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(The FASEB Journal. 1999;13:2299-2310.)
© 1999 FASEB

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

LUCA M. NERI*,{dagger}1, ALBERTO M. MARTELLI{ddagger},2, PAOLA BORGATTI*, MARIA L. COLAMUSSI*, MARCO MARCHISIO* and SILVANO CAPITANI*

* Dipartimento di Morfologia ed Embriologia, Sezione di Anatomia Umana, Università di Ferrara, 44100 Ferrara;
{dagger} Istituto di Citomorfologia Normale e Patologica del CNR., c/o IOR., 40137 Bologna; and
{ddagger} Dipartimento di Morfologia Umana Normale, Università di Trieste, 34138 Trieste, Italy

1Correspondence: Dipartimento di Morfologia ed Embriologia, Sezione di Anatomia Umana, Universitá di Ferrara, via Fossato di Mortara 66, 44100 Ferrara, Italy. E-mail: nrl{at}dns.unife.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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)P3]. 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)P3. 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)P3 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.


Key Words: signal transduction • nuclear translocation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
THE NUCLEUS IS known to be a site for an active lipid metabolism. Although phospholipids are present in the nuclear envelope, evidence suggests that they are also located further inside the nucleus. In the last 10 years several investigations have suggested that they may be involved in signal transduction pathways at the nuclear level and a growing body of evidence supports this hypothesis (1) . Signal transduction pathways must include mechanisms for the initiation of signals at the plasma membrane, a mechanism by which these signals traverse the cytoplasm and induce, finally, a nuclear response (1) . It is becoming evident that not only phosphoinositide hydrolysis plays an important role in cellular response to agonists but also their synthesis when phosphorylated in the D3 position of the inositol ring by phosphatidylinositol 3-kinase (PI 3-K) (1) because the products of this enzyme, constituted of phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P3], phosphatidylinositol-3,4-bisphosphate [PtdIns(3,4)P2], and phosphatidylinositol monophosphate [PtdIns(3)P], are likely to act as second messengers themselves (2) . The enzymes possessing PI 3-K activity have been classified into three different families based on their structure and mechanism of regulation (3) . The most studied class is formed by a heterodimeric complex consisting of a p110 catalytic subunit, which includes the {alpha}, ß, and {delta} isotypes, and of an adapter/regulatory p85 subunit, of which {alpha} and ß isotypes have so far been identified (2) . The function of p85 has been extensively studied and it has been found to form complexes with activated growth factor receptors with intrinsic tyrosine kinase activity, such as insulin-like growth factor-I (IGF-I) and nerve growth factor (NGF), which activate PI 3-K in a number of cells, conferring to this enzyme a role in the regulation of cell growth, differentiation, and apoptosis (4) .

The first report concerning the presence of PI 3-K in the nucleus was published by Neri et al. (5) who demonstrated by immunostaining that an early translocation to the nuclear compartment occurs in NGF-stimulated PC12 cells. This finding was confirmed by other investigators, who reported the presence of nuclear PI 3-K in Saos-2 and HepG2 cells, as well as in rat liver tissue (6 7 8) . More recently, PI 3-K activity has been described to be modulated at the nuclear level by differentiating stimuli. In fact, during granulocytic maturation of HL-60 cells a functionally active PI 3-K is tightly bound to nuclear matrix and increases along the differentiation process (9) .

The presence of PI 3-K within the nucleus raises questions about its function(s) in this cell compartment. A number of reports have hinted at the fact that PtdIns(3,4,5)P3 is likely to have several downstream targets including protein kinase C (PKC)-{zeta} (10) and -{varepsilon} isoforms (11 , 12) , PKB/Akt (13) , and phospholipase C (PLC)-{gamma}1 (14) .

It has previously been established that PKC-{zeta} is involved in the nuclear events related to cell differentiation of rat pheochromocytoma PC12 cells. Indeed, nuclear accumulation of the -{zeta} isozyme by translocation from the cytoplasm was prominent 5–10 min after NGF administration (15) . In this experimental model, PKC-{zeta} phosphorylated a nuclear substrate that has been identified as the 106-kDa protein C23/nucleolin (16) . Our recent findings have shown that a conventional PKC isoform (PKC-{alpha}) is dependent on an increase in the mass of nuclear diacylglycerol (DAG) to translocate to the nucleus (17) . However, because PKC-{zeta} is an atypical isoform that is unaffected by Ca2+ and DAG or phorbol ester (18) , other molecule(s) might be responsible for attracting and activating it at the nuclear level.

