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Ethanol-induced translocation of protein kinase A occurs in two phases: control by different molecular mechanisms
Douglas P Dohrman1, Hui-min Chen, Adrienne S Gordon
1Ernest Gallo Clinic and Research Center, Department of Neurology, University of California-San Francisco, 5858 Horton Street, Emeryville, CA 94608, USA.
Background:
Cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) regulates cellular functions. The specificity of PKA-mediated phosphorylation is determined primarily by PKA localization to sub-cellular sites. Chronic exposure to ethanol causes sustained translocation of the PKA catalytic subunit (C) from the Golgi to the nucleus in NG108-15 cells. Here we find that this is preceded by a transient short-term ethanol-induced translocation of PKA C. Different molecular mechanisms appear to underlie early and late phases of ethanol-induced translocation of PKA subunits.
Methods:
The time course and localization of PKA C and regulatory (RII) subunits was assessed by immunocytochemistry in NG108-15 cells in the presence of ethanol, adenosine receptor (A2) blockade, and inhibitors of PKA activity and RNA and protein synthesis.
Results:
Ethanol induces an early phase (<30 min) of C translocation to the cytoplasm and nucleus. This requires cAMP via adenosine A2 receptor activation. C then returns to the Golgi area after 60 min. A second phase of C translocation occurs during continuing exposure to ethanol (>12 hr). Re-accumulation of nuclear C no longer requires A2 or cAMP. RII also translocates to the nucleus during chronic treatment with ethanol. Both C and RII remain in the nucleus as long as ethanol is present. Unlike the early phase of ethanol induced translocation, the second phase of PKA subunit translocation requires protein and RNA synthesis.
Conclusions:
We identify two distinct phases of ethanol-induced PKA translocation which appear to be regulated by different molecular mechanisms. The first requires A2 signaling and cAMP; the later phase requires RNA and protein synthesis. The two phases of ethanol-induced PKA translocation observed in cell lines may contribute to changes in PKA signaling, cAMP-dependent gene expression, and the initiation and maintenance of sustained drinking behavior in experimental animals.