Distinct effects of fatty acids on translocation of gamma- and epsilon-subspecies of protein kinase C

Abstract

Effects of fatty acids on translocation of the gamma- and epsilon-subspecies of protein kinase C (PKC) in living cells were investigated using their proteins fused with green fluorescent protein (GFP). gamma-PKC-GFP and epsilon-PKC-GFP predominated in the cytoplasm, but only a small amount of gamma-PKC-GFP was found in the nucleus. Except at a high concentration of linoleic acid, all the fatty acids examined induced the translocation of gamma-PKC-GFP from the cytoplasm to the plasma membrane within 30 s with a return to the cytoplasm in 3 min, but they had no effect on gamma-PKC-GFP in the nucleus. Arachidonic and linoleic acids induced slow translocation of epsilon-PKC-GFP from the cytoplasm to the perinuclear region, whereas the other fatty acids (except for palmitic acid) induced rapid translocation to the plasma membrane. The target site of the slower translocation of epsilon-PKC-GFP by arachidonic acid was identified as the Golgi network. The critical concentration of fatty acid that induced translocation varied among the 11 fatty acids tested. In general, a higher concentration was required to induce the translocation of epsilon-PKC-GFP than that of gamma-PKC-GFP, the exceptions being tridecanoic acid, linoleic acid, and arachidonic acid. Furthermore, arachidonic acid and the diacylglycerol analogue (DiC8) had synergistic effects on the translocation of gamma-PKC-GFP. Simultaneous application of arachidonic acid (25 MicroM) and DiC8 (10 microM) elicited a slow, irreversible translocation of gamma-PKC- GFP from the cytoplasm to the plasma membrane after rapid, reversible translocation, but a single application of arachidonic acid or DiC8 at the same concentration induced no translocation. These findings confirm the involvement of fatty acids in the translocation of gamma- and epsilon-PKC, and they also indicate that each subspecies has a specific targeting mechanism that depends on the extracellular signals and that a combination of intracellular activators alters the target site of PKCs.

Figure 1

Comparison of properties of native ε-PKC and its fusion protein with those of GFP. (A) Kinase activities of immunoprecipitated ε-PKC and ε-PKC– GFP were measured in the presence (activated; hatched bar) or absence of (basal; open bar) PS and DO. (B) Fluorescent microscopic photos of CHO-K1 cells expressing ε-PKC or ε-PKC– GFP. Transfected CHO-K1 cells were fixed before or after treatment with 1 μM TPA. Immunoreactivity of ε-PKC (a and d) or ε-PKC– GFP (b and e) was made visible with anti–ε-PKC antibody (red). The fluorescence of ε-PKC–GFP (c and f) was simultaneously observed with a confocal laser scanning fluorescent microscope (green), as described in Materials and Methods. ε-PKC immunoreactivity was translocated from the cytoplasm to the plasma membrane by TPA treatment, both in ε-PKC (a and d) and ε-PKC– GFP (b and e) expressing CHO-K1 cells. The localizations of ε-PKC immunoreactivity and GFP fluorescence were indistinguishable, as seen in b and c. Moreover, localization was indistinguishable after TPA treatment (e and f).

Figure 1

Comparison of properties of native ε-PKC and its fusion protein with those of GFP. (A) Kinase activities of immunoprecipitated ε-PKC and ε-PKC– GFP were measured in the presence (activated; hatched bar) or absence of (basal; open bar) PS and DO. (B) Fluorescent microscopic photos of CHO-K1 cells expressing ε-PKC or ε-PKC– GFP. Transfected CHO-K1 cells were fixed before or after treatment with 1 μM TPA. Immunoreactivity of ε-PKC (a and d) or ε-PKC– GFP (b and e) was made visible with anti–ε-PKC antibody (red). The fluorescence of ε-PKC–GFP (c and f) was simultaneously observed with a confocal laser scanning fluorescent microscope (green), as described in Materials and Methods. ε-PKC immunoreactivity was translocated from the cytoplasm to the plasma membrane by TPA treatment, both in ε-PKC (a and d) and ε-PKC– GFP (b and e) expressing CHO-K1 cells. The localizations of ε-PKC immunoreactivity and GFP fluorescence were indistinguishable, as seen in b and c. Moreover, localization was indistinguishable after TPA treatment (e and f).

Figure 2

Translocation of γ-PKC–GFP induced by saturated fatty acids in CHO-K1 cells. (Top row) Changes induced by 200 μM tridecanoic acid in the fluorescence of γ-PKC–GFP expressed in CHO-K1 cells. γ-PKC–GFP fusion protein is present throughout the cytoplasm within the transfected CHO-K1 cells, and faint fluorescence is seen in the nucleus. The addition of 200 μM tridecanoic acid induced rapid translocation of γ-PKC–GFP fluorescence from the cytoplasm to the plasma membrane, which took place within 20 s of stimulation. Thereafter, γ-PKC– GFP quickly was retranslocated from the membrane to cytoplasm within 2 min, reaching a state similar to that before stimulation. (Second row) Changes in the profiles of GFP intensity on the same axis across a cell treated with 200 μM tridecanoic acid. The axis is between the arrows in the upper left photo. (Third row) Changes produced by 200 μM pentadecanoic acid in the fluorescence of γ-PKC–GFP expressed in CHO-K1 cells. The translocation of γ-PKC–GFP induced by pentadecanoic acid is similar to that of tridecanoic acid. (Bottom row) Changes induced by 200 μM palmitic acid in the fluorescence of γ-PKC–GFP expressed in CHO-K1 cells. The translocation is similar to the translocations of tridecanoic and pentadecanoic acids. Bars, 10 μm.

