This project is focused on the molecular events promoted by interleukin 2 (IL-2) responsible for inducing T cell proliferation. T cell cycle progression is dependent upon an activation threshold, which is hypothesized to be determined by an IL-2-promoted sequential expression of specific genes. In the past grant period, methods were developed to isolate and clone immediate-early genes induced by IL-2, termed cytokine response (CR) genes. To date, little has been uncovered regarding just how signaling via the IL-2 activates immediate-early gene expression. Therefore, for the next grant period we will work retrograde from the novel CR genes identified, to determine the cis-acting response elements and trans-acting transcription factors responsible for CR gene expression (Specific Aim I). Simultaneously, we will identify the signaling molecules and pathways activated via the IL-2R, which culminate in the activation of the transcription factors identified (Specific Aim 2). Methods have been developed for the isolation, sequencing and analysis of the promoter regions of the CR genes. As recommended by the Study Section, we will focus initially on only 2 of the CR genes (CRI and CR5), which appear to be regulated by distinct pathways and factors. To link the IL-2R with the CR gene response elements, luciferase-based promoter-reporter assays are in use together with pharmacologic signal transduction inhibitors and dominant-negative mutants of signaling molecules and transcription factors. Preliminary data indicate that novel, as well as known signaling molecules and transcription factors may be responsible for IL-2-induced immediate-early gene expression. It is anticipated that this approach for the first time will permit a detailed analysis and identification of the biochemical and molecular events initiated by the IL-2R that culminate in moving the cell through the early phases of the cell cycle. Moreover, it is expected that these molecular events have been conserved, and are common to most cytokine mitogenic receptors. Therefore, our results are likely to have direct relevance for disorders of the immune system, developmental abnormalities, and malignancy.
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