Distinct Effects of Fatty Acids on Translocation of gamma - and epsilon -Subspecies of Protein Kinase C

Yasuhito Shirai, Kaori Kashiwagi, Keiko Yagi, Norio Sakai, and Naoaki Saito

Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Kobe 657-8501, Japan

    Abstract
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 µM) and DiC8 (10 µM) 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.

Key words: protein kinase Ctranslocationfatty acidtargetinggreen fluorescent protein
    Introduction
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

PROTEIN kinase C (PKC)1 is a family of serine/threonine protein kinases that is activated in the presence of phospholipid and Ca2+ ions (Nishizuka, 1984, 1988; Parker and Dekker, 1997; Srinivasan and Blundell, 1997). To date, at least 12 subspecies have been identified, and these are divided into three groups based on their structures: conventional, novel, and atypical PKC (Nishizuka, 1988, 1992). The PKC family comprises the regulatory domain in the amino terminus and a catalytic domain in the carboxyl terminus. The conventional PKCs (cPKC), including alpha -, beta I-, beta II-, and gamma -PKC, have two common regions (C1 and C2) in the regulatory domain. The C1 region has two cysteine-rich loops (zinc finger-like motifs) that are the binding site for diacylglycerol (DAG) and phorbol ester (Nishizuka, 1988; Ono et al., 1989). The C2 region binds to calcium (Ono et al., 1989). The novel PKCs (nPKC), delta -, epsilon -, eta -, and theta -PKC, lack the C2 region (Ono et al., 1988b; Hug and Sarre, 1993; Osada et al., 1992). The atypical PKCs (aPKC), zeta - and iota /lambda -PKC, lack the C2 region and have only one cysteine-rich loop in the C1 region (Ono et al., 1989; Selbie et al., 1993; Akimoto et al., 1994). Calcium, phosphatidylserine, and DAG are required for the activation of cPKCs, whereas calcium is not required for the activation of nPKCs. aPKCs are insensitive to both DAG and calcium (Newton, 1997).

cPKCs and nPKCs are translocated from the soluble to the particulate fraction when activated by several stimuli (Kraft et al., 1982; Mochly-Rosen et al., 1990; Jaken, 1996; Ohno, 1997). The molecular mechanism of this translocation, however, has yet to be clarified. A system for monitoring the translocation of gamma -PKC in living cells has recently been developed that uses protein fused with green fluorescent protein (GFP) (Sakai et al., 1997; Oancea et al., 1998). This experimental breakthrough enabled us to investigate the subspecies-specific function of PKC by analyzing the different targeting mechanisms of each subspecies.

Several saturated and unsaturated fatty acids also are reported to potentiate the activity of PKC (McPhail et al., 1984; Murakami et al., 1986; Shinomura et al., 1991; Asaoka et al., 1992; Nishizuka, 1995). For example, saturated fatty acids that have carbon chain lengths of C13 to C18 activate gamma - and epsilon -PKC in vitro (Kasahara and Kikkawa, 1995), and unsaturated fatty acids, such as arachidonic and oleic acid, enhance the kinase activity of several PKC subspecies (Sekiguchi et al., 1988; Lester et al., 1991; Shinomura et al., 1991; Chen and Murakami, 1992). These fatty acid-induced activations of PKCs are enhanced by the presence of diacylglycerol. Furthermore, arachidonic acid-induced translocation of epsilon -PKC and oleic acid-induced translocation of alpha -, beta II-, and delta -PKC have been shown by immunoblot analysis (Khan et al., 1993; Huang et al., 1997). Less information, however, is available on the underlying mechanism of the effects of fatty acids on PKC translocation. To clarify the physiological involvement of fatty acids in PKC signaling pathways, we studied the effects of 11 fatty acids on the translocation of gamma - and epsilon -PKC in living cells that expressed PKC subspecies fused with GFP.

    Materials and Methods
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials

Tridecanoic acid, myristic acid, pentadecanoic acid, palmitic acid, heptadecanoic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid, and docosahexaenoic acid were purchased from Doosan Serdary Research Laboratories (Englewood Cliffs, NJ). 1,2-dioctanoylglycerol (DiC8) and BAPTA-AM were obtained from BIOMOL Research Laboratories (Plymouth Meeting, PA) and Research Biochemicals International (Natick, MA), respectively. All the other chemicals used were of analytical grade.

