INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Protein kinase C (PKC) is a family of phospholipid-dependent serine/threonine protein kinases consisting of at least 10 subspecies that can be classified into three subgroups, classical, novel, and atypical PKC (43, 44, 52, 54). The classical PKC members (, I, II, and ), each of which has a Ca²⁺ binding region (C2 region) and two cysteine-rich regions, are activated by Ca²⁺, phosphatidylserine (PS), and diacylglycerol (DG) or phorbol esters. The novel PKC members (, , , and ), lacking the C2 region, are activated by PS and DG or phorbol esters without Ca²⁺. The atypical PKC members ( and /), which lack the C2 region and have only one cysteine-rich region, are dependent on PS but are not affected by DG, phorbol esters, or Ca²⁺ (52). Although a considerable number of studies have demonstrated the involvement of PKC in various cellular functions (2, 6, 42, 45, 62), the individual roles of each PKC subtype in cellular functions remain unclear. Recent studies in living cells using green fluorescent protein (GFP)-tagged PKC have shown that each PKC subtype has a spatially and temporally different targeting mechanism that is dependent on the extracellular signals contributing to the subspecies-specific functions of PKC (46, 49, 56, 59). Based on these findings, it was proposed that the PKC targeting mechanism is a determinant for the specific function of each PKC subtype in response to various stimuli in various cell types.

Ceramide has recently emerged as an intracellular lipid mediator implicated in various cellular responses, such as programmed cell death, cell differentiation, growth inhibition, and long-term depression of synaptic transmission (5, 18, 19, 24, 47, 51, 64, 65). Ceramide is generated by transient hydrolysis of sphingomyelin, and many reports have indicated that ceramide is produced via receptor-mediated stimulation by various extracellular ligands, including vitamin D₃ (50), gamma interferon (IFN-) (25), tumor necrosis factor alpha (TNF-) (11, 25), interleukin-1 (36), and nerve growth factor (5, 10). Recently, the regulation of PKC activity by ceramide has been reported, but the results are still controversial; ceramide has been shown to activate PKC or inhibit PKC autophosphorylation in renal mesangial cells in vitro (22). Furthermore, it is also reported that ceramide induces the translocation of PKC and PKC from the membrane to the cytosol in human myelogenous leukemia HL-60 cells (57) or of PKC from the cytosol to the membrane in renal mesangial cells and in smooth muscle cells (22, 23). These apparently contradictory results may have been due to differences not only in methods but also in time points and cell types examined, suggesting the necessity to observe the localization of each PKC subtype after ceramide treatment continuously in living cells.

In the present study, we investigated the intracellular movement of GFP-tagged PKC subtypes in living cells after treatment with various stimuli, such as ceramide and IFN-. We also examined the effect of ceramide on the kinase activity of PKC subtypes. We demonstrated here the PKC-specific translocation to the Golgi complex by ceramide and the activation of PKC through tyrosine phosphorylation of the enzyme.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Materials. D-Erythro-C₂-ceramide and D-erythrodihydro-C₂-ceramide were purchased from Biomol Research Laboratories (Plymouth Meeting, Pa.). D-Erythro-C₆-ceramide and 6-{[N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino] hexanoyl} sphingosine (C₆-NBD-ceramide) were purchased from Molecular Probes (Eugene, Oreg.). 12-O-Tetradecanoylphorbol-13-acetate (TPA) was purchased from Sigma Chemical Co. (St. Louis, Mo.). D609 was purchased from Cayman Chemical (Ann Arbor, Mich.). IFN- was a kind gift from Shionogi Research Laboratory and Co., Ltd. (Osaka, Japan). TNF- was purchased from Gibco BRL (Grand Island, N.Y.). Scyphostatin was a kind gift from Takeshi Ogita (Biomedical Research Laboratories, Sankyo Co., Ltd., Tokyo, Japan). Glutathione (GSH) was purchased from Nacalai Tesque Inc. (Kyoto, Japan). Tyrphostin AG490 and genistein were purchased from Calbiochem-Novabiochem Co. (La Jolla, Calif.). Anti-PKC, PKC, and PKC monoclonal antibodies were purchased from Transduction Laboratories (Lexington, Ky.). Anti-PKC and PKC polyclonal antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, Calif.). Anti-PKC polyclonal antibody was purchased from Upstate Biotechnology (Lake Placid, N.Y.) and that used for immunoblotting was produced as described previously (53). Anti-phosphotyrosine antibody, clone 4G10, was purchased from Upstate Biotechnology. Peroxidase-conjugated goat anti-mouse or anti-rabbit immunoglobulin G (IgG) was purchased from Amersham Corp. (Arlington Heights, Ill.). Cy3-labeled goat anti-mouse or anti-rabbit IgG was purchased from Amersham Corp. Calf thymus H1 histone was purchased from Boehringer GmbH (Mannheim, Germany). Myelin basic protein (MBP) was purchased from Sigma Chemical Co. Sodium fluoride (NaF) was purchased from Nacalai Tesque Inc. Sodium orthovanadate was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All the other chemicals used were of analytical grade.