With the above in mind, as an extension of our previous studies, we sought to explore whether nuclear PI 3-K translocation and activation generate PtdIns(3,4,5)P3, which may be related to PKC-{zeta} attraction and/or activation in the nuclear compartment. We found that a rapid and transient enhancement in PI 3-K activity and PtdIns(3,4,5)P3 production 1) occur at the nuclear level early after NGF treatment, 2) precede, and 3) are necessary for PKC-{zeta}, but not PKC-{alpha}, translocation to the nuclear compartment.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Dulbecco’s modified minimum essential medium (DMEM), fetal calf serum, horse serum, 1,2-dioleyl-3-palmitoyl-glycerol, PtdIns, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS), wortmannin, anti-PKC-{alpha} polyclonal antibody, monoclonal antibody to ß-tubulin, normal rabbit IgG, normal goat serum, Cy3-conjugated anti-rabbit and anti-mouse IgG, peroxidase-conjugated anti-rabbit IgG, and bovine serum albumin (BSA) were from Sigma (St. Louis, MO). The enhanced chemiluminescence detection kit was from Boehringer Mannheim, Germany. Protein A-agarose was purchased from Transduction Laboratories, Lexington, KY. [{gamma}-32P]ATP, and [32P]orthophosphate were from Amersham Pharmacia Biotech, Uppsala, Sweden. PtdIns(4)P, PtdIns(4,5)P2, LY 294002, and ET-18-OCH3 were from Calbiochem, La Jolla, CA. PtdIns(3,4,5)P3 was from Alexis Biochemical, Laufelfingen, Switzerland. The Protein Assay kit (detergent compatible) was from Bio-Rad. The two synthetic peptides (RFARKGSLRQKNVHEVKN for PKC-{alpha} and SIYRRGSRRWRKL for PKC-{zeta}), anti-PI 3-K polyclonal and monoclonal antibodies, and NGF were from Upstate Biotechnology, Inc. (Lake Placid, NY). Polyclonal antibody to PKC-{zeta} was from Santa Cruz Biotechnology (Santa Cruz, CA). Silica plates (20 x 20 cm, 0.2 mm thickness DC-Alufolien Kieselgel 60) were from Merck, Darmstadt, Germany.

Cell culture
Rat pheochromocytoma PC12 cells (a kind gift from Dr. L. Green, Columbia University, New York, NY) were cultured in DMEM containing 10% heat-inactivated horse serum and 5% heat-inactivated fetal calf serum. Before stimulation with 100 ng/ml NGF for the indicated times, 70% confluent cells were switched to serum-free medium (DMEM plus 0.5% BSA) overnight. In some cases cells were pretreated for 1 h before NGF exposure with either wortmannin (100 nM) or LY294002 (50 µM). The phospholipase inhibitor ET-18-OCH3 (100 µM) was present starting 5 min before simulation with NGF.

In vivo labeling of polyphosphoinositides, lipid extraction, and thin-layer chromatography
Seventy percent confluent cells cultured overnight in serum-free medium were washed once and incubated with phosphate-free DMEM/0.5% BSA for 15 min, then exposed to the same medium plus carrier-free [32P]orthophosphate (1.0 mCi/ml) at 37°C for 4 h. NGF was added to the cells for the indicated times. Cells (2 x 107), washed twice with ice-cold buffer A (137 mM NaCl, 20 mM Tris, pH 7.4, 1 mM MgCl2, 1 mM CaCl2, and 0.1 mM Na3VO4), or isolated nuclei (from 2 x 107 cells), were transferred to a tube containing 3 ml of chloroform/methanol (1:2) plus 1 mg/ml of butylated hydroxytoluene and 10 µg of a 1:1:1 mixture of PtdIns/PtdIns(4)P/PtdIns(4, 5)P2. After addition of 2.1 ml of chloroform and 2.1 ml of 2.5 M HCl, the lower phase was collected and the upper phase was washed twice with 1 ml of chloroform. The three upper phases were pooled and dried under vacuum. The lipids, dissolved in chloroform, were spotted on a silica plate and separated using chloroform/acetone/methanol/acetic acid/water (80:30:26:24:14). Plates were autoradiographed and the position of the products was compared with migration of unlabeled standards [PtdIns(4)P, PtdIns(4, 5)P2, and PtdIns(3, 4, 5)P3]. Spots of interest were scraped and counted by liquid scintillation.