Cell Culture

COS-7 cells were purchased from the Riken cell bank (Tsukuba, Japan). The CHO-K1 cell strain was a gift from Dr. Nishijima (National Institute of Health, Tokyo, Japan). COS-7 cells were cultured in DME, and CHO-K1 cells in Ham's F12 medium (GIBCO BRL, Grand Island, NY) at 37°C in a humidified atmosphere containing 5% CO2. Both media contained 25 mM glucose, and both were buffered with 44 mM NaHCO3 and supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 µg/ml). The FBS used was not heat inactivated.

Construct of Plasmids Encoding the gamma - and epsilon -PKC-GFP Fusion Proteins

The plasmid bearing humanized GFP-S65A/Y145F cDNA was donated by Dr. Umesono (Kyoto University, Kyoto, Japan). A cDNA fragment encoding GFP with a HindIII site in the 5'-terminal end and an EcoRI site in the 3'-terminal end were obtained by a PCR with pCMX-SAH/Y145F as the template. The sense primer was 5'-AAGCTTATGGTGAGCAAGGGCGAGGAG-3', and the antisense primer was 5'-TTGAATTCCTAGCTAGCTGGCCAGGATCC-3'.

Rat gamma -PKC cDNA was obtained from the cDNA clone of lambda CKRgamma 1 (Ono et al., 1988a). After digestion with EcoRI, an insert fragment encoding rat gamma -PKC was subcloned to an expression plasmid for mammalian cells, pTB 701 (Ono et al., 1988b) (designated BS 55). A cDNA fragment of gamma -PKC with an EcoRI site in the 5' terminus and a HindIII site in the 3' terminus also was produced by a PCR with BS 55 as the template. The sense primer was 5'-TTGAATTCATGGCGGGTCTGGGTCCTGG-3', and the antisense primer was 5'-TTAAGCTTATGGCGGGTCTGGGTCCTGG-3'. The PCR products of both GFP and gamma -PKC were digested with EcoRI and HindIII and then subcloned together into the EcoRI site in pTB701 (BS 336).

A cDNA fragment encoding GFP with MunI-EcoRI-BglII sites in the 5' terminus and a MunI site in the 3' terminus was obtained by PCR with pCMX-SAH/Y145F as the template. The sense primer was 5'-TTTCAATTGAATTCAGATCTATGGTGAGCAAGGGCGAGGAG - 3' , and the antisense primer was 5'-GGCAATTGCTAGCTAGCTGGCCAGGATCC-3'. The PCR product was subcloned into pTB 701 (BS 340). The plasmid bearing rat epsilon -PKC cDNA in pTB 701 was a gift from Dr. Ono (Kobe University) (BS 254). A cDNA fragment of epsilon -PKC with a BglII site in the 5' and 3' termini was produced by a PCR with BS 254 as the template. The sense primer was 5'-TTAGATCTACCATGGTAGTGTTCAATGG-3', and the antisense primer was 5'-TTAGATCTGGGCATCAGGTCTTCACCAAA-3'. The PCR product for epsilon -PKC was digested with BglII and subcloned to the BglII site in BS 340 (BS 394).

Kinase Assay of Native epsilon -PKC and epsilon -PKC-GFP

COS-7 cells were transiently transfected by electroporation with plasmids encoding epsilon -PKC and epsilon -PKC-GFP and then cultured. epsilon -PKC and epsilon -PKC- GFP were immunoprecipitated with anti-epsilon -PKC monoclonal antibody (Transduction Laboratories, Lexington, KY), and their kinase activities were assayed as described elsewhere (Sakai et al., 1997). In brief, the immunoprecipitate was suspended in 120 µl of Dulbecco's PBS (-), and 10 µl of the suspended pellet was used for the kinase assay. Kinase activity measurements of the immunoprecipitated epsilon -PKC and epsilon -PKC-GFP were based on the incorporation of 32P into a fragment of myelin basic protein (Sigma Chemical Co., St. Louis, MO) from [gamma -32P]ATP in the presence of 8 µg/ml phosphatidylserine (PS) and 0.8 µg/ml diolein (DO). Basal activity was measured in the presence of 0.5 mM EGTA instead of PS or DO.

