Elucidating the Molecular Function of the Autism-Associated Kinase Nuak1 in Neurodevelopment and Disease

Abstract ID 97329 Poster Board 356 Background: Dysfunction in kinase signaling is associated with several neurological disorders, including autism spectrum disorder (ASD). Several de novo mutations in NUAK1, which encodes the serine-threonine AMPK-related Nua kinase 1 (NUAK1), have recently been reported to be associated with ASD. NUAK1 is highly expressed in the developing brain, where it regulates terminal axon branching by promoting presynaptic mitochondrial capture. The function of NUAK1 in neurodevelopment requires its catalytic function, as expressing a kinase-dead variant of NUAK1 or pharmacologically inhibiting NUAK1 kinase activity results in a decrease in axon length and branching that is comparable to that seen in NUAK1 knockout neurons. However, the direct phosphorylation substrates of NUAK1 have not been identified. Despite the disease association and developmental role of NUAK1, we currently have a limited understanding of its downstream signaling pathways, and how they impact neuronal development. Aims: We hypothesize that NUAK1 regulates neuronal development through phosphorylation of key substrates that mediate axonal development, and that perturbation in this pathway contributes to the pathogenesis of ASD. In our study we aim to identify the phosphorylation substrates of NUAK1 in the brain, and elucidate how they modulate axonal development. Further, we tested how autism-associated NUAK1 mutations impact its function in neuronal development. Methods: To identify the direct substrates of NUAK1, I have taken a chemical-genetic approach by mutating the gatekeeper residue of NUAK1 to allow the kinase to utilize bulky, N6-substituted ATPɣS analogs. This analog sensitive kinase will be able to utilize this bulky ATPɣS to specifically thiophosphorylate its substrates, and which can be identified through western blotting and LC/MS. I have characterized how ASD-associated mutations in NUAK1 impact its kinase activity using in vitro biochemical assays. Finally, I determined the impact of disease mutations in NUAK1 on its subcellular localization using high resolution confocal microscopy. Results: Using site-directed mutagenesis, I generated gatekeeper residue mutants M132A and M132G. Screening bulky ATPɣS analogs, we optimized the combination of analog-sensitive (AS) NUAK1 and bulky ATPɣS that can efficiently label direct substrates of NUAK1. We found that M132G was the optimal AS-NUAK1, and utilized Phenyl- ATPɣS much more efficiently compared to WT-NUAK1 to label substrates in mouse brain lysate. Using in vitro kinase assays and confocal live cell imaging, we determined that ASD-associated mutations in NUAK1 differentially affect its kinase activity or subcellular localization. Conclusion: Neuronal development is regulated by spatiotemporally precise kinase signaling pathways. In this work, I utilized a chemical genetic strategy to interrogate the molecular function of an understudied ASD-associated kinase NUAK1 during neuronal development, and define the functional consequences of disease mutations in NUAK1. These studies will define the key downstream substrates of NUAK1 in development, potentially revealing mechanisms through which NUAK1 mutations contribute to autism spectrum disorder. This research was supported by the following grants: T32GM007750 (JS).