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. 2014 Apr 1:5:126.
doi: 10.3389/fphys.2014.00126. eCollection 2014.

Deregulation of the actin cytoskeleton and macropinocytosis in response to phorbol ester by the mutant protein kinase C gamma that causes spinocerebellar ataxia type 14

Kazuhiro Yamamoto  1 , Takahiro Seki  2 , Hikaru Yamamoto  3 , Naoko Adachi  4 , Shigeru Tanaka  1 , Izumi Hide  1 , Naoaki Saito  4 , Norio Sakai  1
Affiliations

Deregulation of the actin cytoskeleton and macropinocytosis in response to phorbol ester by the mutant protein kinase C gamma that causes spinocerebellar ataxia type 14

Kazuhiro Yamamoto et al. Front Physiol. .

Abstract

Several missense mutations in the protein kinase Cγ (γPKC) gene have been found to cause spinocerebellar ataxia type 14 (SCA14), an autosomal dominant neurodegenerative disease. γPKC is a neuron-specific member of the classical PKCs and is activated and translocated to subcellular regions as a result of various stimuli, including diacylglycerol synthesis, increased intracellular Ca(2+) and phorbol esters. We investigated whether SCA14 mutations affect the γPKC-related functions by stimulating HeLa cells with TPA (12-O-tetradecanoylpholbol 13-acetate), a type of phorbol ester. Wild-type (WT) γPKC-GFP was translocated to the plasma membrane within 10 min of TPA stimulation, followed by its perinuclear translocation and cell shrinkage, in a PKC kinase activity- and microtubule-dependent manner. On the other hand, although SCA14 mutant γPKC-GFP exhibited a similar translocation to the plasma membrane, the subsequent perinuclear translocation and cell shrinkage were significantly impaired in response to TPA. Translocated WT γPKC colocalized with F-actin and formed large vesicular structures in the perinuclear region. The uptake of FITC-dextran, a marker of macropinocytosis, was promoted by TPA stimulation in cells expressing WT γPKC, and FITC-dextran was surrounded by γPKC-positive vesicles. Moreover, TPA induced the phosphorylation of MARCKS, which is a membrane-substrate of PKC, resulting in the translocation of phosphorylated MARCKS to the perinuclear region, suggesting that TPA induces macropinocytosis via γPKC activation. However, TPA failed to activate macropinocytosis and trigger the translocation of phosphorylated MARCKS in cells expressing the SCA14 mutant γPKC. These findings suggest that γPKC is involved in the regulation of the actin cytoskeleton and macropinocytosis in HeLa cells, while SCA14 mutant γPKC fails to regulate these processes due to its reduced kinase activity at the plasma membrane. This property might be involved in pathogenesis of SCA14.

Keywords: MARCKS; actin cytoskeleton; macropinocytosis; spinocerebellar ataxia type 14; translocation; γPKC.

