Abstract
Eukaryotic protein kinase signaling is a tightly regulated network of integrated phosphorylation events that control cell homeostasis. The microtubule-associated serine/threonine (MAST) kinases form an ancient, highly conserved subfamily of AGC kinases implicated in human diseases such as cancer, diabetes, and neurodevelopmental disorders. Despite their clinical significance, the origins, structure, regulation, and substrate specificity of MAST kinases remain incompletely understood. In this dissertation, I integrate structural, evolutionary, and biochemical approaches to characterize the diversification and regulation of MAST kinases. Bioinformatic analyses reveal that higher-order organisms harbor multiple MAST paralogs and a single MASTL kinase, with conservation of the AGC kinase domain, the domain of unknown function 1908 (DUF1908), and a PDZ binding domain. Although the DUF domain is retained across kingdoms, the PDZ domain is variably lost. MAST proteins exhibit tissue-specific expression and are likely stabilized by protein interactions, as supported by interactome and in silico analyses. Functional assays using Mast2 demonstrate that the DUF1908 domain is essential for kinase activity, while the PDZ domain is dispensable. Regulation diverges from the traditional AGC kinase T-loop phospho-activation, involving a unique T-loop insertion and mTOR-dependent phosphosites critical for activity instead. Proteomic analyses identify potential Mast2 substrates, implicating a role in post-translational modification networks, such as dephosphorylation control via endosulphine-α (ENSA). Collectively, these findings lay the foundation for understanding MAST regulatory mechanisms and identify their biological roles, thus providing a basis for targeted therapeutic strategies and future research into MAST kinase biology.