Isolation of nuclei
This was accomplished as previously reported, with minor changes (19) . Briefly, cells were resuspended in 10 mM Tris-Cl, pH 7.4, 10 mM NaCl, 2 mM MgCl2, 1.0 mM phenylmethylsulfonyl fluoride (PMSF), 10 µg/ml soybean trypsin inhibitor, 1 µg/ml of leupeptin and aprotinin, 1.0 mM Na3VO4, and 20 mM okadaic acid. They were incubated at room temperature for 2 min, cooled in ice water for 5 min, then Nonidet P-40 was added to a final concentration of 1%. After a passage through a 22-gauge needle, the concentration of MgCl2 was adjusted to 5 mM. The samples were centrifuged at 600 x g for 5 min and washed once in 10 mM Tris-Cl, pH 7.4, 5 mM MgCl2, plus protease and phosphatase inhibitors as above. The purity of nuclear preparations was evaluated by Western blot analysis using a monoclonal antibody to ß-tubulin. The absence of immunoreactivity to the cytoskeletal protein in the isolated nuclear preparations confirmed that the isolation procedure produced nuclei of high purity that were free of cytoplasmic contaminants (data not shown).

Protein assay
This was performed with the Bio-Rad Protein Assay (detergent compatible) according to the manufacturer’s instructions.

Immunoprecipitation of PI 3-K
Nuclei (from 2 x 107 cells) were lysed for 30 min at 4°C in 50 mM HEPES, pH 7.9, 100 mM NaCl, 10% glycerol, 10 mM EDTA, 1 mM EGTA, 1% Triton X-100, 0.1 mM Na3VO4, 1 mM PMSF, and 1 µg/ml leupeptin and aprotinin. The suspension was passed several times through a 26-gauge needle, then centrifuged at 12,000 x g for 15 min at 4°C. Lysates (1 ml, containing 500 µg of protein) were pre-cleared by adding 5 µg of normal rabbit IgG and 10 µg of 50% Protein A-agarose, followed by incubation for 1 h at 4°C and centrifugation at 12,000 x g for 10 min at 4°C. Cell lysates were incubated for 2 h at 4°C under constant agitation with 5 µg of polyclonal antibodies to PI 3-K. Ten micrograms of 50% Protein A-agarose was added and incubation proceeded for 1 h at 4°C under constant agitation. The immunoprecipitates were washed three times with lysis buffer, then resuspended in electrophoresis sample buffer.

PI 3-K activity assay
Cells (2 x 107) or nuclei (from 2 x 107 cells) were lysed in 50 mM HEPES, pH 7.4, 10 mM EDTA, 2.0 mm Na3VO4, 10 mM sodium pyrophosphate, 10 mM NaF, and 1% Nonidet P-40 for 15 min at 4°C; immunoprecipitation of PI 3-K was performed as described above using polyclonal antiserum to the 85-kDa regulatory subunit. The immunoprecipitates were washed twice with each of the following buffers: 1) PBS, pH 7.4, containing 1% Nonidet P-40; 2) 100 mM Tris-Cl, pH 7.4, 0.5 M LiCl; 3) 10 mM Tris-Cl, pH 7.4, 100 mM NaCl, 1 mM EDTA. All washing solutions contained 1 mM Na3VO4 (20) . The PI 3-K activity assay was then performed by adding sonicated PtdIns(4,5)P2 (0.5 mg/ml in 10 mm HEPES, pH 7.5, 1 mM EDTA), 10 mM MgCl2, and 50 µM [{gamma}-32P]ATP (10 Ci/mmol). Incubation was for 15 min at room temperature. The reaction was stopped by addition of chloroform/methanol/HCl (200:100:0.75 v/v), followed by two washes with chloroform/methanol/HCl 0.6 N (3:48:47 v/v). The lipid-containing organic phase was resolved on thin-layer chromatography plates developed in isopropanol/acetic acid/H2O (65:1:34), as described previously (21) . The radiolabeled PtdIns(3,4,5)P3 was identified by comparison with standard PtdIns(3,4,5)P3. After autoradiography the spots were excised and quantified by scintillation counting.