Cell culture. HeLa cells and HEK293 cells were purchased from Riken Cell Bank (Tsukuba, Japan). The CHO-K1 cell strain was a gift from Masahiro Nishijima (National Institute of Health, Tokyo, Japan). HeLa cells were cultured in minimum essential medium (Gibco BRL) which was buffered with 44 mM NaHCO₃ and supplemented with 10% fetal bovine serum (FBS) in 5% CO₂ at 37°C in a humidified incubator. HEK293 cells were maintained in minimum essential medium supplemented with 44 mM NaHCO₃ and 10% horse serum in a humidified atmosphere containing 5% CO₂ at 37°C. CHO-K1 cells were cultured as described previously (49, 59). All media were supplemented with penicillin (100 U/ml) and streptomycin (100 µg/ml), and the FBS used was not heat inactivated.

Preparation of PKC-GFP adenovirus. The adenoviral plasmids (pAdEasy-1 and pAdEasy-2) and the shuttle vectors (pShuttle, pShuttle-CMV, pAdTrack, and pAdTrack-CMV) were gifts from Bert Vogelstein (Johns Hopkins University Oncology Center, Baltimore, Md.). Generation of an adenoviral vector including PKC-GFP cDNA was performed according to the methods described on the web site of Johns Hopkins University Oncology Center (http://www.coloncancer.org/adeasy.html). Briefly, the plasmid encoding GFP (BS 348) was digested by BgIII and HindIII. The plasmid encoding PKC (BS390) was digested with XhoI and BamHI, the digested products were subcloned into the XhoI and HindIII sites of the expression vector pShuttle-CMV, and the new plasmid was designated pBsAd010.

Expression of PKC-, PKC-, and PKC-GFP or PKC protein in HeLa cells. GFP was fused to the C terminus of each PKC subtype. For lipofection, plasmids (~5.5 µg) including PKC-, PKC-, and PKC-GFP or PKC cDNA were transfected into 5 × 10⁶ HeLa cells using TransIT-LT2 (Mirus) according to the manufacturer's standard protocol. For adenoviral infection, HeLa cells (5 × 10⁶) were incubated in serum-free medium for 2 h with recombinant PKC-GFP adenovirus with a multiplicity of infection of 10 in a humidified atmosphere containing 5% CO₂ at 37°C. After infection, the viral supernatant was removed, and the cells were cultured in normal serum-containing medium. Adenoviral infection was performed in the immunoblotting experiments and kinase assays. The fluorescence of PKC-, PKC-, and PKC-GFP became detectable 16 h after transfection. All experiments were performed 2 days after transfection.

Observation of PKC-, PKC-, and -PKC-GFP translocation. HeLa cells expressing PKC-, PKC-, and PKC-GFP were spread onto glass-bottomed culture dishes (MatTek, Ashland, Mass.) and cultured for at least 16 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. The fluorescence of GFP was monitored under a confocal laser scanning fluorescence microscope (LSM 410 or 510; Carl Zeiss, Jena, Germany) at a 488-nm excitation wavelength with a 515-nm-long pass or 510- to 525-nm band pass barrier filter. Translocation of the fusion protein was triggered by addition of various stimulators at high concentrations into the HEPES buffer to obtain the appropriate final concentrations. All experiments were performed at 37°C.

Immunostaining of endogenous PKC, PKC, and PKC or expressed PKC. Before and after treatment with 10 µM C₂-ceramide or C₆-NBD-ceramide, HeLa cells were fixed with a fixative containing 4% paraformaldehyde and 0.2% picric acid in 0.01 M PBS (pH 7.4) for 30 min. After washing twice with PBS, the cells were treated with PBS containing 0.3% Triton X-100 and 5% normal goat serum (NGS) for 10 min. The cells were then incubated with the anti-PKC and -PKC monoclonal antibodies (diluted 1:200) or the anti-PKC polyclonal antibody (Upstate Biotechnology) (diluted 1:200) in PBS with 0.03% Triton X-100 (PBS-T) and 5% NGS for 1 h at 25°C. After washing in PBS-T, the cells were incubated with Cy3-labeled goat anti-mouse or anti-rabbit IgG (diluted 1:1,000) for 30 min. After three washes with PBS-T for 10 min each time, the fluorescence of Cy3 was observed under a confocal laser scanning fluorescent microscope with excitation at 588 nm using a 590-nm-long pass barrier filter.

