Increase in nuclear phosphatidylinositol 3-kinase activity and phosphatidylinositol (3,4,5) trisphosphate synthesis precede PKC-zeta translocation to the nucleus of NGF-treated PC12 cells.

We and others have previously demonstrated the existence of an autonomous nuclear polyphosphoinositide cycle that generates second messengers such as diacylglycerol (DAG), capable of attracting to the nucleus specific protein kinase C (PKC) isoforms (Neri et al. (1998) J. Biol. Chem. 273, 29738-29744). Recently, however, nuclei have also been shown to contain the enzymes responsible for the synthesis of the non-canonical 3-phosphorylated inositides. To clarify a possible role of this peculiar class of inositol lipids we have examined the question of whether nerve growth factor (NGF) induces PKC-zeta nuclear translocation in PC12 cells and whether this translocation is dependent on nuclear phosphatidylinositol 3-kinase (PI 3-K) activity and its product, phosphatidylinositol 3,4, 5-trisphosphate [PtdIns(3,4,5)P(3)]. NGF increased both the amount and the enzyme activity of immunoprecipitable PI 3-K in PC12 cell nuclei. Activation of the enzyme, but not its translocation, was blocked by PI 3-K inhibitors wortmannin and LY294002. Treatment of PC12 cells for 9 min with NGF led to an increase in the nuclear levels of PtdIns(3,4,5)P(3). Maximal translocation of PKC-zeta from the cytoplasm to the nucleus (as evaluated by immunoblotting, enzyme activity, and confocal microscopy) occurred after 12 min of exposure to NGF and was completely abrogated by either wortmannin or LY294002. In contrast, these two inhibitors did not block nuclear translocation of the conventional, DAG-sensitive, PKC-alpha. On the other hand, the specific phosphatidylinositol phospholipase C inhibitor, 1-O-octadeyl-2-O-methyl-sn-glycero-3-phosphocholine, was unable to abrogate nuclear translocation of the DAG-insensitive PKC-zeta. These data suggest that a nuclear increase in PI 3-K activity and PtdIns(3,4,5)P(3) production are necessary for the subsequent nuclear translocation of PKC-zeta. Furthermore, they point to the likelihood that PKC-zeta is a putative nuclear downstream target of PI 3-K during NGF-promoted neural differentiation.-Neri, L. M., Martelli, A. M., Borgatti, P., Colamussi, M. L., Marchisio, M., Capitani, S. Increase in nuclear phosphatidylinositol 3-kinase activity and phosphatidylinositol (3,4, 5) trisphosphate synthesis precede PKC-zeta translocation to the nucleus of NGF-treated PC12 cells.