Chemical Genetic Identification of PKC Epsilon Substrates in Mouse Brain

PKC epsilon (PKCε) plays important roles in behavioral responses to alcohol and in anxiety-like behavior in rodents, making it a potential drug target for reducing alcohol consumption and anxiety. Identifying signals downstream of PKCε could reveal additional targets and strategies for interfering with PKCε signaling. We used a chemical genetic screen combined with mass spectrometry to identify direct substrates of PKCε in mouse brain and validated findings for 39 of them using peptide arrays and in vitro kinase assays. Prioritizing substrates with several public databases such as LINCS-L1000, STRING, GeneFriends, and GeneMAINA predicted interactions between these putative substrates and PKCε and identified substrates associated with alcohol-related behaviors, actions of benzodiazepines, and chronic stress. The 39 substrates could be broadly classified in three functional categories: cytoskeletal regulation, morphogenesis, and synaptic function. These results provide a list of brain PKCε substrates, many of which are novel, for future investigation to determine the role of PKCε signaling in alcohol responses, anxiety, responses to stress, and other related behaviors. [Display omitted] •Identification of direct PKCε substrates in mouse brain with chemical genetics.•Utilized AS-PKCε and N6-benzyl-ATPγ for covalent capture of substrates.•Validated direct PKCε substrates using in vitro methods.•Most substrates associated with cytoskeletal, morphological, or synaptic function.•Several substrates involved in alcohol-, stress-, and anxiety-related phenotypes. PKC epsilon (PKCε) plays important roles in behavioral responses to alcohol and in anxiety-like behavior, making it a potential drug target for reducing alcohol consumption and anxiety. Here, a chemical genetic screen and mass spectrometry identified 39 direct substrates of PKCε in mouse brain. They were prioritized using several public databases (LINCS-L1000, STRING, GeneFriends, GeneMANIA) to predict interactions between them and PKCε. Many were novel and broadly fell into three functional categories: cytoskeletal regulation, morphogenesis, and synaptic function.