Codetection of the Golgi complex and PKC-GFP translocated by ceramide. Texas red-conjugated wheat germ agglutinin was used to monitor the Golgi complex. After translocation of PKC-GFP by 10 µM C₂-ceramide, the cells were fixed with a fixative containing 4% paraformaldehyde and 0.2% picric acid in 0.01 M PBS (pH 7.4) for 30 min. After washing twice with PBS, the cells were treated with PBS containing 0.3% Triton X-100 and 5% NGS for 10 min. The cells were then incubated with 1 µg of Texas red-conjugated wheat germ agglutinin (Molecular Probes, Leiden, The Netherlands) per ml for 40 min in PBS-T and 5% NGS. After three washes with PBS-T for 10 min each time, the fluorescence of Texas red and GFP was observed under a confocal laser scanning fluorescent microscope, with excitation at 488 nm using a 510- to 525-nm band pass barrier filter for the former and with excitation at 588 nm using a 590-nm-long pass barrier filter for the latter.

Cell fractionation and immunoprecipitation. For detection of endogenous PKC isozymes in HeLa cells, cultured HeLa cells were harvested with 1 ml of homogenate buffer (250 mM sucrose, 10 mM EGTA, 2 mM EDTA, 20 mM Tris-HCl, 200 µg of leupeptin per ml, 1 mM phenylmethylsulfonyl fluoride, pH 7.4) and centrifuged at 2,000 × g. For immunoblot analysis of tyrosine phosphorylation, we also used the homogenate buffer containing 10 mM NaF and 1 mM Na₃VO₄. The cells were resuspended with 300 µl of homogenate buffer containing 1% Triton X-100 and sonicated (UD-210; Tomy Seiko Co. Ltd., Tokyo, Japan) (output, 5; duty, 50%) 10 times at 4°C, and the supernatant was used after centrifugation at 19,000 × g for 15 min.

Immunoblotting analysis. The same amounts of samples from each fraction were subjected to sodium dodecyl sulfate-7.5% polyacrylamide gel electrophoresis according to the method of Laemmli (28), and the separated proteins were electorophoretically transferred onto polyvinylidene difluoride (PVDF) filters (Millipore Co., Bedford, Mass.). Nonspecific binding sites on the PVDF filters were blocked by incubation with 5% nonfat milk or 2% bovine serum albumin in PBS-T for 18 h. The PVDF filters were then incubated with the anti-PKC monoclonal antibodies (PKC, PKC, or PKC) (diluted 1:1,000), the anti-PKC polyclonal antibodies (PKC, PKC, or PKC) (diluted 1:1,000), the anti-GFP polyclonal antibody (Clontech, Palo Alto, Calif.) (diluted 1:1,000), or anti-phosphotyrosine antibody (diluted 1:1,000) for 1 h at 25°C. After washing in PBS-T, the filters were incubated with peroxidase-conjugated goat anti-mouse or goat anti-rabbit IgG (diluted 1:10,000) for 30 min. After three rinses, the immunoreactive bands were visualized using an enhanced chemiluminescence detection kit (Amersham). After immunoblot using anti-phosphotyrosine antibody, the PVDF filter was washed according to the manufacturer's protocol for reprobing with anti-GFP polyclonal antibody.

Fluorescence photobleaching. Photobleaching experiments were performed as follows. After translocation of PKC-GFP by 10 µM C₂-ceramide, a circular region in the Golgi complex or a square region of perikarya within a plane of the cell was photobleached by scanning for 15 s with an argon laser of the highest power. Recovery and fading of fluorescence in the selected regions were then analyzed by imaging the entire cell by confocal fluorescent microscopy with low laser power at the indicated times after photobleaching. For all of the images, the noise levels were reduced by line scan averaging.

In vitro and in vivo kinase assay of PKC. The immunoprecipitated samples (10 µl of suspended pellet) were used for both in vitro and in vivo kinase assays. In vitro kinase assays of PKC-GFP expressed in HeLa cells were performed as described previously (49). Briefly, the kinase activity in 10 µl of each sample was assayed by measuring the incorporation of ³²P_i into H1 histone from [-³²P]ATP in the presence of PS (8 µg/ml), diolein (DO) (0.8 µg/ml), or C₂-ceramide at various concentrations. For the in vivo kinase assay, endogenous PKC, PKC, or PKC or exogenous PKC-GFP was immunoprecipitated from HeLa cells at various time points after stimulation with 10 µM C₂-ceramide, and then the kinase activities were measured with H1 histone (PKC and PKC) or MBP (PKC) as the substrate without any PKC activators such as PS, DO, or C₂-ceramide.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Expression of PKC isozymes in HeLa cells. The expression of endogenous PKC subtypes in HeLa cells was examined by immunoblotting using subtype-specific antibodies. In HeLa cell lysates, each antibody against PKC, PKC, or PKC detected an immunoreactive band of reasonable molecular weight, while  and  subtypes of PKC were not detected, indicating that the , , and  subtypes of PKC are expressed in HeLa cells (Fig. 1). In this study, therefore, we focused on the localization of PKC, PKC, and PKC but not of PKC or PKC.

