-Stk-transformed fibroblasts express high levels of phosphorylated MEK, ERK, phosphatidylinositol 3-kinase (PI3K), Gab1/2, Akt, Jun kinase (JNK), and STAT3, but unlike virus-infected erythroid cells they fail to express phosphorylated STATs 1 and 5, which may require involvement of the EpoR. In addition, the p38 mitogen-activated protein kinase (MAPK) stress response is suppressed in the transformed fibroblasts. Inhibition of either JNK or the PI3K pathway decreases both monolayer proliferation and anchorage-independent growth of the transformed fibroblasts as does the putative kinase inhibitor luteolin, but inhibition of p38 MAPK has no effect. Our results indicate that sf-Stk is a molecular endpoint of transformation that could be targeted directly or with agents against its downstream effectors.Friend spleen focus-forming virus (SFFV) is a replicationincompetent murine retrovirus that causes a rapid erythroblastosis when injected into mice with its related helper virus Friend MuLV (reviewed in reference 30). Friend SFFV has deletions in its env gene, which 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. The Fv-2 gene encodes one of the key susceptibility factors for SFFV-induced erythroid disease (10, 26). Fv-2, which encodes the receptor tyrosine kinase Stk/RON, is unusual in that it has a second internal promoter, which allows for the transcription of two distinct gene products: full-length Stk and a short form, sf-Stk (8, 26). Full-length Stk is a typical receptor tyrosine kinase with an extracellular ligand binding domain, a transmembrane domain, and an intracellular kinase/ signaling domain. The short form lacks the extracellular ligand binding domain but retains the transmembrane domain and intracellular kinase/signaling domain. Susceptible strains of mice express both full-length Stk and sf-Stk, but resistant strain...

Role of Phosphatidylinositol 3-Kinase in Friend Spleen Focus-Forming Virus-Induced Erythroid Disease

“…Hematopoietic cells infected with SFFV show constitutive activation of PI3-kinase, and SFFV is thought to activate PI3-kinase through sf-Stk or the EpoR primarily via IRS-related adaptor molecules (14,29). In this study, we showed that sf-Stk interacts with p85␣, but the interaction requires sf-Stk kinase activity since a kinase-inactive mutant of sf-Stk fails to interact with p85␣.…”

“…SFFV gp55 is thought to activate these pathways by interacting with either the EpoR or sf-Stk (17,31,43). In several in vitro systems, class IA PI3-kinase has been shown to be activated by Epo through the EpoR (8,20,21) or by SFFV through sf-Stk (5,14). We and others have shown that the PI3-kinase pathway is important for the induction of Epo independence by SFFV (5,29).…”

Activation of the N-Terminally Truncated Form of the Stk Receptor Tyrosine Kinase Sf-Stk by Friend Virus-Encoded gp55 Is Mediated by Cysteine Residues in the Ecotropic Domain of gp55 and the Extracellular Domain of Sf-Stk

“…We have shown that the cysteines in the extracellular domain of Sf-Stk promote the phosphorylation of the receptor by mediating its interaction with gp55. In order to assess other potential roles for these cysteines, we utilized a constitutively active form of Sf-Stk containing an M330T mutation in the kinase domain which was reported to induce Epo ind colony formation in the absence of FVP (27). We introduced the M330T mutation into both SfStk and Sf-StkC4A (Sf-StkM330T and Sf-StkC4AM330T) and transfected these constructs into 293 cells in the presence or absence of gp55.…”

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

“…NIH 3T3/sf-Stk/SFFV cells express higher levels of phosphorylated STAT3, MEK1, ERK 1/2, Gab 1/2, Akt, PI3K and JNK 1/2. 27 These effectors all contribute to growth/survival and are frequently upregulated in cancers. These effectors also have the potential to be activated downstream of the EpoR.…”