Differential nuclear localization of protein kinase C isoforms in neuroblastoma × glioma hybrid cells

Abstract

The protein kinase C (PKC) a, p and E isoforms have distinct nuclear localizations in neuroblastoma X glioma hybrid cells NG 108-15. We found by immunoblotting that PKC a, pII, 6 and E are the predominant isoforms in these cells. In contrast to other neuronal cell lines, none of these isoforms is down-regulated during differentiation.Confocal immunofluorescence microscopy revealed that in undifferentiated cells PKC a is located in the cytoplasm and in the nucleus excluding nucleoli. In differentiated cells PKC a was almost exclusively located in the cytoplasm. Stimulation of the cells with phorbol ester resulted in translocation to the plasma membrane. PKC PII was not detectable in the nuclei. PKC 6 was found in the nucleoli and in the cytoplasm, in differentiated cells particularly in the neurites. Phorbol ester failed to induce a translocation to other compartments. PKC E was localized with the nuclear-pore complexes at the nuclear envelope. In differentiated cells after stimulation with phorbol ester, partial translocation to the plasma membrane was observed.Protein kinase C (PKC) plays a key role in many signaltransduction pathways and cellular processes (for review see Nishizuka, 1986;Lester and Epand, 1992). Ten isoforms of PKC are found in mammals, showing characteristic patterns of expression in different tissues. Four isoforms (called PKC A forms), PKC a, PI, pII and 7, are phospholipid and Ca2+ dependent in contrast to the B forms such as PKC 6, E and cwhich are Ca2+ independent (Stabel and Parker, 1991). After an increase of intracellular Ca2+ levels the PKC A isoforms were shown to be translocated to the plasma membrane. Binding to the plasma membrane via phospholipids enables activation by the second messenger diacylglycerol (Huang, 1989).There is evidence that members of the PKC family respond differently to various combinations of Caz+ and phospholipid-degradation products. This could be one of the reasons for distinct intracellular distribution and activation patterns (Asaoka et al., 1992).Several investigators have observed translocation to other cell compartments including the cell nucleus in addition to the plasma membrane. With fibroblast cells, Leach et al. (1992) observed translocation of PKC a to the cell nucleus after treatment with phorbol ester or a-thrombin. In promyelocytic leukemia (HL 60) cells, stimulation with the PKC activator bryostatin leads to a selective translocation of PKC P but not PKC a to the nucleus (Hocevar and Fields, 1991).In liver, PKC BI1 was found in the nuclei (Rogue et al., 1990 found PKC a and PKC y as the 'membrane inserted' and constitutively active form.The current view is that each cell type expresses a specific set of PKC isoforms, which are localized and regulated in a cell-specific manner. It is not yet understood how this differential expression, localization, and regulation is achieved. The specific localizations of PKC isoforms imply specific functions for each cell type. Due to the known effects of PKC activation on nuclear events (induction of proli...

Ethanol Alters the Subcellular Localization of δ- and ε Protein Kinase C in NG108–15 Cells

“…Consistent with this possibility, ethanol-induced increases in extractable PKC activity were reported in NG108 -15 cells (9) and other cell types (11,12). However, in addition to ␦-and ⑀-PKC, other PKC isozymes are also present in these cells (8,19); they could account for the increases in PKC activity. To determine whether ␦-and ⑀-PKC are activated by ethanol, it will be necessary to develop antibodies to distinguish between the active and inactive states of specific PKC isozymes (20) .…”

“…Localization of PKC isozymes to specific sites and translo- cation on activation to new sites also occurs in NG108 -15 cells grown in 10% fetal calf serum (19). ␦-PKC was found to be localized to neurites, the nucleus, and cytoplasm and ⑀-PKC to the cytoplasm and the perinuclear area, specifically the nuclear envelope.…”

Bradykinin induces rise of free calcium in nuclei of neuroblastoma × glioma hybrid NG 108‐15 cells

“…PKC was also detected without stimulation in nuclei of different origins, e.g., liver and brain and references cited therein). We also observed PKC isozymes in nuclei of NG 108-I5 cells (Beckmann et al, 1994). Nuclear PKC may be activated by Ca2+ and DAG, and it is conceivable that repeated activation can lead to the occurrence of a permanently active, membrane-inserted form of PKC as it was shown to be present in brain nuclei .…”

The Role of Protein Kinase C in Cellular Tolerance to Ethanol

“…3A). Beckmann et al (28) did not detect ␤ 1 in NG108-15 cells. This may reflect the difference in culture medium in which the cells are grown.…”

“…Recent evidence suggests that the localization of intracellular signaling enzymes determines their specificity of action (29 -31). PKA (32), protein phosphatases 1 and 2A (30), and specific isozymes of PKC (33) have unique locations within a number of cell lines including NG108-15 cells (28). Ethanol has been shown to cause translocation of PKC activity from cytosolic to membrane fractions in astroglial cells (34), human lymphocytes (5), and epidermal keratinocytes (6).…”