View larger version (17K): [in this window] [in a new window]   FIG. 1.   Immunoblotting analysis of endogenous PKC subtypes in HeLa cells. Total cell lysates (25 µg) extracted from HeLa cells (lanes 1) were separated by sodium dodecyl sulfate-7.5% polyacrylamide gel electrophoresis, transferred onto nitrocellulose membranes, and stained with antibodies against each PKC subtype. Rat brain homogenate was used as a positive control for PKC, PKC, PKC, and PKC, and recombinant PKC expressed in CHO-K1 cells was used as a positive control for PKC (lanes 2). The , , and  subtypes of PKC were detected with reasonable molecular masses in HeLa cells. The results shown are representative of two independent experiments.

Translocation of PKC, PKC, and PKC induced by C₂-ceramide and phorbol ester. When PKC- and PKC-GFP were expressed in HeLa cells, intense and homogeneous fluorescence of PKC- and PKC-GFP was observed throughout the cytoplasm, with no signals detected in the nucleus (Fig. 2A and C). In contrast, faint but significant fluorescence of PKC-GFP was seen in the nucleus in addition to intense fluorescence in the cytoplasm, and occasionally, PKC-GFP was found more densely in the perinuclear region than in the surrounding cytoplasm (Fig. 2A and C). Localization of the fluorescence did not change for at least 60 min when observed under a confocal laser scanning fluorescence microscope without stimulation.

View larger version (50K): [in this window] [in a new window]   FIG. 2.   Ceramide- or TPA-induced translocation of PKC subtypes in HeLa cells. (A) C2-ceramide (C2-Cer)-induced translocation of PKC-, PKC-, and PKC-GFP overexpressed in HeLa cells. PKC-, PKC-, and PKC-GFP were seen throughout the cytoplasm of HeLa cells, and faint signals for PKC-GFP were also seen in the nucleus. The addition of 10 µM C2-ceramide induced translocation of PKC-GFP but not of PKC- or PKC-GFP from the cytoplasm to the perinuclear region. (B) Immunocytochemical localization of endogenous PKC, PKC, and PKC before and after C2-ceramide treatment in HeLa cells. Endogenous PKC, PKC, and PKC were visualized by immunostaining with anti-PKC, PKC, or PKC antibodies and Cy3-labeled secondary antibodies. The addition of 10 µM C2-ceramide induced the accumulation of endogenous PKC but not of PKC or PKC to the perinuclear region. (C) TPA-induced translocation of PKC-, PKC-, and PKC-GFP overexpressed in HeLa cells. TPA at 1 µM induced translocation of PKC-GFP from the cytoplasm to the plasma membrane. TPA at 1 µM induced translocation of PKC-GFP from the cytoplasm and nucleoplasm to the plasma membrane and nuclear membrane, respectively. Application of 1 µM TPA failed to induce translocation of PKC-GFP. The results shown are representative of three independent experiments. Bars, 10 µm.

View larger version (49K): [in this window] [in a new window]   FIG. 3.   Characterization of the ceramide-induced translocation of PKC-GFP. (A) C2-dihydroceramide (DH-C2-Cer) (10 µM), an inactive ceramide, failed to induce translocation of PKC-GFP. (B) Preincubation with D609 (200 µg/ml) for 30 min did not affect the C2-ceramide (C2-Cer)-induced translocation of PKC-GFP. (C) After translocation of PKC-GFP to the perinuclear region by treatment with 10 µM C2-ceramide for 20 min, 1 µM TPA irreversibly induced translocation of all the PKC-GFP to the plasma membrane within 3 min. The results shown are representative of three independent experiments. Bars, 10 µm.

View larger version (37K): [in this window] [in a new window]   FIG. 4.   Ceramide-induced translocation of PKC-GFP assessed by immunoblotting analysis. With immunoblotting analysis, PKC-GFP was detected as a 110-kDa band that was more abundant in the cytosolic fraction (c). C2-ceramide treatment (10 µM, 20 min) induced translocation of PKC-GFP from the cytosolic fraction to the particulate fraction (p). No degradation products were detected before or after treatment with C2-ceramide (left panel). The level of PKC-GFP in the total homogenate was not changed after ceramide treatment for 20 min (right panel). The results shown are representative of three independent experiments.

