Brain-specific serine/threonine-protein kinase 1 is a substrate of protein kinase C epsilon involved in sex-specific ethanol and anxiety phenotypes.

Protein kinase C epsilon (PKCε) regulates behavioural responses to ethanol and plays a role in anxiety-like behaviour, but knowledge is limited on downstream substrates of PKCε that contribute to these behaviours. We recently identified brain-specific serine/threonine-protein kinase 1 (BRSK1) as a substrate of PKCε. Here, we test the hypothesis that BRSK1 mediates responses to ethanol and anxiety-like behaviours that are also PKCε dependent. We used in vitro kinase assays to further validate BRSK1 as a substrate of PKCε and used Brsk1-/- mice to assess the role of BRSK1 in ethanol- and anxiety-related behaviours and in physiological responses to ethanol. We found that BRSK1 is phosphorylated by PKCε at a residue identified in a chemical genetic screen of PKCε substrates in mouse brain. Like Prkce-/- mice, male and female Brsk1-/- mice were more sensitive than wild-type to the acute sedative-hypnotic effect of alcohol. Unlike Prkce-/- mice, Brsk1-/- mice responded like wild-type to ataxic doses of ethanol. Although in Prkce-/- mice ethanol consumption and reward are reduced in both sexes, they were reduced only in female Brsk1-/- mice. Ex vivo slice electrophysiology revealed that ethanol-induced facilitation of GABA release in the central amygdala was absent in male Brsk1-/- mice similar to findings in male Prkce-/- mice. Collectively, these results indicate that BRSK1 is a target of PKCε that mediates some PKCε-dependent responses to ethanol in a sex-specific manner and plays a role distinct from PKCε in anxiety-like behaviour.

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.

Apremilast regulates acute effects of ethanol and other GABAergic drugs via protein kinase A-dependent signaling.

Phosphodiesterase type 4 (PDE4) inhibitors prevent hydrolysis of cyclic adenosine monophosphate and increase protein kinase A (PKA)-mediated phosphorylation. PDE4 inhibitors also regulate responses to ethanol and GABAergic drugs. We investigated mechanisms by which the PDE4 inhibitor, apremilast, regulates acute effects of ethanol and GABAergic drugs in male and female mice. Apremilast prolonged the sedative-hypnotic effects of gaboxadol, zolpidem, and propofol but did not alter etomidate effects, and unexpectedly shortened the sedative-hypnotic effects of diazepam. Apremilast prolonged rotarod ataxia induced by zolpidem, propofol, and loreclezole, shortened recovery from diazepam, but had no effect on ataxia induced by gaboxadol or etomidate. The PKA inhibitor H-89 blocked apremilast's ability to prolong the sedative-hypnotic effects of ethanol, gaboxadol, and propofol and to prolong ethanol- and propofol-induced ataxia. H-89 also blocked apremilast's ability to shorten the sedative-hypnotic and ataxic effects of diazepam. The ß1-specific antagonist, salicylidene salicylhydrazide (SCS), produced faster recovery from ethanol- and diazepam-induced ataxia, but did not alter propofol- or etomidate-induced ataxia. SCS shortened the sedative-hypnotic effects of ethanol and diazepam but not of propofol. In Xenopus oocytes, a phosphomimetic (aspartate) mutation at the PKA phosphorylation site in ß1 subunits decreased the maximal GABA current in receptors containing α1 or α3, but not α2 subunits. In contrast, phosphomimetic mutations at PKA sites in ß3 subunits increased the maximal GABA current in receptors containing α1 or α2, but not α3 subunits. The GABA potency and allosteric modulation by ethanol, propofol, etomidate, zolpidem, flunitrazepam, or diazepam were not altered by these mutations. We propose a model whereby apremilast increases PKA-mediated phosphorylation of ß1-and ß3-containing GABAA receptors and selectively alters acute tolerance to ethanol and GABAergic drugs.