Rare Neurological Disease-Associated Pseudokinases - PROJECT SUMMARY/ABSTRACT Protein kinases regulate a wide range of cellular processes by transferring phosphate from ATP to protein substrates (phosphorylation), thereby modulating their function, stability, and interactions. The human kinome consists of over 500 protein kinases, including a subset known as pseudokinases—proteins that adopt a kinase- like fold but lack key catalytic residues. Traditionally, pseudokinases have been considered inactive, functioning primarily as scaffolds or allosteric regulators. However, our laboratory has discovered several pseudokinases that are catalytically active, catalyzing reactions beyond phosphorylation, such as AMPylation, polyglutamylation, and mRNA capping via RNAylation. These findings highlight the catalytic versatility of the kinase fold and suggest that pseudokinases may possess diverse, uncharacterized biochemical activities. This proposal aims to explore the catalytic potential of three rare disease-associated pseudokinases—SCYL1, SCYL2, and TBCK—which have been implicated in neurodevelopmental and neurodegenerative disorders, including autosomal recessive spinocerebellar ataxia-21 (SCAR21/CALFAN), neurogenic arthrogryposis multiplex congenita 4, and TBCK syndrome. Understanding the biochemical and cellular functions of these pseudokinases will provide critical insights into disease mechanisms and uncover new enzymatic activities within this protein class. Thus, we propose to determine whether SCYL1, SCYL2, and TBCK exhibit catalytic activity using a multidisciplinary approach combining biochemistry and mass spectrometry, an approach we have successfully used to uncover catalytic activity in other pseudokinases. We will also assess whether the active site residues of SCYL1, SCYL2, and TBCK are essential for their cellular functions. Using inducible knockdown cell lines, we will evaluate the ability of wild-type and mutant constructs to rescue defects in Golgi morphology, trafficking, and cell proliferation. These studies will clarify the functional importance of the pseudokinase domain and its potential enzymatic activity in cellular processes and disease pathology. The outcomes of this proposal will establish whether SCYL1, SCYL2, and TBCK possess enzymatic activity and determine the functional significance of their active sites in cellular processes. Additionally, the generated tools, including purified proteins and engineered cell lines, will serve as valuable resources for future studies. Ultimately, these results will lay the foundation for an R01 proposal aimed at identifying the physiological substrates of these pseudokinases and elucidating their roles in rare neurological diseases.