Intracellular movement of ceramide. To compare the time course of PKC-GFP translocation with permeation of ceramide into cells, we monitored the movement of ceramide in HeLa cells using a fluorescent analogue of ceramide, C₆-NBD-ceramide. After application of 10 µM C₆-NBD-ceramide to HeLa cells, the fluorescence of C₆-NBD-ceramide was first detected on the plasma membrane at 1 min, and weak signals were in the perinuclear region, and then the intensity of the fluorescence on the plasma membrane gradually increased until 10 min. Obvious accumulation of the fluorescence was seen in the perinuclear region at 3 min, and the intensity of fluorescence was markedly increased at 10 min (Fig. 5A, top row). The C₆-NBD-ceramide accumulated at the perinuclear region was not altered by TPA treatment (1 µM, 15 min) (data not shown). C₆-ceramide also induced the translocation of PKC-GFP similarly to the effect of C₂-ceramide. The accumulation of PKC-GFP was first seen at 1 min and was apparent 3 min after treatment with C₆-ceramide, and the intensity of GFP fluorescence in the perinuclear region increased until 10 min (Fig. 5A, bottom row) and reached a maximum at 20 min (data not shown).

View larger version (58K): [in this window] [in a new window]   FIG. 5.   Localization of fluorescent ceramide (C6-NBD-ceramide) in HeLa cells. (A) C6-NBD-ceramide (NBD-C6-Cer) at 10 µM rapidly accumulated on the plasma membrane and perinuclear region of HeLa cells 1 min after the treatment, and the fluorescence in the perinuclear region increased significantly until 10 min (top row). C6-ceramide (C6-Cer)-induced translocation of PKC-GFP showed a similar time course to that of C6-NBD-ceramide (bottom row). (B) HeLa cells transfected with PKC were fixed after treatment with 10 µM C6-NBD-ceramide for 20 min. Cells were immunostained with anti-PKC monoclonal antibody and with Cy3-labeled IgG as secondary antibody to make the expressed PKC visible. The localization of C6-NBD-ceramide (NBD) is shown in green (left) and of PKC is shown in red (center). On the merged image, the overlapped signals of C6-NBD-ceramide and Cy3 appear in yellow (right). The results shown are representative of three independent experiments. Bars, 10 µm (A, top row) and 20 µm (A, bottom row, and B).

Translocation of PKC-GFP induced by IFN-. The effect of IFN-, which hydrolyzes sphingomyelin to generate ceramide, on the translocation of PKC-GFP was investigated in HeLa cells, since these cells are known to express IFN- receptors (33). IFN- at 100 U/ml induced significant PKC-GFP translocation from the cytoplasm to the perinuclear region within 5 min, and the intensity of fluorescence increased in the perinuclear region until 30 min (Fig. 6A, top row). We examined the influence of serum deprivation on the translocation of PKC-GFP induced by IFN-. When the culture medium was replaced with serum-free medium, IFN- induced the same translocation of PKC-GFP as seen in the presence of FBS (Fig. 6A, bottom row). Serum deprivation did not alter the localization of PKC-GFP until at least 60 min after treatment (data not shown). The application of 100 U of IFN- per ml failed to induce PKC- or PKC-GFP translocation for at least 60 min (Fig. 6B and C).

View larger version (55K): [in this window] [in a new window]   FIG. 6.   Effects of IFN- on PKC subtype translocation in HeLa cells. (A) Treatment with IFN- (100 U/ml) induced translocation of PKC-GFP from the cytoplasm to the perinuclear region within 5 min after treatment of HeLa cells in culture medium containing FBS (top row). The translocation of PKC-GFP was not altered by eliminating FBS from the culture medium (bottom row). (B) IFN- (100 U/ml) did not affect the localization of PKC-GFP expressed in HeLa cells. (C) IFN- (100 U/ml) did not affect the localization of PKC-GFP expressed in HeLa cells. The results shown are representative of three independent experiments. Bars, 10 µm.

View larger version (54K): [in this window] [in a new window]   FIG. 7.   Effects of sphingomyelinase inhibitors on IFN--induced translocation of PKC-GFP. (A) Treatment with Mg2+-free HEPES [Mg() HEPES] buffer containing 0.5 mM EDTA for 30 min blocked the IFN- (100 U/ml)-induced translocation of PKC-GFP (top row). IFN- induced translocation of PKC-GFP in normal HEPES buffer containing 1 mM Mg2+ (bottom row). (B) IFN--induced translocation of PKC-GFP was inhibited by pretreatment with 50 µM scyphostatin (Scypho.) for 15 min. However, scyphostatin did not inhibit the 10 µM C2-ceramide (C2-Cer)-induced translocation of PKC-GFP (top row). GSH treatment (5 mM, 30 min) also abolished IFN-- but not C2-ceramide-induced translocation of PKC-GFP (bottom row). The results shown are representative of three independent experiments. Bars, 10 µm.

