A Phosphatidylinositol 3-Kinase Docking Site in the Cytoplasmic Tail of the Jaagsiekte Sheep Retrovirus Transmembrane Protein Is Essential for Envelope-Induced Transformation of NIH 3T3 Cells

Abstract

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of a transmissible lung cancer of sheep known as ovine pulmonary carcinoma. Recently, we have found that the expression of the JSRV envelope (Env) is sufficient to transform mouse NIH 3T3 cells in classical transformation assays. To further investigate the mechanisms of JSRV oncogenesis, we generated a series of envelope chimeras between JSRV and the JSRVrelated endogenous retroviruses of sheep (enJSRVs) and assessed them in transformation assays. Chimeras containing the exogenous JSRV SU region and the enJSRV TM region were unable to transform NIH 3T3 cells. Additional chimeras containing only the carboxy-terminal portion of TM (a region that we previously identified as VR3) of the endogenous envelope with SU and the remaining portion of TM from the exogenous JSRV were also unable to transform NIH 3T3 cells. The VR3 region includes the putative membrane-spanning region and cytoplasmic tail of the JSRV TM glycoprotein; this suggested that the cytoplasmic tail of the JSRV Env mediates transformation, possibly via a cell signaling mechanism. Mutations Y590 and M593 in the cytoplasmic tail of the JSRV envelope were sufficient to inhibit the transforming abilities of these constructs. Y590 and Jaagsiekte sheep retrovirus (JSRV) is the etiological agent of a contagious lung cancer of sheep known as ovine pulmonary carcinoma (OPC) or sheep pulmonary adenomatosis (4,16,30,36). OPC is a naturally occurring disease in most countries of the world and is characterized by the transformation of the differentiated epithelial cells of the lungs, type II pneumocytes and Clara cells (17,33). OPC is being extensively studied for its similarities with a particular type of human lung cancer known as bronchioloalveolar carcinoma (BAC) (17,21,23,33). The causes of BAC are unknown. BAC is not strongly associated with cigarette smoking, and its incidence appears to be increasing in the United States (1,5,6,51). Thus, the JSRV/ OPC model can offer a foundation for understanding the molecular mechanisms of type II pneumocytes and Clara cell transformation, especially considering the role played by retroviruses in the discovery of cellular oncogenes (46). M593 are part of a Y-X-X-M motif that is recognized by the phosphatidylinositol 3-kinase (PI-3K). PI-3K initiates a cell signaling pathway that inhibits apoptosis and is required for a number of mitogens during the G 1 -to-S-phase transition of the cell cycle. PI-3K activatesCell transformation by JSRV is offering a new paradigm for retroviral oncogenesis (45). The JSRV genome does not contain a cell-derived oncogene, which would suggest that JSRV induces cell transformation by insertional activation of protooncogenes (46). However, the incubation period in experimentally inoculated lambs can be as short as 2 to 3 weeks (47, 52), which appears too short for insertional activation to occur. Our recent study showed that the expression of the envelope (Env) protein of JSRV is able to transform rodent fibroblasts in vitro (27). Thus, ...

Hyaluronidase 2 negatively regulates RON receptor tyrosine kinase and mediates transformation of epithelial cells by jaagsiekte sheep retrovirus

“…tyrosine residue at position 590 (Y590) of the intracellular portion of the JSRV Env is essential for transformation of NIH 3T3 cells by Env, implying phosphorylation of Y590 and subsequent activation of Akt by PI3-kinase bound to Y590 (5,10). In contrast, we have not found phosphorylation of Y590 in MDCK (Fig.…”

“…6) is quite different from that proposed for 208F rat and NIH 3T3 mouse fibroblasts, where the cytoplasmic tail of the Env protein is thought to interact with PI3-kinase to stimulate the PI3-kinase͞Akt pathway leading to transformation (10,11). Indeed, mouse Hyal2 seems to play no role in transformation of NIH 3T3 mouse cells, because the Hyal2 protein from mouse NIH 3T3 cells functions very poorly as a receptor for JSRV and binds JSRV Env very weakly if at all, and overexpression of the mouse Hyal2 does not suppress transformation by JSRV Env, as expected if Hyal2 functions as a tumor suppressor (27).…”

“…The cytoplasmic tail of Env, in particular a YXXM protein motif that is a putative docking site for phosphatidylinositol 3-kinase (PI3-kinase) after tyrosine phosphorylation, is important for fibroblast transformation, which seems to be mediated through the PI3-kinase͞Akt pathway (10,11). Transformation has not been explored yet in epithelial cells, the target for JSRV disease, and the mechanism of transformation might be different compared with that in fibroblasts.…”

Jaagsiekte Sheep Retrovirus (JSRV): from virus to lung cancer in sheep

“…Mutations of the YXXM motif of the JSRV-env gene abolish cell transformation of NIH-3T3 [43,61] and rat 208F and RK3E cells [38]. Akt activation was observed in different cell lines transformed by JSRV (Fig.…”

“…In addition to the TM region, deletions of the SU (surface) glycoprotein (going from the signal peptide to the junction between the SU and TM subunits) also abolish transformation induced by the envelope, suggesting that multiple domains of SU may be involved in cellular transformation. The cytoplasmic tail of TM, composed of 43 amino acids, is essential for the transformation process in MDCK and NIH-3T3 cells [43,61]. This region contains a peptidic YXXM motif (Fig.…”