Western blot analysis
Proteins (from 107 nuclei) separated on 7.5% polyacrylamide gels (22) were transferred to nitrocellulose sheets using a semi-dry blotting apparatus. Sheets were saturated in phosphate-buffered saline (PBS) containing 5% normal goat serum and 4% BSA for 60 min at 37°C (blocking buffer), then incubated overnight at 4°C in blocking buffer containing primary antibodies. After four washes in PBS containing 0.1% Tween-20, they were incubated for 30 min at room temperature with peroxidase-conjugated anti-rabbit IgG, diluted 1:3,000 in PBS-Tween-20, and washed as above. Bands were visualized by the enhanced chemiluminescence method.

In vitro assay for nuclear PKC activity
Isolated nuclei (from 2 x 107 cells) were lysed and subjected to immunoprecipitation as above using anti-PKC-{alpha} or -{zeta} antibodies. The immunoprecipitates were incubated at 30°C for 10 min in 20 mM Tris-Cl, pH 7.4, 10 mM MgCl2, 10 µM ATP, 10 µg of the appropriate synthetic peptide, 5 µCi of [{gamma}-32P]ATP, in the presence of 1.2 mM CaCl2, 40 µg/ml PS, and 3.3 µM dioleylglycerol (which was omitted for assaying the DAG-independent PKC-{zeta}). An equal volume of Laemmli’s sample buffer was added and proteins were separated using 18% SDS-PAGE. Gels were then stained with Coomassie blue R-250, dried, and autoradiographed. The radioactive bands were excised and counted using a liquid scintillation counter.

Measurement of DAG produced in vivo
The assay was performed according to Divecha et al. (23) , using DAG kinase enzyme purified from rat brain. DAG was extracted from nuclei, dissolved in 20 µl of CHAPS (9.2 mg/ml), and sonicated at room temperature for 15 s. After the addition of 80 µl of reaction buffer (50 mM Tris acetate, pH 7.4, 80 mM KCl, 10 mM Mg-acetate, 2 mM EGTA), the assay was started by the addition of 20 µl of DAG kinase enzyme followed by 80 µl of reaction buffer containing 5 µM ATP, and 1 µCi of [{gamma}-32P]ATP. Incubation was for 1 h at room temperature, then phosphatidic acid was extracted, chromatographed, autoradiographed, and its radioactivity counted in a liquid scintillation system. Standard curves were obtained as reported by Divecha et al. (23) , using 1,2-dioleyl-3-palmitoyl-glycerol as substrate.

In situ immunofluorescence
Cultures of PC12 cells (control and NGF-treated) growing on coverslips coated with rat tail collagen were washed twice in cold PBS, pH 7.2, fixed with freshly prepared 4% paraformaldehyde (30 min at room temperature), and permeabilized with 0.2% Triton X-100 in PBS (10 min). Samples were reacted either with a polyclonal (diluted 1:80) or a monoclonal (diluted 1:40) antibody directed against the 85-kDa subunit of PI 3-K with identical results. Otherwise stated, pictures have been obtained with the monoclonal antibody. The anti-PKC-{alpha}, anti-PKC-{zeta}, and anti-PI 3-K antibodies were used at a dilution of 1:100 in 2% BSA, 3% NGS in PBS. The secondary antibody was a Cy3-conjugated anti-rabbit or anti-mouse IgG, diluted 1:500. All incubations were carried out at 37°C. Finally, the coverslips were mounted in glycerol containing 1,4-diazabicyclo [2.2.2] octane to retard fading, using additional coverslips as spacers to preserve the three-dimensional structure of cells.