View larger version (56K): [in this window] [in a new window]   FIG. 8.   Effects of tyrosine kinase inhibitors on IFN--induced translocation of PKC-GFP. IFN--induced translocation of PKC-GFP was inhibited by pretreatment with 100 µM genistein for 30 min. However, genistein did not alter 10-µM ceramide (C2-Cer)-induced translocation of PKC-GFP (top row). Similarly, preincubation with tyrphostin AG490 (100 µM) for 30 min abolished IFN--but not C2-ceramide-induced translocation of PKC-GFP (bottom row). The results shown are representative of three independent experiments. Bars, 10 µm.

Target site of PKC-GFP in response to ceramide. To identify the intracellular compartment where PKC-GFP accumulated in response to C₂-ceramide, the Golgi complex was visualized with Texas red-conjugated wheat germ agglutinin in HeLa cells expressing PKC-GFP after ceramide treatment. As shown in Fig. 9, intense GFP fluorescence was present in the perinuclear region in addition to moderate fluorescence throughout the cytoplasm. Texas red fluorescence accumulated in the perinuclear region and was also seen on the nuclear membrane. Merged images showed that the fluorescence of GFP and that of Texas red were colocalized in the perinuclear region, indicating that PKC-GFP is targeted to the Golgi complex in response to ceramide. Colocalization of PKC-GFP and Texas red-conjugated wheat germ agglutinin was also seen after stimulation with C₆-ceramide or IFN- (data not shown).

View larger version (21K): [in this window] [in a new window]   FIG. 9.   Colocalization of PKC-GFP and wheat germ agglutinin binding sites in PKC-GFP-expressing HeLa cells treated with ceramide. HeLa cells transfected with PKC-GFP were fixed after treatment with 10 µM C2-ceramide for 20 min. Cells were treated with Texas red-conjugated wheat germ agglutinin (WGA) to make the Golgi complex visible. The localization of PKC-GFP is shown in green (left). The Golgi complex is shown in red (center). On the merged image, overlapping GFP and Texas red signals appear yellow (right). The results are representative of four independent experiments. Bar, 10 µm.

FRAP of PKC-GFP translocated by ceramide. We investigated the interaction of PKC-GFP with the Golgi complex by fluorescence recovery after photobleaching (FRAP). We measured the fluorescence recovery of the PKC-GFP in the bleached area and also the fluorescence fading in the unbleached area after photobleaching with an argon laser at 488 nm. As shown in Fig. 10, after treatment with 10 µM C₂-ceramide for 30 min, photobleaching of a circular area in the Golgi complex abolished the fluorescence of PKC-GFP in the circle. The GFP fluorescence in the circle recovered within 40 s to a level similar to that in the unbleached Golgi complex. The recovery of fluorescence was significantly faster than the translocation of PKC-GFP induced by ceramide (Fig. 5A). In contrast, the fluorescence in the unbleached perikarya faded gradually. Photobleaching was also applied to a square area in perikarya (Fig. 10). GFP fluorescence of the bleached area (perikarya) rapidly recovered (within 30 s), and the fluorescence in the Golgi complex rapidly faded.

View larger version (19K): [in this window] [in a new window]   FIG. 10.   FRAP of PKC-GFP after translocation induced by ceramide. (A) Fluorescence recovery of PKC-GFP after photobleaching of the Golgi complex (a to c) or of the cytoplasm (d to f). The images were obtained before (a and d) and 0 s (b and e), 132 s (c), or 38 s (f) after photobleaching. The bleached areas are shown in red circles (a to c) and orange squares (d to f). The blue squares (a to c) and green circles (d to f) show the areas where fluorescence fading was measured. (B and C) Measurement of fluorescence recovery of PKC-GFP after photobleaching of the Golgi complex (B) or of the cytoplasm (C). Time-dependent recovery (red circle and orange square) of fluorescence in the bleached areas and fading (blue square and green circle) of the fluorescence in the unbleached areas are shown as percentages of the fluorescence before bleaching. Arrowheads (a to f) indicate the time points of the pictures in panel A. The results shown are representative of three independent experiments.

Changes in kinase activity of PKC by C₂-ceramide, in vitro and in vivo. The effects of C₂-ceramide on the kinase activity of PKC-GFP were examined by an in vitro kinase assay. As shown in Fig. 11A, C₂-ceramide at 10 µM failed to activate PKC-GFP in vitro. In the presence of PS and DO, the kinase activity of PKC-GFP was increased 2.9-fold, and C₂-ceramide inhibited the activation of PKC-GFP by PS and DO. The activity of PKC-GFP in the presence of the cofactors was dose-dependently inhibited by C₂-ceramide, and the maximal level was seen at 10 µM (31% inhibition).

