Identification of Novel Substrates of Protein Kinase C epsilon Using a Chemical Genetic Strategy

Alcohol use disorder (AUD) is a major public health problem in the United States, and only three drugs are approved to treat it: Disulfuram, naltrexone, and acamprosate. However, these drugs have small effect sizes and suffer from patient compliance issues. Hence, there is a need to develop new medications to treat AUD. We have previously identified Protein Kinase C epsilon (PKCε) as a target for developing AUD medications. Working under the hypothesis that PKCε signal transduction pathways contain additional AUD drug targets, we sought to identify direct substrates of PKCε in the brain that regulate alcohol consumption. We used a chemical-genetic approach with an ATP analog-specific version of PKCε (AS-PKCε) bearing a mutation in the enzyme’s ATP binding pocket. This mutation allows AS-PKCε to efficiently utilize bulky ATP analogs to deliver a chemical tag to unknown substrates. We used ATP analogs containing a thiophosphate at the γ-phosphate position to label substrates with a tag that is resistant to phosphatases. Using this method, we identified 65 candidate PKCε substrates in the brain. As PKCε has been implicated in neurotransmitter release and neurite outgrowth, we were particularly intrigued by 5 substrates involved in both processes: Synapsin 1, brain serine/threonine kinase 1(Brsk1/ SAD-B), neuromodulin (GAP43), microtubule associated protein tau (MAPT), and alpha adducin. In validation studies we confirmed that Brsk1, GAP43, and MAPT could be phosphorylated by PKCε in vitro. Furthermore, we found that thiophosphorylation in intact cells could be achieved and that there was a strong trend towards increased levels of thiophosphorylation in synapsin 1 when PKCε was activated.