Transfection of PKCs and Fusion Proteins to Cultured CHO-K1 Cells

Plasmids (~5.5 µg) encoding gamma - or epsilon -PKC-GFP (BS 336 and BS 394) were transfected to 5 × 106 CHO-K1 cells by lipofection using TransITTM-LT2 (Mirus Co., Madison, WI) according to the manufacturer's standard protocol. The transfected cells were cultured at 37°C to obtain the optimal GFP fluorescence and then used for immunoblotting, immunostaining, and the observation of translocation.

Immunostaining of CHO-K1 Cells Expressing epsilon -PKC and Its Fusion Protein

Before and after translocation was induced by 1 µM TPA, cells expressing epsilon -PKC or epsilon -PKC-GFP were fixed with 4% paraformaldehyde and 0.2% picric acid in 0.1 M PBS for 30 min. After three washes with 0.1 M PBS, the cells were treated with PBS containing 0.3% Triton X and 10% normal goat serum (NGS) for 20 min. They then were incubated sequentially, first with anti-epsilon -PKC monoclonal antibody (diluted 1:1,000) for 40 min in PBS containing 0.03% Triton X (PBS-T) and then with Cy3-labeled goat anti-mouse IgG (Amersham Corp., Arlington Heights, IL) for 30 min at room temperature. Fluorescence of epsilon -PKC-like immunoreactivity was observed under a confocal laser scanning fluorescent microscope (model LSM 410 invert; Carl Zeiss, Jena, Germany) at 588-nm argon excitation with a 590-nm-long pass barrier filter. GFP fluorescence was observed at 488-nm argon excitation with a 510-525-nm-band pass barrier filter.

Observation of gamma - and epsilon -PKC-GFP Translocation

CHO-K1 cells expressing gamma - and epsilon -PKC-GFP were spread on glass-bottomed culture dishes (MatTek Corp., Ashland, MA) and incubated for 16-60 h before observation. The culture medium was replaced with normal Hepes buffer composed of 135 mM NaCl, 5.4 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 5 mM Hepes, and 10 mM glucose, pH 7.3. In the experiment with Ca2+ chelators, the cells were washed with Hepes buffer without CaCl2 (Ca2+-free Hepes buffer) and then incubated for 30 min with 2.5 mM EGTA and 30 µM BAPTA-AM in Ca2+-free Hepes buffer. Translocation of the fusion protein was triggered by the addition of the various stimulants to the Hepes buffer to obtain the appropriate final concentration. All experiments were done at 37°C.

The fluorescence of GFP was monitored under a confocal laser scanning fluorescent microscope at 488-nm argon excitation with a 515-nm-long pass barrier filter. Lastly, the changes in fluorescence were quantified using confocal software.

Codetection of the Golgi Network and epsilon -PKC-GFP Translocated by the Stimulation of Arachidonic Acid

Texas red-conjugated wheat germ agglutinin was used to monitor the Golgi network. After induction of the translocation of epsilon -PKC-GFP by 100 µM arachidonic acid, the cells were fixed and treated with 0.3% Triton X and 10% NGS as described above, after which the cells were incubated with 0.5 µg/ml Texas red-conjugated wheat germ agglutinin (Molecular Probes, Leiden, Netherlands) in PBS-T for 30 min. Finally, the fluorescence of Texas red and GFP were observed under a confocal laser scanning fluorescent microscope, the former at 588-nm argon excitation using a 590-nm-long pass barrier filter and the latter at 488-nm argon excitation using a 510-525-nm-band pass barrier filter.

    Results
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Properties of epsilon -PKC and Its Fusion Protein with GFP

Enzymological and immunochemical properties of epsilon -PKC- GFP and native epsilon -PKC were examined. Fig. 1 A shows that both the immunoprecipitated epsilon -PKC and epsilon -PKC-GFP had basal kinase activity and that both were activated at ~3.3-fold the basal level in the presence of PS and DO. These findings indicate that epsilon -PKC-GFP also is dependent on PS and DO and that its enzymological property is very similar to that of the native epsilon -PKC.