View larger version (26K): [in this window] [in a new window]   FIG. 11.   Effects of ceramide on kinase activity of PKC subspecies, in vitro and in vivo. (A) Effects of C2-ceramide on kinase activity of PKC-GFP assessed by in vitro kinase assay. Kinase activities of the immunoprecipitated PKC-GFP were measured in the presence of various concentrations of C2-ceramide (C2-Cer) or activators of PKC such as PS and DO. Data are expressed as percentages of the control level. Statistical significance: *, P < 0.05 versus kinase activity of PS and DO. (B) Changes in kinase activity of PKC-GFP in HeLa cells after C2-ceramide treatment assessed by in vivo kinase assay. PKC-GFP was immunoprecipitated from HeLa cells overexpressing PKC-GFP at various time points after ceramide treatment. The kinase activity of PKC-GFP was assayed with H1 histone as the substrate without any activators such as PS or DO. Data are expressed as percentages of the control level (the kinase activity before stimulation). (C) Effects of C2-ceramide on kinase activity of endogenous PKC subtypes assessed by in vivo kinase assay. Endogenous PKC, PKC, and PKC were immunoprecipitated from HeLa cells before and after C2-ceramide (C2-Cer) treatment. The kinase activity of PKC, PKC, and PKC was assayed with H1 histone (PKC and PKC) or MBP (PKC) as the substrate without any activators such as PS or DO. Data are expressed as percentages of the control level (the kinase activity of each PKC subtype immunoprecipitated from untreated cells). Statistical significance: *, P < 0.01 versus kinase activity of control. All results represent the means and standard errors of more than three determinations.

Tyrosine phosphorylation of PKC-GFP after treatment with C₂-ceramide in HeLa cells. It has been reported that PKC is activated by tyrosine phosphorylation after various stimulations (8, 9, 17, 27, 30, 31, 60). To elucidate whether or not the ceramide-induced activation of PKC-GFP is through tyrosine phosphorylation, we investigated the effect of tyrosine kinase inhibitor on the C₂-ceramide-induced activation of PKC-GFP. Treatment with 10 µM C₂-ceramide induced significant activation of PKC-GFP, while treatment with 10 µM C₂-dihydroceramide for 20 min did not induce significant activation of PKC-GFP (Fig. 12A). The activation of PKC-GFP by C₂-ceramide was abolished by the pretreatment with 200 µM genistein, a nonspecific tyrosine kinase inhibitor (Fig. 12A). To determine whether or not PKC-GFP was tyrosine phosphorylated after the treatment with C₂-ceramide, tyrosine phosphorylation of PKC-GFP was analyzed by immunoblotting using an anti-phosphotyrosine antibody. Although C₂-dihydroceramide did not cause any tyrosine phosphorylation of PKC-GFP, PKC-GFP was significantly tyrosine phosphorylated by treatment with C₂-ceramide (Fig. 12B). In addition, pretreatment with 200 µM genistein effectively blocked the tyrosine phosphorylation of PKC-GFP induced by C₂-ceramide. Immunoblotting with the anti-GFP antibody revealed that similar amounts of PKC-GFP were immunoprecipitated in all samples.

View larger version (21K): [in this window] [in a new window]   FIG. 12.   Effects of a tyrosine kinase inhibitor on kinase activity and tyrosine phosphorylation of PKC-GFP after C2-ceramide treatment. (A) Effect of genistein on the ceramide-induced activation of PKC-GFP assessed by in vivo kinase assay. After pretreatment with genistein (200 µM), the PKC-GFP was immunoprecipitated from ceramide (C2-Cer)- or C2-dihydroceramide (DH-C2-Cer)-treated cells. The kinase activities of the immunoprecipitated PKC-GFP were assayed with H1 histone as the substrate without any activators such as PS or DO. Data are expressed as percentages of the control level (the kinase activity of PKC-GFP from untreated cells). Statistical significance: *, P < 0.01 versus kinase activity of control. (B) Effect of genistein on tyrosine phosphorylation of PKC-GFP in transfected HeLa cells treated with C2-ceramide. The PKC-GFP was prepared as described for panel A, and tyrosine phosphorylation of PKC-GFP was analyzed by immunoblotting using anti-phosphotyrosine (anti-p-Tyr) antibody (top row). Immunoblotting of the same membrane was performed using anti-GFP polyclonal antibody as described in Materials and Methods (bottom row). All results represent the means and standard errors of more than three determinations.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

We have studied the targeting mechanism of PKC subtypes in living cells using GFP fusion proteins to elucidate the individual function of each PKC subtype. In CHO-K1 cells overexpressing PKC-, PKC-, and PKC-GFP, spatial and temporal targeting varied depending on PKC subtype and extracellular stimulus (49, 56, 59). We further demonstrated that PKC translocation to the specific intracellular compartment (PKC targeting) is necessary for the recognition and phosphorylation of the substrates in the compartment (48). This subtype-and stimulus-specific targeting strongly suggested that the targeting mechanisms of PKC subtypes determine their individual roles in cell signaling pathways. In the present study, we examined the effects of ceramide on PKC translocation to clarify how ceramide is involved in various cell responses, especially in PKC-mediated signaling pathways.

