Department of Molecular and Pharmacological Neuroscience, Graduate School of Biomedical and Health Sciences, Hiroshima University Hiroshima, Japan.
Department of Molecular and Pharmacological Neuroscience, Graduate School of Biomedical and Health Sciences, Hiroshima University Hiroshima, Japan ; Department of Chemico-Pharmacological Sciences, Graduate School of Pharmaceutical Sciences, Kumamoto University Kumamoto, Japan.
Front Physiol. 2014 Apr 1;5:126. doi: 10.3389/fphys.2014.00126. eCollection 2014.
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.
几种蛋白激酶 Cγ（γPKC）基因突变已被发现可导致脊髓小脑共济失调 14 型（SCA14），这是一种常染色体显性神经退行性疾病。γPKC 是经典 PKC 中的神经元特异性成员，并且由于各种刺激而被激活并转位到亚细胞区域，包括二酰基甘油合成、细胞内 Ca（2+）增加和佛波醇酯。我们通过用 TPA（12-O-十四烷酰佛波醇 13-乙酸酯）刺激 HeLa 细胞来研究 SCA14 突变是否影响与 γPKC 相关的功能，TPA 是一种佛波醇酯。野生型（WT）γPKC-GFP 在 TPA 刺激后 10 分钟内转位到质膜，随后发生核周转位和细胞收缩，这是 PKC 激酶活性和微管依赖性的。另一方面，尽管 SCA14 突变体 γPKC-GFP 表现出类似的质膜转位，但随后的核周转位和细胞收缩对 TPA 的反应明显受损。转位的 WT γPKC 与 F-肌动蛋白共定位，并在核周区域形成大的囊泡结构。在表达 WT γPKC 的细胞中，FITC-葡聚糖（一种巨胞饮作用的标志物）的摄取在 TPA 刺激下得到促进，并且 FITC-葡聚糖被 γPKC 阳性囊泡包围。此外，TPA 诱导 MARCKS 的磷酸化，MARCKS 是 PKC 的膜底物，导致磷酸化的 MARCKS 转位到核周区域，表明 TPA 通过 γPKC 激活诱导巨胞饮作用。然而，TPA 未能激活巨胞饮作用并触发表达 SCA14 突变体 γPKC 的细胞中磷酸化 MARCKS 的转位。这些发现表明，γPKC 参与调节 HeLa 细胞中的肌动蛋白细胞骨架和巨胞饮作用，而 SCA14 突变体 γPKC 由于其在质膜上的激酶活性降低而无法调节这些过程。这种特性可能与 SCA14 的发病机制有关。