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

Immunostaining with anti-epsilon -PKC antibodies showed that both epsilon -PKC and epsilon -PKC-GFP were expressed in the cytoplasm of CHO-K1 cells before stimulation (Fig. 1 B, a and b) and intense immunoreactivities on the plasma membrane after treatment with 1 µM TPA (Fig. 1 B, d and e). Within the same cells, localization of the GFP fluorescence of epsilon -PKC-GFP (Fig. 1 B, b) corresponded to that of the epsilon -PKC-like immunoreactivity (Fig. 1 B, c). Colocalization of GFP and epsilon -PKC-like immunoreactivity also was observed after treatment with TPA (Fig. 1 B, e and f). These results indicate that in response to TPA, both native epsilon -PKC and epsilon -PKC-GFP were translocated from the cytoplasm to the plasma membrane without proteolysis. This was confirmed by immunoblotting using anti-epsilon -PKC antibody (data not shown).

Effects of Fatty Acids on the Translocation of gamma - and epsilon -PKC-GFP

The effect of a single application of a saturated fatty acid (such as tridecanoic acid, myristic acid, pentadecanoic acid, palmitic acid, heptadecanoic acid, or stearic acid) on the subcellular localization of gamma -PKC-GFP was investigated. All the saturated fatty acids examined induced the translocation of gamma -PKC-GFP at 200 µM. Fig. 2 shows sequential pictures of the typical translocation of gamma -PKC- GFP induced by saturated fatty acids. When gamma -PKC-GFP was expressed in CHO-K1 cells, intense fluorescence was present throughout the cytoplasm, and faint fluorescence was present in the nucleus. The presence of gamma -PKC-GFP in the nucleus was obvious because the nucleolus appeared as a small black circle. Stimulation with 200 µM tridecanoic acid, pentadecanoic acid, or palmitic acid caused translocation of the gamma -PKC-GFP in the cytoplasm to the plasma membrane at 20 s, but there was a return to the cytoplasm within 2 min. These fatty acids, however, had no effect on the gamma -PKC-GFP in the nucleus. These fluorescence changes were confirmed by the profiles of GFP intensity shown in Fig. 2. Myristic acid, heptadecanoic acid, and stearic acid also induced translocation of gamma -PKC-GFP similar to that shown in Fig. 2 (data not shown).


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Fig. 2.   Translocation of gamma -PKC-GFP induced by saturated fatty acids in CHO-K1 cells. (Top row) Changes induced by 200 µM tridecanoic acid in the fluorescence of gamma -PKC-GFP expressed in CHO-K1 cells. gamma -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 gamma -PKC-GFP fluorescence from the cytoplasm to the plasma membrane, which took place within 20 s of stimulation. Thereafter, gamma -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 gamma -PKC-GFP expressed in CHO-K1 cells. The translocation of gamma -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 gamma -PKC-GFP expressed in CHO-K1 cells. The translocation is similar to the translocations of tridecanoic and pentadecanoic acids. Bars, 10 µm.

Unsaturated fatty acids also caused the translocation of gamma -PKC-GFP. Oleic and arachidonic acid at 200 µM induced a very rapid, transient translocation of gamma -PKC-GFP from the cytoplasm to the plasma membrane similar to that of saturated fatty acids (Fig. 3). Linolenic and docosahexaenoic acids induced gamma -PKC-GFP translocation similar to that of oleic and arachidonic acids (data not shown). Linoleic acid at 200 µM, however, occasionally translocated gamma -PKC-GFP to different components, but at a lower concentration (100 µM), it induced a translocation from the cytoplasm to the membrane similar to that of oleic and arachidonic acids (Fig. 3). After rapid translocation to the membrane caused by treatment with 200 µM linoleic acid, the gamma -PKC-GFP on the plasma membrane faded slightly, and at 1 min it appeared as an accumulation of dots throughout the cytoplasm. Finally, gamma -PKC-GFP again appeared as an accumulation of patchy dots on the plasma membrane and on the nuclear membrane. It is noteworthy that the target site for gamma -PKC-GFP on stimulation with linoleic acid depends on the concentration.


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Fig. 3.   Translocation of gamma -PKC-GFP induced by unsaturated fatty acids. (Top row) Changes induced by 200 µM oleic acid in the fluorescence of gamma -PKC-GFP. Oleic acid also induced very rapid, transient translocation of gamma -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 gamma -PKC-GFP. Linoleic acid at a low concentration (100 µM) induced translocation of gamma -PKC-GFP from the cytoplasm to the plasma membrane as did oleic acid. At 200 µM, however, it caused a different translocation of gamma -PKC-GFP. After rapid translocation to the membrane at 30 s, the gamma -PKC-GFP on the plasma membrane faded slightly. gamma -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 gamma -PKC- GFP. The translocation of gamma -PKC-GFP induced by arachidonic acid is similar to that induced by saturated fatty acid and oleic acid. Bars, 10 µm.