Since HeLa cells express IFN- receptors coupled to the sphingomyelin-ceramide pathway, we used HeLa cells that endogenously express PKC, PKC, and PKC for the present study. Among these three endogenously expressed PKC subtypes, only PKC was translocated to the Golgi complex by a permeable ceramide analogue. Immunoblotting analysis indicated that ceramide induced translocation of PKC from the cytosol to the particulate fraction, suggesting that PKC was associated with the Golgi membrane after ceramide treatment. Immunocytochemical studies also revealed that ceramide translocated endogenous PKC but not PKC or PKC to the Golgi complex. The results of the present study in living cells suggested that only PKC, at least in HeLa cells, is responsible for the ceramide-induced cellular responses, although it is possible that other PKC subtypes expressed at undetectable levels in HeLa cells are also involved in the responses or that ceramide acts on PKC without translocation of PKC to specific subcellular compartments. Shirai et al. demonstrated that among PKC, PKC, and PKC, only PKC was insensitive to various fatty acids, including arachidonic acid, which induced translocation of PKC to the Golgi complex (59). Ceramide also translocated PKC but not PKC to the Golgi complex in the present study (data not shown). Since ceramide translocates both PKC and PKC to the Golgi complex and arachidonic acid translocates only PKC but not PKC, it is suggested that arachidonic acid-induced translocation of PKC to the Golgi complex may occur by a mechanism different from that involved in ceramide-induced translocation of PKC and PKC to the Golgi complex. Previous biochemical studies, however, showed that PKC and PKC have ceramide-binding abilities and that treatment with ceramide translocated PKC as well as PKC from the membrane to the cytosol fraction (57). It was also reported that ceramide induced translocation of PKC from the cytosol to the membrane fraction (22) and that PKC was translocated to the perinuclear region by ceramide (16). In the present study using living HeLa cells, neither PKC nor PKC responded to ceramide 60 min after treatment. The precise reason for this discrepancy is not clear, but it may have been due to differences in the cell types or experimental conditions used.

Many studies have shown that ceramide is produced through sphingomyelin hydrolysis after exposure to various extracellular stimuli, including IFN- (25) and TNF- (11, 25). As shown in Fig. 6, physiological receptor stimulation by IFN- evoked translocation of only PKC, but not PKC or PKC, from the cytoplasm to the Golgi complex as seen when treated with ceramide. Since Mg²⁺-dependent neutral sphingomyelinase inhibitors such as scyphostatin and GSH inhibited IFN-- but not ceramide-induced translocation of PKC, it is likely that the translocation of PKC occurred downstream of the Mg²⁺-dependent neutral sphingomyelinase pathway. Furthermore, the chelation of extracellular Mg²⁺ completely blocked the translocation of PKC, demonstrating that the Mg²⁺-dependent neutral sphingomyelinase is activated outside the plasma membrane. Since D609, an inhibitor of SMS, did not alter the ceramide-induced translocation of PKC or that induced by IFN-, ceramide generated by hydrolysis of sphingomyelin, but not sphingomyelin generated from ceramide, induced PKC translocation. Although serum deprivation has been reported to induce sphingomyelin hydrolysis and generation of ceramide within 10 h after treatment (24), the serum deprivation did not alter localization of PKC, at least within the 60-min observation period in the present study. Furthermore, because IFN- induced translocation both in the presence and absence of serum, translocation of PKC did not occur through an unknown effect of serum. The TNF- receptor is also known to be expressed in HeLa cells (13, 38), and TNF- also induced similar but slower translocation of PKC (data not shown). From the present findings that both AG490, a JAK2 inhibitor, and genistein, a tyrosine kinase inhibitor, completely blocked IFN--induced translocation of PKC, it is likely that Mg²⁺-dependent neutral sphingomyelinase is activated downstream of the IFN- receptor-JAK pathway and that ceramide is subsequently produced, leading to translocation of PKC to the Golgi complex, although the detailed pathway between JAK2 and Mg²⁺-dependent sphingomyelinase is currently unclear.

Ceramide is widely used as a marker for the Golgi complex, as ceramide accumulates in this organelle (32). As shown in Fig. 5, C₆-NBD-ceramide accumulated to the perinuclear region with a time course similar to that of C₆-ceramide-induced translocation of PKC, and finally, ceramide and PKC accumulated to the same compartment, the Golgi complex (Fig. 5B). This simultaneous translocation of PKC with ceramide to the Golgi complex suggested that the translocation of PKC was due to its association with ceramide accumulating in the Golgi complex. However, NBD-ceramide was transiently accumulated on the plasma membrane just after application, but ceramide treatment did not cause translocation of PKC to the plasma membrane. These observations suggested that ceramide may act on PKC only at the Golgi complex but not at the plasma membrane. Al