Mitogens play an integral role in resistance, proliferation, and survival of human tumors. Unraveling the intricate signal transduction pathways that regulate tumorigenesis is a critical step in identifying new therapeutic targets for disruption to impede the progression of cancer. It is not clear as to what extent tumor derived mitogens such as IGF-1 and EGF-1 signal to activate the oncogenic activity of the murine double minute protein (Mdm2). Mdm2 plays a role in tumorigenesis in part through the disruption of the tumor suppressor protein p53. It has been assumed that Mdm2 nuclear localization was a passive process requiring only the nuclear localization sequence (NLS). We show that IGF-1 activated PI3-K/Akt signaling induces the phosphorylation and nuclear translocation of Mdm2, which disrupts p53 activity and facilitates p53 degradation. The p53-Mdm2 complex is exported from the nucleus for both proteins to be degraded. Thus, it seems likely that there is a second signal transduction pathway responsible for mediating Mdm2 nuclear export. We hypothesize that Mdm2 nuclear export is regulated by post-translational modifications mediated by the mitogen activated protein kinase pathway, and culminates in the inactivation of p53.
The specific aims i n this proposal are designed to ascertain how signal transduction pathways directly regulate Mdm2 and its activity to block p53 function. [Aim 1] To elucidate the signal transduction pathway that mediates nuclear export of Mdm2 by post-translational modifications. We will use selective pharmacological inhibitors to signaling kinases, and dominant negative kinases to block nuclear export of Mdm2. We will determine post-translational modifications to Mdm2 by mass spectrometry in response to growth factor simulation and pharmacological blockade of growth factor stimulated signaling pathways. The identification of phosphorylation sites required for export will be alter to a non-phosphorylatable amino acids to confirm the requirement of these sites to promote Mdm2 nuclear export. [Aim 2] This specific aim is to characterize the effect of Mdm2 nuclear export on p53 activity and stability in response to growth factors and DNA damage. Preventing Mdm2 nuclear export should have a dramatic effect on p53 activity. We will examine p53 transcriptional activity when Mdm2 is unable to exit the nucleus. We will also determine if p53 ubiquitation mediated by Mdm2 occurs in the nucleus or cytoplasm and in what compartment the p53-Mdm2 complex is degraded. Biologically, we will test if Mdm2 rendered in the nucleus can attenuate p53 dependent apoptosis in response to DNA damage. Considering that nuclear Mdm2 is prevalent in human cancer, upon completion of this proposal we will understand the contribution of signal transduction pathways in promoting Mdm2 nuclear export and potentially how to regulate export of nuclear Mdm2 to increase p53 dependent apoptosis. ? ? ?
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