The Role of Phosphatidylinositol 3-Kinase, Rho Family GTPases, and STAT3 in Ros-induced Cell Transformation

Abstract

Using loss-of-function mutants of Ros and inducible epidermal growth factor receptor-Ros chimeras we investigated the role of various signaling pathways in Rosinduced cell transformation. Inhibition of the mitogenactivated protein kinase (MAPK) pathway with the MEK (MAP/extracellular signal-regulated kinase kinase) inhibitor PD98059 had little effect on the Ros-induced monolayer and anchorage-independent growth of chicken embryo fibroblasts and NIH3T3 cells even though more than 70% of the MAPK was inhibited. In contrast, inhibiting the phosphatidylinositol 3-kinase (PI3K) pathway with the drug LY294002, a dominant negative mutant of PI3K, ⌬p85, or the phosphatidylinositol phosphatase PTEN (phosphatase and tensin homologue deleted in chromosome ten) resulted in a dramatic reduction of v-Ros-and epidermal growth factor receptor-Ros-promoted anchorage-independent growth of chicken embryo fibroblasts and NIH3T3 cells, respectively. Parallel and downstream components of PI3K signaling such as the Rho family GTPases (Rac, Rho, Cdc42) and the survival factor Akt were all shown to contribute to Ros-induced anchorage-independent growth, although Rac appeared to be less important for Ros-induced colony formation in NIH3T3 cells. Furthermore, the transformation-attenuated v-Ros mutants F419 and DI could be complemented by constitutively active mutants of PI3K and Akt. Finally, we found that overexpressing a constitutively active mutant of STAT3 (STAT3C) conferred a resistance to the inhibition of Ros-induced anchorage-independent growth by LY294002, suggesting a possible overlap of functions between PI3K and STAT3 signaling in mediating Ros-induced anchorage-independent growth.The proto-oncogene c-Ros encodes a receptor protein-tyrosine kinase (RPT kinase) 1 that shares structural homology with the sevenless protein of Drosophila melanogaster (1). In addition, the protein-tyrosine kinase (PT kinase) domain of c-Ros shares greater sequence and structural homology with those of the insulin receptor family members than any other RPT kinases (1, 2). Although its ligand remains unknown, c-Ros expression is specific and highly regulated. c-Ros has been found to be expressed in a variety of organs/tissues, with relatively high levels in the kidney and intestine. c-Ros protein has been shown to be localized in the epithelial cells of the kidney collecting duct and intestinal villi in chicken (3). v-Ros is the oncogenic form of c-Ros and was identified as the transforming gene of the avian sarcoma virus UR2 (4). Comparison of the v-Ros oncogene with c-Ros revealed that all but 21 nucleotides immediately upstream of the transmembrane domain of c-Ros were deleted and replaced by the 150 amino acids of the retroviral p19 gag sequence (5) (Fig. 1A). v-Ros codes for a gag-Ros fusion protein with the p19 gag sequence protruding extracellularly (6). Aside from enhanced monolayer and anchorageindependent growth, UR2-infected chicken embryo fibroblasts (CEF) display a distinct transforming characteristic with an extremely elongated morph...

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