Relevance of Akt phosphorylation in cell transformation induced by Jaagsiekte sheep retrovirus

Abstract

Expression of the JSRV envelope (Env) is sufficient to transform immortalized rodent fibroblasts. A putative docking site for the PI3-K kinase (Y(590)-X-X-M(593)) in the cytoplasmic tail of the transmembrane domain of the JSRV Env is a major determinant of viral-induced cell transformation. Akt is constitutively phosphorylated in rodent fibroblasts transformed by the JSRV Env. However, recent data suggest that Y590 and M593 are not necessary for JSRV Env-induced transformation of the immortalized chicken fibroblasts cell line DF-1. In this study we found that JSRV-induced transformation of DF-1 cells is Akt-independent. In addition, a replication-competent avian vector expressing the JSRV Env (RCASBP(A)+JE) was also able to induce transformation of primary chicken embryo fibroblasts (CEF). Vectors expressing JSRV Env Y590 mutants were still able to induce CEF cells transformation but not as efficiently as the vectors expressing the wild-type Env. In CEF cells, as in DF-1 cells, only the expression of the wild-type Env induced constitutive phosphorylation of Akt. Thus, in chicken cells, the degree of transformation induced by the JSRV Env is maximum in the presence of Y590 and Akt phosphorylation. We addressed the significance of Akt phosphorylation in rat 208F cells transformed by the JSRV Env and showed that Akt is indeed activated and shows kinase activity. Inhibitors of the PI-3K/Akt pathway reproducibly decreased the transformation efficiency of the JSRV Env. In vivo, we found phosphorylated Akt only in nasal tumors induced by the enzootic nasal tumor virus (ENTV), a JSRV-related beta-retrovirus. No evidence of Akt phosphorylation was found in lung tumor sections of sheep affected by pulmonary adenocarcinoma. As a whole, these results suggest that the activation of the PI-3K/Akt pathway contributes to the process of JSRV-induced cell transformation but most likely is not the primary determinant both in vitro and in vivo.

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

“…The transforming property of the JSRV envelope has been demonstrated in vitro in various cell lines including murine NIH 3T3 fibroblasts [44], rat 208F fibroblasts [70], avian DF-1 fibroblasts [4,93], bronchial human BEAS-2B epithelial cells [16], canine kidney MDCK epithelial cells [43], and rat kidney RK3E cells [46]. The oncogenic property of the JSRV envelope has also been shown in vivo in an immuno-deficient mouse model [87] and recently in sheep [9].…”

“…In JSRV-transformed cells, phosphorylation of the Y590 residue of the intra-cytoplasmic TM tail, or interaction between the p85 subunit of PI3K and the envelope (a prerequisite for Akt activation), have never been shown [42,43]. Mutations of the YXXM motif do not abolish Akt phosphorylation, so that the exact role of this motif remains to be determined [42,43,93]. Moreover, implication of the YXXM motif may differ between cell types in in vitro experiments [4,36,41,42,61,93]; while it is essential for transformation of NIH 3T3 fibroblasts, it is not required for transformation of avian DF-1 fibroblasts.…”

Oncogenic transformation by the jaagsiekte sheep retrovirus envelope protein

“…Despite these important correlations, no interaction between JSRV Env protein and PI3K/p85 has been observed, indicating JSRV Env may activate PI3K/Akt indirectly, possibly through as-yet unidentified cellular adaptor molecules or by other signaling pathways that are directly triggered by JSRV Env. Interestingly, however, Akt phosphorylation was not Oncogenesis by the JSRV envelope protein S-L Liu and AD Miller detected in the lung sections of sheep OPA, nor in the JSRV Env-transformed DF-1 cells in vitro, yet was present in the ENTV-induced nasal tumors of sheep (Zavala et al, 2003). These data indicate that the Akt phosphorylation in the Env-transformed cells may be cell-type dependent.…”

“…The first is Akt pathway that is either dependent or independent of PI3K. This pathway is activated in Env-transformed fibroblasts derived from rodent (NIH 3T3 and 208F) Alberti et al, 2002;Chow et al, 2003;Maeda et al, 2003Maeda et al, , 2005Liu et al, 2003b) and chicken (CEF and DF-1) (Allen et al, 2002;Zavala et al, 2003), as well as in MDCK dog epithelial cells (Liu and Miller, 2005). The second is Raf-MEK-MAPK pathway, which is activated in Env-transformed NIH 3T3 mouse fibroblasts and RK3E rat epithelial cells (Maeda et al, 2005).…”

“…First, Akt is activated in several Env-transformed cell lines, as shown by Akt phosphorylation and elevated in vitro Akt kinase activity Zavala et al, 2003;Liu et al, 2003b;Liu and Miller, 2005). Second, PI3K specific inhibitor LY294002 or wortmannin inhibits transformation by JSRV Env in these cells, and inhibits their Akt phosphorylation and in vitro kinase activity in a dosedependent manner Zavala et al, 2003;Liu et al, 2003b).…”