The importance of the proper regulation of Ras is underscored by the observation that approximately 30% of all human cancers involve Ras mis-regulation. Therefore, characterization of the mechanism by which Ras activates its effectors may present potential targets for cancer prevention and therapeutic treatments. Furthermore, understanding how Ras signaling influences a cell to choose between alternate fates, is likely to have important implications to non-cancerous versus cancerous cellular decisions. The Kinase Suppressor of Ras (KSR) is a recently identified signaling molecule whose ability to modulate Ras signal transduction is dependent upon its levels of expression. Investigation into the mechanisms underlying KSR-mediated regulation of Ras activity will utilize the murine model of T cell development since this system has been invaluable in the analysis of Ras regulation. In addition, it is amenable to genetic manipulation and the abnormal activation of Ras signaling in this system leads to T cell lymphomas. Furthermore, as it is a mammalian model, results obtained in this system are highly likely to be applicable to human diseases. The over-expression of KSR in developing T cells inhibits development and Ras signaling in a manner similar to that of a dominant-negative form of Ras. To further investigate KSR's role in Ras-mediated T cell development, three aspects of KSR function will be addressed. The first goal is to identify the elements necessary for the observed KSR gain-of-function effect by mutational analyses as well as determining the consequences of KSR loss-of-function during T cell development using the RNA interference technique. The second objective is to analyze how different levels of KSR expression affect Ras-mediated T cell survival. This will be performed by using a lentiviral-based approach to modulate the number of KSR integrants in transgenic animals, thereby producing an in vivo dose response curve to examine expression levels.
The third aim entails using subcellular localization techniques and confocal microscopy analysis to determine if KSR's spatial localization, like that of Ras and its effectors, is important for T cell activation. These studies will reveal the mechanism by which KSR modulates Ras activity in T cells and are likely to produce insight into two processes into which appropriate Ras regulation is essential: cancer and cell fate choices. Future objectives are two-fold: first, the consequences of KSR-mediated Ras regulation during cancer will be investigated, focusing on the process of cancer in T cell lymphomas; and secondly, the participation of KSR in other cell fate choices in developing thymocytes will be analyzed.
