Dopamine and Ethanol Cause Translocation of PKC Associated with RACK: Cross-Talk between cAMP-Dependent Protein Kinase A and Protein Kinase C Signaling Pathways

We found previously that neural responses to ethanol and the dopamine D2 receptor (D2) agonist 2,10,11-trihydroxy-Npropylnorapomorphine hydrobromide (NPA) involve both protein kinase C ( PKC) and cAMP-dependent protein kinase A (PKA). However, little is known about the mechanism underlying ethanoland D2-mediated activation of PKC and the relationship to PKA activation. In the present study, we used a new PKC antibody, 14E6, that selectively recognized active PKC when not bound to its anchoring protein RACK (receptor for activated C-kinase), and PKC isozyme-selective inhibitors and activators to measure PKC translocation and catalytic activity. We show here that ethanol and NPA activated PKC and induced translocation of both PKC and its anchoring protein, RACK to a new cytosolic site. The selective PKC agonist, pseudoRACK, activated PKC but did not cause translocation of the PKC/ RACK complex to the cytosol. These data suggest a step-wise activation and translocation of PKC after NPA or ethanol treatment, where PKC first translocates and binds to its RACK and subsequently the PKC/ RACK complex translocates to a new subcellular site. Direct activation of PKA by adenosine-3 ,5 -cyclic monophosphorothioate, Sp-isomer (Sp-cAMPS), prostaglandin E1, or the adenosine A2A receptor is sufficient to cause PKC translocation to the cytosolic compartment in a process that is dependent on PLC activation and requires PKA activity. These data demonstrate a novel crosstalk mechanism between PKC and PKA signaling systems. PKA and PKC signaling have been implicated in alcohol rewarding properties in the mesolimbic dopamine system. Crosstalk between PKA and PKC may underlie some of the behaviors associated with alcoholism. Intracellular signal transduction cascades linked to protein kinase C (PKC) have been implicated in drug abuse (Hodge et al., 1999; Olive et al., 2000; Choi et al., 2002; Olive and Messing, 2004; Newton and Messing, 2006). In particular, the isozyme PKC mediates an intracellular response to ethanol (Gordon et al., 1997, 2001) and is associated with excessive drinking. PKC knockout mice exhibit decreased alcohol consumption in two bottle-choice and operant selfadministration paradigms (Hodge et al., 1999; Olive et al., 2000). In addition, conditional expression of PKC in the basal forebrain, amygdala, and cerebellum of PKC knockout mice restored the wild-type response to alcohol (Choi et al.,