Chemical genetic screen for substrates of autism risk gene NUAK1 uncovers its role in ARF6 mediated dendritic spine maturation

Dysregulation of signaling by Novel (Nua) Kinase 1 (NUAK1) is associated with autism spectrum disorder, tissue fibrosis, and cancer progression. Direct phosphorylation targets of NUAK1 in the brain are unknown, hindering mechanistic understanding of its role in neurodevelopment. Here, we demonstrate that autism-associated NUAK1 variants differentially impact catalytic activity and subcellular distribution. We engineered ATP-analog sensitive NUAK1 and utilized its specificity towards bulky analogs to identify over 30 hitherto unknown direct phosphorylation targets of NUAK1 in the brain. We demonstrate that Pleckstrin Homology and Sec7-domain containing protein 3 (PSD3) is a bona fide phosphorylation target of NUAK1. PSD3, a guanine nucleotide exchange factor (GEF) for ARF6 GTPase, is phosphorylated by NUAK1 at Ser476. In neurons, expression of phosphodeficient PSD3 (S476A) leads to enhanced dendritic spine maturation in an ARF6-dependent fashion. Mechanistically, NUAK1 suppresses ARF6 activation through PSD3 phosphorylation. Abolishing Ser476 phosphorylation leads to aberrant ARF6 activation and increased generation of PI(4,5)P2. Our study reveals direct neuronal substrates of an autism risk gene NUAK1 and further delineates a mechanism through which NUAK1 phosphorylation of PSD3 controls ARF6 activation and dendritic spine maturation.