Translocation of gamma -PKC-GFP was also examined in response to various concentrations of the fatty acids (Table I). Of the 11 fatty acids tested, gamma -PKC-GFP was most sensitive to pentadecanoic acid; ~38% of the cells showed marked translocation in response to 50 µM pentadecanoic acid. Tridecanoic acid, palmitic acid, heptadecanoic acid, linoleic acid, and docosahexaenoic acid at 100 µM caused translocation in more than 40% of the cells, whereas the other fatty acids required more than 200 µM.

                              
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Table I
Effectiveness of Fatty Acids in Inducing the Translocation of gamma - and epsilon -PKC-GFP

The sensitivity of epsilon -PKC-GFP to fatty acids differed from that of gamma -PKC-GFP (Table I). Except for tridecanoic, linoleic, and arachidonic acids, the fatty acids in general had little effect on the translocation of epsilon -PKC- GFP. As shown in Table I, only five fatty acids (tridecanoic, pentadecanoic, linoleic, arachidonic, and docosahexaenoic acids) induced translocation when used alone at 200 µM. The other fatty acids required more than 300 µM for activity or failed to translocate epsilon -PKC-GFP. Tridecanoic acid was the most effective for the epsilon -PKC-GFP translocation, being more effective for epsilon -PKC-GFP than gamma -PKC-GFP (Table I).

epsilon -PKC-GFP fluorescence was detected in the cytoplasm. Occasionally, fairly intense fluorescence was present in the perinuclear region (Fig. 4 A, second row), but no epsilon -PKC-GFP was present within the nucleus before stimulation. Except for palmitic acid, the saturated fatty acids induced rapid, transient translocation of epsilon -PKC-GFP from the cytoplasm to the membrane (Fig. 4 A). An addition of 200 µM tridecanoic and pentadecanoic acid caused rapid movement of epsilon -PKC-GFP to the membrane and a return to the cytoplasm at 2 min. Of the five unsaturated fatty acids tested, the translocation of epsilon -PKC-GFP was reproducibly induced by linoleic, arachidonic, or docosahexaenoic acid at 200 µM. The translocation induced by docosahexaenoic acid was similar to that induced by the saturated fatty acids in Fig. 4 A (data not shown). Linoleic and arachidonic acids, however, induced a unique translocation of epsilon -PKC-GFP (Fig. 4 B). Neither linoleic nor arachidonic acid at 200 µM induced rapid, reversible translocation of epsilon -PKC-GFP to the plasma membrane, but both induced slow, irreversible accumulation of epsilon -PKC-GFP to perinuclear regions. The accumulation of epsilon -PKC-GFP in the perinuclear region was evident 3 min after the stimulations with linoleic and arachidonic acids and was still detectable 15 min after stimulation (data not shown). Arachidonic acid-induced translocation occurred in the presence of 10 µM of indomethacine, which inhibits the generation of eicosanoids from arachidonic acid (data not shown) (Flower et al., 1985).


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Fig. 4.   Translocation of epsilon -PKC-GFP induced by saturated and unsaturated fatty acids. (A) Translocation of epsilon -PKC-GFP induced by saturated fatty acids. (Top row) Changes induced by 200 µM tridecanoic acid in the fluorescence of epsilon -PKC-GFP expressed in CHO-K1 cells. epsilon -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 epsilon -PKC-GFP fluorescence from the cytoplasm to the plasma membrane, within 20 s of stimulation. Thereafter, epsilon -PKC-GFP was rapidly retranslocated from the membrane to the cytoplasm. (Second row) Changes induced by 200 µM pentadecanoic acid in the fluorescence of epsilon -PKC-GFP. Fairly intense fluorescence is present in the perinuclear region before the stimulation. The translocation of epsilon -PKC- GFP induced by pentadecanoic acid is similar to that of tridecanoic acid. (B) Translocation of epsilon -PKC- GFP induced by unsaturated fatty acids. (Third row) Changes induced by 200 µM linoleic acid in the fluorescence of epsilon -PKC-GFP. The addition of 200 µM linoleic acid induced slow translocation of epsilon -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 epsilon -PKC-GFP. epsilon -PKC-GFP fluorescence in the cytoplasm has faded, and intense fluorescence is present in the perinuclear area at 1 min. The accumulation of epsilon -PKC-GFP at the perinuclear area is still detectable at 3 min. Bars, 10 µm.

