Subtype-specific translocation of the delta subtype of protein kinase C and its activation by tyrosine phosphorylation induced by ceramide in HeLa cells - PubMed (original) (raw)

Subtype-specific translocation of the delta subtype of protein kinase C and its activation by tyrosine phosphorylation induced by ceramide in HeLa cells

T Kajimoto et al. Mol Cell Biol. 2001 Mar.

Abstract

We investigated the functional roles of ceramide, an intracellular lipid mediator, in cell signaling pathways by monitoring the intracellular movement of protein kinase C (PKC) subtypes fused to green fluorescent protein (GFP) in HeLa living cells. C(2)-ceramide but not C(2)-dihydroceramide induced translocation of delta PKC-GFP to the Golgi complex, while alpha PKC- and zeta PKC-GFP did not respond to ceramide. The Golgi-associated delta PKC-GFP induced by ceramide was further translocated to the plasma membrane by phorbol ester treatment. Ceramide itself accumulated to the Golgi complex where delta PKC was translocated by ceramide. Gamma interferon also induced the delta PKC-specific translocation from the cytoplasm to the Golgi complex via the activation of Janus kinase and Mg(2+)-dependent neutral sphingomyelinase. Photobleaching studies showed that ceramide does not evoke tight binding of delta PKC-GFP to the Golgi complex but induces the continuous association and dissociation of delta PKC with the Golgi complex. Ceramide inhibited the kinase activity of delta PKC-GFP in the presence of phosphatidylserine and diolein in vitro, while the kinase activity of delta PKC-GFP immunoprecipitated from ceramide-treated cells was increased. The immunoprecipitated delta PKC-GFP was tyrosine phosphorylated after ceramide treatment. Tyrosine kinase inhibitor abolished the ceramide-induced activation and tyrosine phosphorylation of delta PKC-GFP. These results suggested that gamma interferon stimulation followed by ceramide generation through Mg(2+)-dependent sphingomyelinase induced delta PKC-specific translocation to the Golgi complex and that translocation results in delta PKC activation through tyrosine phosphorylation of the enzyme.

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Figures

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 αPKC, δPKC, ɛPKC, and ζPKC, and recombinant ηPKC expressed in CHO-K1 cells was used as a positive control for ηPKC (lanes 2). The α, δ, and ζ subtypes of PKC were detected with reasonable molecular masses in HeLa cells. The results shown are representative of two independent experiments.

FIG. 2

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

FIG. 3

Characterization of the ceramide-induced translocation of δPKC-GFP. (A) C2-dihydroceramide (DH-C2-Cer) (10 μM), an inactive ceramide, failed to induce translocation of δPKC-GFP. (B) Preincubation with D609 (200 μg/ml) for 30 min did not affect the C2-ceramide (C2-Cer)-induced translocation of δPKC-GFP. (C) After translocation of δPKC-GFP to the perinuclear region by treatment with 10 μM C2-ceramide for 20 min, 1 μM TPA irreversibly induced translocation of all the δPKC-GFP to the plasma membrane within 3 min. The results shown are representative of three independent experiments. Bars, 10 μm.

FIG. 4

Ceramide-induced translocation of δPKC-GFP assessed by immunoblotting analysis. With immunoblotting analysis, δPKC-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 δPKC-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 δPKC-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.

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 δPKC-GFP showed a similar time course to that of C6-NBD-ceramide (bottom row). (B) HeLa cells transfected with δPKC were fixed after treatment with 10 μM C6-NBD-ceramide for 20 min. Cells were immunostained with anti-δPKC monoclonal antibody and with Cy3-labeled IgG as secondary antibody to make the expressed δPKC visible. The localization of C6-NBD-ceramide (NBD) is shown in green (left) and of δPKC 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).

FIG. 6

Effects of IFN-γ on PKC subtype translocation in HeLa cells. (A) Treatment with IFN-γ (100 U/ml) induced translocation of δPKC-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 δPKC-GFP was not altered by eliminating FBS from the culture medium (bottom row). (B) IFN-γ (100 U/ml) did not affect the localization of αPKC-GFP expressed in HeLa cells. (C) IFN-γ (100 U/ml) did not affect the localization of ζPKC-GFP expressed in HeLa cells. The results shown are representative of three independent experiments. Bars, 10 μm.

FIG. 7

Effects of sphingomyelinase inhibitors on IFN-γ-induced translocation of δPKC-GFP. (A) Treatment with Mg2+-free HEPES [Mg(−) HEPES] buffer containing 0.5 mM EDTA for 30 min blocked the IFN-γ (100 U/ml)-induced translocation of δPKC-GFP (top row). IFN-γ induced translocation of δPKC-GFP in normal HEPES buffer containing 1 mM Mg2+ (bottom row). (B) IFN-γ-induced translocation of δPKC-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 δPKC-GFP (top row). GSH treatment (5 mM, 30 min) also abolished IFN-γ- but not C2-ceramide-induced translocation of δPKC-GFP (bottom row). The results shown are representative of three independent experiments. Bars, 10 μm.

FIG. 8

Effects of tyrosine kinase inhibitors on IFN-γ-induced translocation of δPKC-GFP. IFN-γ-induced translocation of δPKC-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 δPKC-GFP (top row). Similarly, preincubation with tyrphostin AG490 (100 μM) for 30 min abolished IFN-γ-but not C2-ceramide-induced translocation of δPKC-GFP (bottom row). The results shown are representative of three independent experiments. Bars, 10 μm.

FIG. 9

Colocalization of δPKC-GFP and wheat germ agglutinin binding sites in δPKC-GFP-expressing HeLa cells treated with ceramide. HeLa cells transfected with δPKC-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 δPKC-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.

FIG. 10

FRAP of δPKC-GFP after translocation induced by ceramide. (A) Fluorescence recovery of δPKC-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 δPKC-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.

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 δPKC-GFP assessed by in vitro kinase assay. Kinase activities of the immunoprecipitated δPKC-GFP were measured in the presence of various concentrations of C2-ceramide (C2-Cer) or activators of δPKC 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 δPKC-GFP in HeLa cells after C2-ceramide treatment assessed by in vivo kinase assay. δPKC-GFP was immunoprecipitated from HeLa cells overexpressing δPKC-GFP at various time points after ceramide treatment. The kinase activity of δPKC-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 αPKC, δPKC, and ζPKC were immunoprecipitated from HeLa cells before and after C2-ceramide (C2-Cer) treatment. The kinase activity of αPKC, δPKC, and ζPKC was assayed with H1 histone (αPKC and δPKC) or MBP (ζPKC) 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.

FIG. 12

Effects of a tyrosine kinase inhibitor on kinase activity and tyrosine phosphorylation of δPKC-GFP after C2-ceramide treatment. (A) Effect of genistein on the ceramide-induced activation of δPKC-GFP assessed by in vivo kinase assay. After pretreatment with genistein (200 μM), the δPKC-GFP was immunoprecipitated from ceramide (C2-Cer)- or C2-dihydroceramide (DH-C2-Cer)-treated cells. The kinase activities of the immunoprecipitated δPKC-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 δPKC-GFP from untreated cells). Statistical significance: ∗, P < 0.01 versus kinase activity of control. (B) Effect of genistein on tyrosine phosphorylation of δPKC-GFP in transfected HeLa cells treated with C2-ceramide. The δPKC-GFP was prepared as described for panel A, and tyrosine phosphorylation of δPKC-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.

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Subtype-specific translocation of the delta subtype of protein kinase C and its activation by tyrosine phosphorylation induced by ceramide in HeLa cells - PubMed (original) (raw)