6533b82cfe1ef96bd128f68f

RESEARCH PRODUCT

Expression of protein kinase C gene family members is temporally and spatially regulated during neural development in vitro.

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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.