Immunostaining with anti-gamma -PKC and -epsilon -PKC antibodies confirmed that these fatty acid-induced translocations occurred in CHO-K1 cells that expressed native gamma - and epsilon -PKC (data not shown).

Target Site of epsilon -PKC-GFP on Stimulation with Arachidonic Acid

To identify the compartment in which epsilon -PKC-GFP accumulated in response to arachidonic acid, the Golgi network was made visible with Texas red-conjugated wheat germ agglutinin after arachidonic acid induced the translocation of epsilon -PKC-GFP.

GFP fluorescence was present throughout the cytoplasm, but not in the nucleus, and was most intense in the perinuclear region (Fig. 5, left). The fluorescence of the two Texas red-stained cells shown in a micrograph (Fig. 5, center) indicates that one was the cell detected by GFP fluorescence and the other was a cell that expressed no epsilon -PKC-GFP. Intense Texas red fluorescence was present around the nucleus. An overlapping image shows that the Texas red fluorescence and GFP colocalized in the perinuclear region (Fig. 5, right).


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Fig. 5.   Colocalization of epsilon -PKC-GFP and wheat germ agglutinin binding sites in epsilon -PKC-GFP-expressing CHO-K1 cells treated with arachidonic acid. CHO-K1 cells transfected with epsilon -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 epsilon -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.

Effect of Ca2+ Chelators on Fatty Acid-induced Translocation of gamma - and epsilon -PKC-GFP

To clarify whether the fatty acid-induced translocations of gamma - and epsilon -PKC-GFP require Ca2+ ions, we examined the effects of Ca2+ chelators on the translocations. Pretreatment with 2.5 mM EGTA and 30 µM BAPTA-AM blocked the translocation of gamma -PKC-GFP induced by docosahexaenoic acid, but not that of epsilon -PKC-GFP (Fig. 6). Similarly, tridecanoic acid-induced translocation of gamma -PKC was inhibited by Ca2+ chelators, whereas translocation of epsilon -PKC occurred in the presence of Ca2+ chelators (data not shown). These findings suggest that the fatty acid-induced translocation of epsilon -PKC-GFP is independent of Ca2+, whereas Ca2+ is necessary for the fatty acid-induced translocation of gamma -PKC.


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Fig. 6.   Effects of Ca2+ chelators on fatty acid-induced translocation of gamma - and epsilon -PKC- GFP. (Control) CHO-K1 cells expressing gamma - and epsilon -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 gamma - and epsilon -PKC-GFP. (Ca2+ 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 gamma -PKC-GFP but not that of epsilon -PKC-GFP.

Synergistic Effect of Arachidonic Acid and Diacylglycerol on the Translocation of gamma - and epsilon -PKC-GFP

As shown in Fig. 7, DiC8 at 10 µM caused very rapid translocation of gamma -PKC-GFP to the plasma membrane at 15 s, but the translocated gamma -PKC-GFP returned to the initial state within 3 min (Fig. 7 A), and thereafter no translocation occurred. The application of 1 µM DiC8 alone, however, failed to induce the gamma -PKC-GFP translocation (Fig. 7 B). Similarly, use of arachidonic acid alone at 200 µM, but not at 25 µM, caused translocation of gamma -PKC-GFP (Table I). Simultaneous applications of 1 µM DiC8 and 25 µM arachidonic acid induced remarkable translocation of gamma -PKC-GFP. 15 s after the coapplication, there was very faint translocation to the plasma membrane, similar to that induced by a single application of a low concentration of DiC8 or arachidonic acid. Subsequently, about 3 min after stimulation, slow translocation of gamma -PKC-GFP to the plasma membrane became evident. This late phase translocation was unidirected and irreversible (Fig. 7 C). This synergistic action occurred in the presence of 10 µM indomethacine, indicating that arachidonic acid itself contributed to the unidirected and irreversible translocation of gamma -PKC-GFP (data not shown). In contrast, the translocation of epsilon -PKC-GFP was neither markedly enhanced nor altered by the simultaneous application of arachidonic acid and DiC8.


