Summary The PI3K/Akt signaling is one of most important oncogenic events in human cancers. It regulates many aspects of biological functions including cell proliferation, survival, and metabolism important for cancer initiation and progression. While extensive efforts have been made in the last three decades to understand the downstream effectors responsible for the biological and oncogenic processes regulated by PI3K/Akt signaling, the upstream signals mediating PI3K/Akt signaling activation upon diverse growth factor stimulation is not well understood. Understanding and defining the upstream mechanisms responsible for PI3K/Akt signaling activation will not only provide new insight into how PI3K/Akt signaling activation is orchestrated, but also offer novel paradigms and therapeutic targets for cancer intervention. Although it has been well established that PIP3 is critical for the membrane recruitment and subsequent activation of Akt, our recent studies provide the evidence that Akt undergoes methylation and subsequent non-proteolytic K63-linked ubiquitination, which are crucial for Akt membrane recruitment and subsequent phosphorylation and activation upon stimulation with diverse growth factors, opening up a new frontier for Akt signaling regulation. Of note, we identified SETDB1 as a methyltransferase for Akt K64 methylation and TRAF6 ligase as an upstream E3 ligase triggering K63-linked ubiquitination and activation of Akt, and these events are required for cancer progression. However, the outstanding questions remained to be addressed are how SETDB1 and TRAF6 are activated or recruited to the Akt complex upon growth factor treatment to trigger Akt methylation and subsequent Akt ubiquitination and activation, thus promoting oncogenic processes. The goal of this study is to dissect the upstream regulatory mechanisms by which growth factors activate and recruit SETDB1 and TRAF6 ligase to Akt complex to elicit Akt methylation and subsequent Akt ubiquitination, define the mechanism by which Akt ubiquitination facilitates Akt membrane localization and activation, and finally explore the role of these regulatory modes in cancer development and develop small molecule inhibitors targeting these regulatory mechanisms. Our preliminary results revealed that SETDB1 and TRAF6 undergo novel posttranslational modifications, which are crucial for methylation, ubiquitination and activation of Akt by growth factors and oncogenic activity. We hypothesized that SETDB1 and TRAF6 undergo the novel posttranslational modification upon growth factor treatment, which recruits SETDB1 to the Akt complex and activates TRAF6 E3 ligase to facilitate Akt methylation and subsequent Akt ubiquitination and activation, thus leading to tumorigenesis. Our innovative hypothesis has been formulated based on our preliminary results and prior research. We proposed three specific aims to validate this provocative and paradigm-shifting concept using cutting-edge technologies includin...