Oehrlein S A, Maelicke A, Herget T
Johannes Gutenberg-University, Laboratory of Molecular Neurobiology, Institute of Physiological Chemistry and Pathobiochemistry, Mainz, Germany.
Eur J Cell Biol. 1998 Dec;77(4):323-37. doi: 10.1016/S0171-9335(98)80091-5.
We used primary cultures of rat hippocampal neurons and PCC7-Mz1 cells to correlate the expression of the protein kinase C (PKC) gene family with specific events during neural differentiation. Multipotent PCC7-Mz1 embryonic carcinoma stem cells develop into a tissue-like pattern of neuronal, fibroblast-like and astroglial cells by all-trans retinoic acid (RA) treatment. Western blot analyses demonstrate that PKCalpha, betaI, gamma, theta, mu, lambda, and zeta were constitutively expressed but the expression of PKCbetaII, delta, epsilon, and eta was up-regulated three days after addition of RA when cells mature morphologically. While the protein levels of the PKC isoforms betaII, delta and eta decreased after d6, when the major phenotypical alterations of the developing neurons were completed, PKCepsilon expression remained at a high level. Immunofluorescence studies demonstrated that PKCalpha, lambda and zeta were constantly expressed in stem cells and the arising cell types. PKCdelta was detected in all differentiated cell types, whereby PKCbetaII, gamma, epsilon, and zeta were solely found in the neuronal derivatives with PKCgamma predominantly located in the nuclei. PKCeta was weakly expressed at the Golgi complex of stem cells but expanded throughout the entire somata of all developing neurons. In contrast, PKCbetaII was abundant only in the somata of a minor fraction of all neurons (approximately 2.5%). Also, PKCepsilon was exclusively synthesized by a subpopulation of neurons (40+/-5%), where it was localized in the somata and in the axons. PKCzeta was persistently expressed in two forms, the full-length PKCzeta and the constitutively active, proteolytic product PKMzeta, reasoning that permanent PKCzeta activity is important for PCC7-Mz1 physiology. Fractionation of extracts from undifferentiated and differentiating PCC7-Mz1 cells revealed that the conventional cPKCalpha was partly and the cPKCbetaI and the novel nPKCs delta and epsilon were mainly membrane bound, implying that they were also in an active state. However, when using the PKC substrate MARCKS (myristoylated alanine-rich C kinase substrate) to monitor cellular PKC activity, we observed that activation of PKC by phorbol ester was required for complete MARCKS phosphorylation and its translocation from the membrane to the cytoplasm. Our data show that the cell type-specific expression, subcellular localization and activation of PKCs are regulated in an isoform-specific manner during neurogenesis suggesting that they are involved in the control of neural development and in particular in neuronal differentiation.
我们使用大鼠海马神经元原代培养物和PCC7-Mz1细胞，将蛋白激酶C（PKC）基因家族的表达与神经分化过程中的特定事件相关联。多能性PCC7-Mz1胚胎癌干细胞通过全反式视黄酸（RA）处理发育成神经元、成纤维细胞样和星形胶质细胞的组织样模式。蛋白质印迹分析表明，PKCalpha、betaI、gamma、theta、mu、lambda和zeta组成性表达，但在添加RA三天后，当细胞形态成熟时，PKCbetaII、delta、epsilon和eta的表达上调。虽然在第6天发育中的神经元主要表型改变完成后，PKC同工型betaII、delta和eta的蛋白质水平下降，但PKCepsilon表达仍维持在高水平。免疫荧光研究表明，PKCalpha、lambda和zeta在干细胞及衍生细胞类型中持续表达。在所有分化细胞类型中均检测到PKCdelta，而PKCbetaII、gamma、epsilon和zeta仅在神经元衍生物中发现，其中PKCgamma主要位于细胞核中。PKCeta在干细胞的高尔基体中弱表达，但在所有发育中的神经元的整个胞体中广泛分布。相比之下，PKCbetaII仅在所有神经元的一小部分（约2.5%）的胞体中大量存在。此外，PKCepsilon仅由一部分神经元（40±5%）合成，其定位于胞体和轴突中。PKCzeta以两种形式持续表达，即全长PKCzeta和组成性活性蛋白水解产物PKMzeta，这表明PKCzeta的持续活性对PCC7-Mz1细胞生理功能很重要。对未分化和正在分化的PCC7-Mz1细胞提取物进行分级分离显示，传统的cPKCalpha部分与膜结合，cPKCbetaI以及新型nPKCs delta和epsilon主要与膜结合，这意味着它们也处于活性状态。然而，当使用PKC底物MARCKS（肉豆蔻酰化富含丙氨酸的C激酶底物）监测细胞PKC活性时，我们观察到佛波酯激活PKC是MARCKS完全磷酸化及其从膜向细胞质转位所必需的。我们的数据表明，在神经发生过程中，PKC的细胞类型特异性表达、亚细胞定位和激活以同工型特异性方式受到调节，这表明它们参与神经发育的控制，特别是神经元分化。