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Fig. 7.   Synergistic effect of arachidonic acid and the diacylglycerol analogue on the translocation of gamma -PKC- GFP. (A) The addition of 10 µM DiC8, a diacylglycerol analogue, induced rapid translocation of gamma -PKC- GFP from the cytoplasm to the plasma membrane. (B) DiC8 at 1 µM did not translocate gamma -PKC-GFP. (C) A coaddition of 25 µM arachidonic acid and 1 µM DiC8 induced rapid translocation of gamma -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 gamma -PKC-GFP remained on the membrane even 10 min after treatment. Bars, 10 µm.

    Discussion
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Abstract
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Materials & Methods
Results
Discussion
References

Using gamma - and epsilon -PKC proteins fused with GFP, we showed the fatty acid-induced translocation of PKCs in living cells in real time. In order for the proteins' movement to be visible by monitoring the fluorescence of GFP fused to the protein, the fusion proteins must have the same properties as native PKCs. Furthermore, because PKCs are reported to be proteolysed by proteases such as calpain (Kishimoto et al., 1983), we examined the enzymological and immunochemical properties of gamma - and epsilon -PKC-GFP. Because gamma -PKC-GFP is reported to have the same properties as native gamma -PKC (Sakai et al., 1997), we analyzed the properties of epsilon -PKC-GFP by a kinase assay, immunostaining, and Western blotting and found that (a) the activity of epsilon -PKC-GFP was dependent on PS and DO (Fig. 1 A); (b) the epsilon -PKC-GFP images immunostained with anti-epsilon -PKC antibody coincided with the fluorescences of GFP before and after TPA stimulation (Fig. 1 B); and (c) no epsilon -PKC- GFP-degraded product was found in the immunoblot analysis, even after TPA treatment (data not shown). These findings indicate that the GFP fluorescence fused to epsilon -PKC can be used as a marker for native epsilon -PKC.

We investigated the effects of saturated fatty acids with carbon chain lengths of C13 to C18 and unsaturated fatty acids on the translocation of gamma - and epsilon -PKC-GFP because these fatty acids are known to potentiate the activity of PKC subspecies (Kasahara and Kikkawa, 1995). Of the 11 fatty acids examined, the translocation of gamma -PKC was induced more effectively by the saturated than the unsaturated fatty acids, whereas the kinase activity was enhanced more effectively by the unsaturated fatty acids (Shinomura et al., 1991). Saturated fatty acids enhanced the kinase activity of both gamma - and epsilon -PKC by the synergistic action of diacylglycerol, and epsilon -PKC was more sensitive to saturated fatty acids than gamma -PKC (Shinomura et al., 1991; Kasahara and Kikkawa, 1995). The translocation of epsilon -PKC, however, was less sensitive to the saturated fatty acids than was that of gamma -PKC and was not induced synergistically in the presence of arachidonic acid and DiC8. These discrepancies may be due to the different methods used to detect the effects of the fatty acids; the translocation of PKC was monitored after the extracellular fatty acid application, whereas PKC activity was assayed by applying the fatty acids directly to the PKCs in vitro. Taking into account that translocation was induced by phorbol ester, even in the presence of a PKC inhibitor (Sakai et al., 1997), an increase in the kinase activity of PKC does not always correspond to the translocation of PKC.

Except for linoleic acid at a high concentration, all the fatty acids induced similar translocations of gamma -PKC: rapid, reversible translocation from the cytoplasm to the plasma membrane. In contrast, both arachidonic and linoleic acids generated the slow translocation of epsilon -PKC from the cytoplasm to the perinuclear area, and saturated fatty acids induced a translocation of epsilon -PKC similar to that of gamma -PKC. The target site of the arachidonic acid-induced translocation of epsilon -PKC was identified as the Golgi network. This is consistent with previous reports showing that epsilon -PKC was localized to the Golgi via its zinc-finger domain (Lehel et al., 1995) and that arachidonic acid stimulated epsilon -PKC redistribution in heart cells (Huang et al., 1997). These findings indicate that the target site of epsilon -PKC differs in response to the fatty acid used and strongly suggest that the mechanism of translocation induced by arachidonic acid differs for gamma - and epsilon -PKC. These interpretations are supported by the different effects of fatty acids on the kinase activities of PKC subspecies in vitro (Shinomura et al., 1991; Koide et al., 1992; Kasahara and Kikkawa, 1995). Saturated fatty acids with carbon chain lengths of C12 to C14 activated alpha -, beta -, gamma -, and epsilon -PKC, but not delta -PKC. Phorbol ester synergically enhanced the activity of alpha -, beta -, and gamma - PKCs when simultaneously treated with fatty acids, whereas it suppressed the activity of epsilon -PKC.

