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Abstract
Cells expressing oncogenic Ras proteins transmit a complex set of signals that ultimately result in constitutive activation of signaling molecules, culminating in unregulated cellular function. Although the role of oncogenic Ras in a variety of cellular responses including transformation, cell survival, differentiation, and migration is well documented, the direct Ras/effector interactions that contribute to the different Ras biological end points have not been as clearly defined. Observations by other groups in which Ras-dependent transformation can be blocked by expression of either dominant negative forms of Phosphatidylinositol (PI) 3-kinase or PTEN, a 3-phosphoinositide-specific phosphatase, support an essential role for PI 3-kinase and its lipid products in the transformation process. These observations coupled with the in vitro observations that the catalytic subunits of PI 3-kinase, the p110 isoforms, bind directly to Ras-GTP foster the implication that a direct interaction between an oncogenic Ras protein and PI 3-kinase are causal in the oncogenicity of mutant Ras proteins. Using an activated Ha-Ras protein (Y64G/Y71G/F156L) that fails to interact with PI 3-kinase, we demonstrate that oncogenic Ha-Ras does not require a direct interaction with PI 3-kinase to support anchorage-independent growth of IEC-6 epithelial cells. We do find, however, that IEC-6 cells expressing an oncogenic Ha-Ras protein that no longer binds PI 3-kinase are greatly impaired in their ability to migrate toward fibronectin.
Highlights
The Ras proteins exert their effects on cells by directly associating with downstream effector molecules, thereby initiating the activation of the signaling networks responsible for Rasmediated biological events
1 The abbreviations used are: PI, phosphatidylinositol; EGF, epidermal growth factor; EGFR, EGF receptor; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; MOPS, 4-morpholinepropanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]1-propanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; TBS, Tris-buffered saline; MAPK, mitogen-activated protein kinase; these putative effector molecules, three major candidates, Raf-1, RalGDS, and PI 3-kinase, have been implicated as direct downstream Ras effectors that may contribute to Ras-dependent transformation [3,4,5,6,7,8,9]
Using 10T1/2 fibroblasts transformed by minimal levels of G12V Ha-Ras, we observe that Raf-1 preferentially associates with c-N-Ras and not the ectopically expressed oncogenic HaRas [12]. We have extended these observations by demonstrating that G12V Ha-Ras induces the secretion of EGF receptor (EGFR) ligands that promote the activation of c-N-Ras1⁄7Raf-1 complexes and, up-regulate MAPK activity [18]
Summary
The Ras proteins exert their effects on cells by directly associating with downstream effector molecules, thereby initiating the activation of the signaling networks responsible for Rasmediated biological events. Of Cancer Research, 237 Fulham Rd., London SW3 6JB, UK These putative effector molecules, three major candidates, Raf-1, RalGDS, and PI 3-kinase, have been implicated as direct downstream Ras effectors that may contribute to Ras-dependent transformation [3,4,5,6,7,8,9]. Raf-1 is the only known effector, that has been detected in stable complexes with Ras in vivo, at physiological levels of expression (10 –12) The consequences of such Ras/Raf interactions on cellular function have been extensively studied and found to result in the activation of the Raf/MEK/MAPK cascade. Ectopic expression of PTEN in Ras transformed cells reduces the number of foci in a focus formation assay It is not clear, whether the role of PI 3-kinase in Ras-dependent transformation arises from a direct association with Ras-GTP or through other unidentified indirect mechanisms.
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