Chemical Approaches to Cell Signaling Enzymes

Abstract This is a competing renewal application of our longstanding R01 Grant CA74305 that concerns the development and application of chemical approaches to enhance the understanding of cell signaling pathways and the attachment, removal, and function of protein post-translational modifications (PTMs). In the next cycle, we plan to investigate how ubiquitination regulates two key cancer-related signaling enzymes, PTEN and Akt, how several cancer-connected E3 ubiquitin (Ub) ligases target and catalyze Ub transfer to their protein substrates, and how mTORC2 phosphorylates its major substrate Akt. PTEN is one of the most commonly mutated tumor suppressor genes and catalyzes the conversion of phospholipid phosphatidylinositol-3,4,5-triphosphate (PIP3) to PIP2 and in this way antagonizes the action of the PI3-kinase/Akt oncogenic signaling pathway. Akt is a protein Ser/Thr kinase that is activated by PIP3 and drives tumor formation. Akt is an intensively studied cancer therapeutic target. E3 Ub ligases are a large family (>600 members) of enzymes that target Akt, PTEN, and thousands of cellular substrates for Lys ubiquitination. Our research program will continue to develop and employ protein semisynthetic methods including ubiquitin hydrazide installation to generate mimics of mono-ubiquitinated proteins and covalent E3-Ub-substrate ternary complexes. Our three specific aims are: 1. Elucidate the basis of PTEN and Akt regulation by Lys ubiquitination; 2. Delineate the catalytic mechanisms and protein substrate recognition of key E3 ubiquitin ligases; 3. Determine the molecular basis of mTORC2 mediated- phosphorylation of Akt. With site-specifically modified signaling proteins in hand, we will integrate kinetic assays, structural analysis, binding measurements, and cell-based studies to clarify key regulatory and signaling features. Upon completion of this research effort, we will broaden the knowledge of how Lys ubiquitination targets critical cancer- related proteins and influences their functions. We will also deepen our understanding of how the multisubunit mTORC2 complex is able to activate Akt. Moreover, these studies should pave the way for new therapeutic strategies to combat pathway dysregulation in cancer. This research program will also enable the training of the next generation of biochemical investigators with an interest in cancer.