Some fatty acids mobilize intracellular Ca2+ (Gamberucci et al., 1997; Schaloske et al., 1998). We therefore clarified whether fatty acid translocation depends on an increase in the intracellular Ca2+ concentration. Docosahexaenoic and tridecanoic acids induced translocation of epsilon -PKC in the presence of Ca2+ chelators, indicating that the fatty acid-induced translocation of epsilon -PKC is independent of the increase in the intracellular Ca2+ concentration, although none of the fatty acids were tested in the presence of Ca2+ chelators. In contrast, neither docosahexaenoic nor tridecanoic acid caused the translocation of gamma -PKC in the presence of Ca2+ chelators. This suggests that fatty acids cause the translocation of gamma -PKC through an increase in the intracellular Ca2+ concentration, but it does not exclude the possibility that they directly act on gamma - and epsilon -PKC and induce their translocation. Intracellular Ca2+ appears to be indispensable for gamma -PKC translocation because the DiC8-induced translocation of gamma -PKC was blocked by Ca2+ chelators (data not shown).

Furthermore, arachidonic acid increases the activities of gamma -PKC and epsilon -PKC by the synergistic action of DiC8 (Kasahara and Kikkawa, 1995). As shown in Fig. 7, arachidonic acid also increased translocation sensitivity to DiC8 and then induced the additional, unidirectional translocation of gamma -PKC. In contrast, synergistic translocation of epsilon -PKC could not be induced by arachidonic acid and DiC8. Oancea et al. (1998) also detected no synergistic effect of arachidonic acid on DiC8-induced translocation of the first cystein-rich region (Cys-1) of gamma -PKC, but they did show that arachidonic acid inhibited the DiC8-induced translocation of the Cys-1 of PKC to the plasma membrane. These findings suggest that the synergistic effect of arachidonic acid on gamma -PKC activity occurs through an unknown mechanism that differs from the targeting mechanism by arachidonic acid of Cys-1 (or epsilon -PKC) to the Golgi complex.

The anchoring proteins of PKC, RACKs, have recently been identified (Mochly-Rosen et al., 1991). RACKs bind activated PKC (Jaken, 1997) and are thought to be involved in PKC translocation. To clarify the mechanism of PKC translocation, it is necessary to examine the effects of these anchoring proteins on PKC translocation using GFP-labeled PKC in living cells.

In conclusion, each PKC subspecies has a different targeting mechanism that depends on the extracellular stimuli used, i.e., the fatty acid. Moreover, the synergistic actions of intracellular activators have an important role in targeting, thereby contributing to the subspecies-specific function.

    Footnotes

Received for publication 22 May 1998 and in revised form 10 August 1998.

   Y. Shirai and K. Kashiwagi equally contributed to the performance of all the experiments and writing of the manuscript.
   Address all correspondence to Naoaki Saito, Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan. Tel.: 81-78-803-1251. Fax: 81-78-803-0993. E-mail: naosaito{at}kobe-u.ac.jp

We thank Dr. Yasutomi Nishizuka for his helpful discussions of our work.

This work was supported by grants from the Ministry of Education, Science, Sports, and Culture of Japan, the Yamanouchi Foundation for Research on Metabolic Disorders, and the Kato Memorial Bioscience Foundation.

    Abbreviations used in this paper

cPKC, nPKC, and aPKC, conventional, novel, and atypical PKCs; DAG, diacylglycerol; DiC8, 1,2-dioctanoylgrecerol; DO, diolein; GFP, green fluorescent protein; PKC, protein kinase C; PS, phosphatidylserine; TPA, 12-o-tetradecanoylphorbol 13-acetate.

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