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Figures

Figure 1
Figure 1
γ PKC is translocated around the centrosome in microtubule- and PKC kinase activity-dependent manners. (A) Time-lapse imaging of γPKC-GFP expressing HeLa cells treated with TPA. γPKC-GFP expressing cells were treated with 100 nM TPA for 1 h. These images were obtained from the same cells at each time point (0, 15, 30, and 60 min) after TPA treatment. The translocations of γPKC-GFP to the plasma membrane (15 min) and to the perinuclear region (30 and 60 min) were observed, accompanied by a reduction in cell size (30 and 60 min). Bar = 10 μm. (B) γPKC translocation around the centrosome. Representative fluorescence images (γPKC-GFP, γ-tubulin, and merged) of γPKC-GFP-expressing HeLa cells treated with 100 nM TPA for 60 min, followed by immunostaining with an anti-γ-tubulin antibody, a marker of the centrosome. Translocated γPKC-GFP was localized around the centrosome. Bar = 10 μm. (C–E) γPKC translocation around the centrosome and cell shrinkage were dependent on microtubules and PKC kinase activity. (C) Representative fluorescence images of γPKC-GFP-expressing cells treated with TPA alone (left), TPA plus 1 μg/ml nocodazole, an inhibitor of tubulin polymerization (center) and TPA plus 2.5 μg/ml Gö6976, an inhibitor of cPKC (right). Nocodazole and Gö6976 were added 20 min prior to treatment with 100 nM TPA. The cells were fixed 30 min after TPA treatment. Nocodazole and Gö6976 prevented the TPA-induced perinuclear translocation of γPKC-GFP and cell shrinkage. Bar = 10 μm. (D) The percentage of γPKC-GFP-positive HeLa cells with perinuclear translocation after 30 min of TPA treatment, shown in (C). The data represent the mean ± standard error from four independent experiments. (E) The cell size of the GFP-positive HeLa cells after TPA treatment for 30 min, shown in (C). The data represent the mean ± standard error. The number of quantified cells in each treatment is indicated in the column. **p < 0.01, ***p < 0.001 (unpaired t-test).
Figure 2
Figure 2
SCA14 mutant γ PKC impairs the perinuclear translocation and cell size reduction in response to TPA treatment. (A) Representative fluorescence images of GFP (left), WT γPKC-GFP (center) or S119P γPKC-GFP (right)-expressing HeLa cells treated with TPA for 30 min. In response to TPA treatment for 30 min, WT γPKC-GFP exhibited membrane translocation, followed by perinuclear translocation and a reduction in cell size. In contrast, S119P mutant γPKC-GFP did not exhibit perinuclear translocation and remained at the plasma membrane. In cells expressing GFP alone, TPA did not induce the translocation of GFP or cell shrinkage. Scale bar = 10 μm. (B, C) Quantification of the TPA-induced perinuclear translocation of γPKC-GFP and cell shrinkage. (B) The percentage of γPKC-GFP-positive HeLa cells with perinuclear translocation, shown in (A). WT and mutant (S119F, S119P, and G128D) γPKC-GFP were expressed in HeLa cells and were stimulated with TPA for 30 min. After fixation, the percentage of cells with perinuclear translocation was evaluated. The data represent the mean ± standard error from three independent experiments. (C) The cell size of the GFP-positive HeLa cells after TPA treatment, shown in (A). GFP, WT, and mutant γPKC-GFP were expressed in HeLa cells and were stimulated with TPA for 30 min. After fixation, the cell size was measured from the GFP fluorescence images. The data represent the mean ± standard error. The number of quantified cells in each treatment is indicated in the column. **p < 0.01, ***p < 0.001 (unpaired t-test).
Figure 3
Figure 3
γ PKC, but not SCA14 mutant γ PKC, mediates the TPA-induced remodeling of the actin cytoskeleton and the activation of macropinocytosis. (A) Representative images (DIC, γPKC-GFP, F-actin, and merged) of γPKC-GFP-expressing HeLa cells treated with TPA for 30 min. The lower panels show high magnification images of the boxed areas in the merged images in the upper panels. F-actin was visualized by staining with 100 nM phalloidin-TRITC. Perinuclear γPKC-GFP was colocalized with F-actin around many large vesicles (> 0.2 μm in a diameter). The scale bars in the upper and lower panels are 10 and 2 μm, respectively. (B) FITC-dextran uptake in γPKC-HT-expressing cells in response to TPA treatment. FITC-dextran, a fluorescent marker for macropinocytosis, was simultaneously incubated with cells expressing γPKC-HT for 30 min during TPA treatment. Representative images (γPKC-HT, FITC-dextran and merged) are shown. The far right image shows the high magnification image of the boxed area in the merged image. FITC-dextran was surrounded by γPKC-HT. The scale bars in the merged and high magnification images are 5 and 2 μm, respectively. (C) FITC-dextran uptake in WT and mutant (S119P and G128D) γPKC-HT-expressing cells in response to TPA treatment. Cells expressing γPKC-HT were simultaneously incubated for 30 min with FITC-dextran and TPA. Representative images (γPKC-HT and FITC-dextran) are shown. TPA enhanced the uptake of FITC-dextran in cells expressing WT γPKC-HT, but not in cells expressing mutant γPKC-HT. Scale bar = 5 μm.
Figure 4
Figure 4
γ PKC, but not SCA14 mutant γ PKC, mediates the TPA-induced perinuclear translocation of phosphorylated MARCKS. (A) Perinuclear translocation of phosphorylated MARCKS (p-MARCKS) 30 min after TPA treatment. Representative images (γPKC-GFP, p-MARCKS and merged) are shown. Cells expressing γPKC-GFP were treated with TPA for 30 min, followed by immunostaining with an anti-p-MARCKS antibody. The far right image shows the high magnification image of the boxed area in the merged image. p-MARCKS was strongly colocalized with the perinuclear γPKC-GFP, especially around the vesicles. The scale bars of the merged and high magnification images are 5 and 2 μm, respectively. (B) Localization of p-MARCKS in cells expressing GFP alone, WT and S119P γPKC-GFP 30 min after TPA treatment. Representative images (p-MARCKS and GFP) are shown. As described above, p-MARCKS was colocalized with WT γPKC-GFP in the perinuclear region. In contrast, obvious p-MARCKS immunostaining was not observed in cells expressing GFP alone or mutant γPKC-GFP. Scale bar = 10 μm.
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