Chemoenzymatic Protein Semisynthesis Approaches Toward Cell Signaling Enzymes

ABSTRACT Protein phosphorylation plays a key role in numerous cellular processes through a dynamic balance between protein kinases and phosphatases. In many disease conditions, this balance is inclined toward kinase hyperactivation and/or phosphatase inactivation, which are highly related to changes of their posttranslational modifications (PTMs). Being able to introduce multiple PTMs on such specific signaling enzymes in a chemically well-defined manner is both impactful and transformative to understand cell signaling pathways and the function of PTMs. In previous study, by employing protein semisynthesis methods to generate homogenous protein kinase Akt forms as substrates or stoichiometrically phosphorylated at Ser473 as calibration standards, we provided a detailed portrait of how mTOR Complex 2 (mTORC2) but not mTORC1 can selectively recognize and phosphorylate Akt Ser473 to activate this key signaling kinase. In this proposal, we continue to develop and utilize protein semisynthesis methods to elucidate the regulation of two key signaling enzymes including protein kinase S6K1 and the heterotrimeric phosphatase PP2A by PTMs and other allosteric mechanisms. S6K1 is a crucial downstream effector of mTORC1, and is critically regulated by the phosphorylation of a cluster of Ser/Thr residues (Ser411, Ser418, Thr421 and Ser424) in the C-terminal autoinhibitory domain (CTD). Yet how the CTD phosphorylation modulates S6K1 structure and function has been poorly defined. The PP2A phosphatase heterotrimer is responsible to the vast majority Ser/Thr phosphatase activity in eukaryotic cells, and its assembly has been linked to changes in the C-terminal PTMs of the C subunit including Thr304 and Tyr307 phosphorylations and Leu309 methylation. However, the function of these PTMs has yet to be fully characterized, and remain a great of interest in the field. It is also very little known how PP2A recruits its substrates, limiting our understanding of PP2A-regulated signaling. We will produce these two enzymes containing site-specific PTMs and their non-hydrolyzable analogs, and will integrate kinetic assays, structural analysis, binding measurements, and cell-based studies to clarify the structural and catalytic features. Moreover, we have developed a novel proximity crosslinking method using nanobodies as proximity-directing agents for analyzing PP2A interactome in different cellular conditions in response to various stimuli. Successful completion of this project will not only provide a detailed molecular understanding of how these two signaling enzymes regulated by specific PTMs, but inspire novel therapeutic strategies combat the diseases associated with their dysregulation.