The overall goal of the proposed research is to elucidate the mechanisms by which the PI 3-kinase/Akt/GSK-3 signaling pathway regulates cell growth and survival. Both Akt and GSK-3 phosphorylate multiple transcription factors, but the overall program of gene expression controlled by PI 3-kinase/Akt/GSK-3 signaling remains to be fully understood. During the past grant period, global expression profiling combined with computational/experimental analysis of transcription factor binding sites has been used to characterize gene regulation by PI 3-kinase/Akt/GSK-3 signaling, both following growth factor stimulation of quiescent cells and during the continuous proliferation of cells maintained in serum. These studies identified CREB as a key transcription factor targeted by GSK-3 in quiescent cells, and have further implicated members of the AP-1, NFkB and NFAT transcription factor families as GSK-3 targets. A distinct set of genes, enriched in regulators of programmed cell death, is controlled by continuous PI 3-kinase signaling in proliferating cells. Computational analysis of upstream regulatory regions combined with chromatin immunoprecipitation assays indicate that FOXO, NFkB and Myc/Mad/Max transcription factors play major roles in regulation of these genes. In addition, computational analysis of the 3'untranslated regions of their mRNAs suggests that mRNA turnover is also involved in gene regulation by PI 3-kinase signaling. These studies will be continued with the goal of elucidating the program of gene regulation controlled by PI 3-kinase/Akt/GSK-3 signaling, including regulation of mRNA stability. The proposed experiments will proceed according to the following specific aims. 1. Characterization of the transcriptional program regulated by GSK-3 in quiescent cells. The roles of AP-1, NFkB and NFAT transcription factors will be investigated by chromatin immunoprecipitation and RNA interference. Computational analysis will be expanded to identify transcription factor binding sites in predicted enhancers as well as promoter regions, and the role of GSK-3 in regulation of delayed primary response as well as immediate-early genes will be studied. 2. Transcriptional regulation by PI 3-kinase/Akt/GSK-3 signaling in proliferating cells. RNA interference and transient transfection assays will be used to determine the roles of FOXO, NFkB and Myc/Mad/Max transcription factors in PI 3-kinase-dependent gene expression in proliferating cells. The role of GSK-3 in gene regulation during cell proliferation will also be investigated. 3. Regulation of mRNA stability. The role of mRNA degradation in gene regulation by PI 3-kinase will be investigated by determining the effect of PI 3-kinase/Akt/GSK-3 signaling on the stability of mRNAs that are down-regulated following inhibition of PI 3-kinase.
Mutations affecting the PI 3-kinase/Akt/GSK-3 signaling pathway are among the most common abnormalities leading to the development of human cancer, and components of this pathway are active targets for drug development. The present proposal seeks to further understand the mechanism by which this pathway regulates cell survival. Discoveries of novel targets of PI 3-kinase signaling may provide new targets for cancer treatment.
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