Ethanol Alters the Subcellular Localization of δ- and ε Protein Kinase C in NG108–15 Cells

Abstract

SUMMARYProtein kinase C (PKC) has been shown to regulate the ethanol sensitivity of membrane-bound receptors and transporters, but little is known about the molecular mechanisms underlying this regulation. PKC is a family of isozymes that translocate to new intracellular sites on activation. Here we present immunochemical data showing that ethanol causes translocation of ␦-and ⑀-PKC to new intracellular sites. Ethanol causes translocation of ␦-PKC from the Golgi to the perinucleus; this translocation is similar to that induced by activation of PKC with phorbol esters.In contrast, ⑀-PKC translocation caused by ethanol is different from that induced by phorbol esters; ethanol causes translocation of ⑀-PKC from the perinucleus to the cytoplasm, whereas phorbol ester activation causes translocation of ⑀-PKC to the nucleus. Because the substrate specificity of these kinases is determined by their site of localization, ethanol-induced translocation of ␦-and ⑀-PKC to new intracellular sites may explain some of the pleiotropic effects of ethanol on cellular functions.PKC, a family of isozymes that mediates multiple cellular functions, has been shown to regulate the effects of ethanol on receptors (1-6) and membrane-bound transporters (7), but the mechanism underlying this regulation is unknown. Ethanol alters the amount, activity, and subcellular distribution of PKC. Increased amounts of ␣-, ␦-, and ⑀-PKC in NG108 -15 cells (8) and of ␦-and ⑀-PKC in PC12 cells (9) are found after chronic ethanol exposure, and there is increased activity of PKC in NG108 -15 and PC12 cells (9). PKC activity is also increased in human platelets (10), lymphocytes (11), and epidermal keratinocytes (12) after acute ethanol exposure. Moreover, ethanol causes translocation of PKC activity from cytosolic to membrane fractions in astroglial cells (13), human lymphocytes (11), and epidermal keratinocytes (12).On activation, each PKC isozyme translocates from a specific intracellular site to another (14). Recent evidence suggests that the specificity of substrate phosphorylation of each isozyme is determined by its localization (14). Ethanol-induced activation and translocation of specific PKC isozymes to new intracellular sites could therefore account for many of the pleiotropic effects of ethanol on cell functions. To test this hypothesis, we carried out studies on the localization of ␦-and ⑀-PKC in NG108 -15 neuroblastoma ϫ glioma hybrid cells. We report here that ethanol causes translocation of ␦-and ⑀-PKC to new intracellular sites in these cells. Materials and MethodsCell culture. NG108-15 neuroblastoma ϫ glioma hybrid cells were seeded in single-chamber slides in defined media consisting of Dulbecco's modified Eagle's medium/Ham's F-12 medium (3:1); 0.1 mM hypoxanthine; 1.0 M aminopterin; 12 mM thymidine; 25 mM HEPES, pH 7.4; trace elements (0.5 nM MnCl 2 , 0.5 nM [NH 4 ] 6 Mo 7 O 24 , 0.25 nM SnCl 4 , 25 nM Na 3 VO 4 , 5 nM CdSO 4 , 0.25 nM NiSO 4 , 15 nM H 2 SeO 3 , 25 nM Na 2 SiO 3 ); bovine insulin (5 g/ml); human transferrin (50 g/ml);...

Activation of ɛ protein kinase C correlates with a cardioprotective effect of regular ethanol consumption

“…These data suggest an isozyme-specific role for PKC in producing ethanol's longterm cardioprotective effect against ischemia-reperfusion injury. This observation is consistent with recent studies in other cell types demonstrating ethanol-induced activation of PKC (40)(41)(42)(43)(44).…”

“…Such sustained translocation was proposed to occur by Downey and coworkers (59). Ethanol-induced activation of PKC has been observed in other cell types (40)(41)(42)(43)(44). For example, in neural crest-derived PC12 cells, ethanol-induced activation of PKC enhances nerve growth factor-induced signaling and neurite outgrowth (40).…”

Ethanol Regulation of D1 Dopamine Receptor Signaling is Mediated by Protein Kinase C in an Isozyme-Specific Manner

“…EtOH treatment has also been shown to promote the subcellular translocation of specific PKC isozymes. Chronic EtOH exposure (48 h) promotes translocation of PKCd and PKCe between specific intracellular compartments (Gordon et al, 1997). The subcellular redistribution of PKCe but not PKCd is also evident in cellular fractions prepared from cardiac myocytes (Miyamae et al, 1998).…”

State-specific Monoclonal Antibodies Identify an Intermediate State in Epsilon Protein Kinase C Activation

“…After 24 h, the culture medium was changed to the above medium containing 80 M ascorbic acid, and cells were cultured an additional 72 h. Myocytes were then incubated in a defined medium (M-199 with penicillin and streptomycin, 2 g/ml vitamin B 12 , and 10 g/ml each of insulin and transferrin) for 24 h, after which experiments were initiated. NG108-15 Cell Culture-NG108 -15 cells were cultured as described (28), and maintained in a serum-free defined medium for 6 days prior to immunofluorescence or immunoprecipitation experiments. Experiments with the dopamine receptor agonist NPA were carried out with NG108 cells stably transfected with the dopamine D2 receptor, NG108-15/D2 (29).…”