ABSTRACT Two recent studies found that sleep and wake have opposing effects on the brain phosphoproteome. Prolonged wakefulness results in hyperphosphorylation of many proteins in the brain, whereas sleep promotes dephosphorylation of the brain proteome. A gain-of-function mutation in salt-inducible kinase 3 (sik3) results in a brain state similar to that of prolonged wakefulness and is associated with hyperphosphorylation of a subset of the proteins that are hyperphosphorylated in wild-type animals during prolonged wakefulness. These observations suggest that protein phosphorylation may play a role in the accumulation of sleep pressure during wakefulness that dissipates during sleep, and that this phosphorylation is mediated in part by sik3, although it is clear that additional protein kinases are involved. However, the identity of these protein kinases remains unknown. Based on a large (>500,000 subjects) human genome-wide association study (GWAS) that identified many loci in the human genome that are associated with human sleep variation and sleep disorders, we are performing a targeted genetic screen using zebrafish to identify the relevant gene in each locus. Four of the genes targeted in this screen are listed as “Understudied Proteins” as part of the NIH “Illuminating the Druggable Genome” (IDG) project. We have found that loss-of-function mutations in the zebrafish ortholog of one of these human genes results in decreased sleep. We will test the hypotheses that (1) the three other IDG genes targeted in the screen also regulate sleep, and that (2) these genes play roles in changes in the brain phosphoproteome that are associated with sleep or wakefulness. We will test hypothesis (1) by assaying each mutant for sleep phenotypes using locomotor activity and arousal threshold assays. For genes in which hypothesis (1) is correct, we will test hypothesis (2) by characterizing the zebrafish brain phosphoproteome in response to gain- and loss- of-function genetic perturbations of each gene. These experiments will provide the first demonstrated functions for the IDG genes being tested, characterize effects of these protein kinases on the brain phosphoproteome, and explore an exciting new mechanism that may underlie sleep homeostasis. Based on these experiments and human GWAS data, this project will also reveal the basis for some of the observed variation in human sleep and some human sleep disorders and provide new druggable targets to treat these disorders.