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

We and others have previously demonstrated the existence of an autonomous n uclear 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, 29738-2974 4). Recently, however, nuclei have also been shown to contain the enzymes r esponsible for the synthesis of the non-canonical 3-phosphorylated inositid es. To clarify a possible role of this peculiar class of inositol lipids we have examined the question of whether nerve growth factor (NGF) induces PK C-zeta nuclear translocation in PC12 cells and whether this translocation i s dependent on nuclear phosphatidylinositol 3-kinase (PI 3-K) activity and its product, phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P-3]. N GF increased both the amount and the enzyme activity of immunoprecipitable PI 3-K in PC12 cell nuclei. Activation of the enzyme, but not its transloca tion, was blocked by PI 3-K inhibitors wortmannin and LY294002. Treatment o f 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 abr ogated by either wortmannin or LY294002. Zn 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-phosphocoline was unable to abrogate nuclear translocation of the DAG-insensitive PKC-zeta. These da ta 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-ze ta. Furthermore, they point to the likelihood that PKC-zeta is a putative n uclear downstream target of PI 3-K during NGF-promoted neural differentiati on.