Figure 3

Translocation of γ-PKC–GFP induced by unsaturated fatty acids. (Top row) Changes induced by 200 μM oleic acid in the fluorescence of γ-PKC–GFP. Oleic acid also induced very rapid, transient translocation of γ-PKC–GFP from the cytoplasm to the plasma membrane that was similar to that induced by saturated fatty acid. (Second and third rows) Changes induced by 100 and 200 μM linoleic acid in the fluorescence of γ-PKC–GFP. Linoleic acid at a low concentration (100 μM) induced translocation of γ-PKC–GFP from the cytoplasm to the plasma membrane as did oleic acid. At 200 μM, however, it caused a different translocation of γ-PKC–GFP. After rapid translocation to the membrane at 30 s, the γ-PKC–GFP on the plasma membrane faded slightly. γ-PKC–GFP is seen as dots throughout the cytoplasm at 1 min, after which it appears on the plasma membrane as patchy dots and on the nuclear membrane. (Bottom row) Changes induced by 200 μM arachidonic acid in the fluorescence of γ-PKC– GFP. The translocation of γ-PKC–GFP induced by arachidonic acid is similar to that induced by saturated fatty acid and oleic acid. Bars, 10 μm.

Figure 4

Translocation of ε-PKC–GFP induced by saturated and unsaturated fatty acids. (A) Translocation of ε-PKC–GFP induced by saturated fatty acids. (Top row) Changes induced by 200 μM tridecanoic acid in the fluorescence of ε-PKC–GFP expressed in CHO-K1 cells. ε-PKC–GFP fusion protein is present throughout the cytoplasm but not in the nucleus. The addition of 200 μM tridecanoic acid induced rapid translocation of ε-PKC–GFP fluorescence from the cytoplasm to the plasma membrane, within 20 s of stimulation. Thereafter, ε-PKC–GFP was rapidly retranslocated from the membrane to the cytoplasm. (Second row) Changes induced by 200 μM pentadecanoic acid in the fluorescence of ε-PKC–GFP. Fairly intense fluorescence is present in the perinuclear region before the stimulation. The translocation of ε-PKC– GFP induced by pentadecanoic acid is similar to that of tridecanoic acid. (B) Translocation of ε-PKC– GFP induced by unsaturated fatty acids. (Third row) Changes induced by 200 μM linoleic acid in the fluorescence of ε-PKC–GFP. The addition of 200 μM linoleic acid induced slow translocation of ε-PKC–GFP fluorescence from the cytoplasm to the perinuclear region. Intense dotlike fluorescence is present near the nucleus at 3 min. (Bottom row) Changes induced by 200 μM arachidonic acid in the fluorescence of ε-PKC–GFP. ε-PKC–GFP fluorescence in the cytoplasm has faded, and intense fluorescence is present in the perinuclear area at 1 min. The accumulation of ε-PKC–GFP at the perinuclear area is still detectable at 3 min. Bars, 10 μm.

Figure 5

Colocalization of ε-PKC–GFP and wheat germ agglutinin binding sites in ε-PKC–GFP–expressing CHO-K1 cells treated with arachidonic acid. CHO-K1 cells transfected with ε-PKC–GFP were fixed after treatment with 100 μM arachidonic acid. Cells were treated with Texas red–conjugated wheat germ agglutinin to make the Golgi network visible. The localization of ε-PKC–GFP is shown (at left in green) by making GFP visible. The Golgi network is shown in the center (red). The overlapping images of GFP and Texas red appear as yellow. Bar, 10 μm.

Figure 6

Effects of Ca²⁺ chelators on fatty acid–induced translocation of γ- and ε-PKC– GFP. (Control) CHO-K1 cells expressing γ- and ε-PKC– GFP were incubated in normal Hepes buffer for 30 min, and then docosahexaenoic acid was added to the buffer to give 200 μM. Images were recorded before treatment and at 30 s after treatment (stimulated). Docosahexaenoic acid–induced translocation occurred for both γ- and ε-PKC–GFP. (Ca^{2 +} chelators) After the cells had been incubated for 30 min with 2.5 mM EGTA and 30 μM BAPTA-AM in Ca²⁺-free Hepes buffer, docosahexaenoic acid was challenged as in the control. Images were recorded before treatment and 30 s after treatment (stimulated). Treatment with Ca²⁺ chelators blocked the docosahexaenoic acid–induced translocation of γ-PKC–GFP but not that of ε-PKC–GFP.

Figure 7

Synergistic effect of arachidonic acid and the diacylglycerol analogue on the translocation of γ-PKC– GFP. (A) The addition of 10 μM DiC8, a diacylglycerol analogue, induced rapid translocation of γ-PKC– GFP from the cytoplasm to the plasma membrane. (B) DiC8 at 1 μM did not translocate γ-PKC–GFP. (C) A coaddition of 25 μM arachidonic acid and 1 μM DiC8 induced rapid translocation of γ-PKC–GFP followed by delayed, irreversible translocation. A low concentration of DiC8 induced rapid, reversible translocation within 15 s when applied with a low concentration of arachidonic acid. After the rapid translocation, a second translocation occurred at 3 min, and γ-PKC–GFP remained on the membrane even 10 min after treatment. Bars, 10 μm.