Progression of cells through the cell division cycle is controlled by interconnecting signal transduction pathways that result in transient activation and repression of growth-responsive any step in the pathway depends on the completion of a prior step, thus providing multiple points where the cascade can be blocked. A detailed understanding of the regulatory pathways of neoplastic cells. The first step towards controlling cell growth is to identify pathways, which when deregulated, lead to neoplasia. One such pathway contains the c-raf gene. c-RAF is a serine/threonine kinase that can be activated by over a dozen different mitogens, suggesting that c-RAF is of central importance in cell growth. In fact, studies of c-RAF function have shown a correlation between c-RAF activation and transformation and between c-RAF inhibition and constraints on cell growth. Because c-RAF function will have pleiotropic effects on cell growth and is unlikely to lead to useful therapeutic intervention. What is required is to understand the individual pathways leading from c-RAF so that specific neoplastic properties of transformed cells can reverted. Therefore, a logical second step is to identify cell cycle-regulated genes that are indirectly controlled by c-RAF activation. We have shown that constitutive expression of an activated v-RAF in quiescent cells is sufficient to elicit an increase in transcription from both early (egr2) and late (cad ad rep-3b) serum-response genes. However, several important questions are left unresolved by these experiments. First, it is not known if c-RAF is involved in the normal activation of theses promoters after growth stimulation. Second, a complete c-RAF-activated pathway from early to late response promoters has not yet been traced. To answer these questions, we propose to determine if c-RAF is critical for the cell cycle-regulated transcriptional activation of v-RAF responsive promoters (Specific Aim 1), to determine the role of the early response EGR2 transcription factor in cell proliferation and in the activation of the late response cad or rep-3b (Specific Aim 2), and to characterize the activation of the late response cad or rep-3b promoters (Specific Aim 3). Our preliminary experiments have identified several new components (EGR2,REP-3b, and CAD) of an important signal transduction pathway. Our proposed experiments will attempt to connect the individual members to other components of the c-RAF pathways. Using the knowledge gained in these experiments, we can direct our future experiments toward specific inactivation of different components of the pathway. These future studies should provide insight about novel ways to revert specific neoplastic properties of transformed cells.