Confocal laser scanning microscope (CLSM) and image processing analysis
Samples were imaged by a LSM410 CLSM (Zeiss, Oberckochen, Germany). This confocal system was coupled with a 1 mW HeNe ion laser as light source, used to reveal Cy3 signal with a 543-nm wavelength. Samples were observed with a x100, 1.3 numerical aperture, PlanNeofluar objective lens. Images were acquired, frame by frame, with a scanning mode format of 512 x 512 pixels. Digitized optical sections (0.5 µM), i.e., Z series of confocal data (‘stacks’) were transferred from the CLSM to the graphics workstation Indy (Silicon Graphics, Mountain View, CA) and stored with a scanning mode format of 512 x 512 pixels and 256 gray levels. The image processing was performed using the ImageSpace software (Molecular Dynamics, Sunnyvale, CA). To reduce the unwanted background noise generated by the photomultiplier signal amplification, all the image stacks were treated with a three-dimensional filter (Gaussian filtering) that was carried out on each voxel, with a mask of three pixels in the x, y, and z direction (3 x 3 x 3). Photographs were taken by a digital video recorder Focus ImageCorder Plus (Focus Graphics, Foster City, CA) using ASA 100 TMax black and white film (Kodak Limited, Rochester, NY).

Statistical analysis
Data are means from three different experiments and are expressed as mean ± SD. The asterisk indicates significant differences (P<0.01) in a Student’s paired t test. All of the other differences were found to be not significant with P>0.01.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Measurement of PI 3-K activity and PtdIns(3,4,5)P3 levels in intact PC12 cells stimulated with NGF
As shown in Table 1 exposure of PC12 cells to NGF produced an increase in immunoprecipitable PI 3-K activity. It should be borne in mind that the polyclonal antibody against the p85 subunit of PI 3-K we employed in this study is directed against the NH2 terminus SH2 domain, a region that is common to both {alpha} and ß isoforms of the regulatory subunit. The maximum stimulation occurred after 5 min of NGF exposure, when PI 3-K activity was elevated to ~9.5-fold over the basal level of activity. However, after 10 min of stimulation the activation was greatly reduced (~2.4-fold) and after 15 min the activity was almost the same as at the beginning of stimulation. To investigate changes in the levels of PtdIns(3,4,5)P3 elicited by NGF, we measured the in vivo levels of this inositol lipid. Cells were prelabeled with [32P]orthophosphate, exposed to NGF for various times, the lipids extracted, and the incorporated radioactivity measured by scintillation counting. As shown in Table 1 , 5 min after NGF exposure, 32P incorporation into PtdIns(3,4,5)P3 increased to levels that were approximately eightfold over the basal level. At 15 min the level of PtdIns(3,4,5)P3 declined to near control levels.


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  		Table 1. PI 3-K activity and PtdIns(3,4,5)P3 levels in intact PC12 cells stimulated with NGFa


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  		Table 5. Effect of different inhibitors on the production of nuclear DAG in vivo after 6 min of NGF stimulationa

NGF induces nuclear translocation of PI 3-K
Western blotting analysis of nuclei from NGF-treated PC12 cells showed an early translocation of PI 3-K that was maximal after 9 min of stimulation. After 30 min the nuclear increase was reduced close to the basal level (Fig. 1A ). When the cells were pretreated with either wortmannin (100 nM) or LY294002 (50 µM), which are potent PI 3-K inhibitors, the NGF-dependent nuclear translocation of PI 3-K was still detectable (Fig. 1B ).



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  		Figure 1. NGF induces nuclear translocation of PI 3-K. A) Western blotting analysis for PI 3-K in nuclei isolated from unstimulated cells (lane 1), and from cells exposed to NGF for 6 (lane 2), 9 (lane 3), 15 (lane 4), and 30 min (lane 5). B) Western blotting analysis for PI 3-K in nuclei isolated from cells pretreated for 1 h with inhibitors. Lane 1, 100 nM wortmannin only; lane 2, 100 nM wortmannin plus NGF for 9 min; lane 3, 50 µM LY294002 only; lane 4, 50 µM LY294002 plus 9 min of NGF exposure. Eighty micrograms of nuclear protein was blotted to each lane.

Activity changes in nuclear PI 3-K were monitored by immunoprecipitation of the enzyme and in vitro phosphorylation of exogenous polyphosphoinositides. NGF treatment resulted in a nearly 11-fold increase in nuclear PI 3-K activity that reached its peak at 9 min of treatment, paralleling enzyme translocation from the cytoplasm (Fig. 2 ). If the cells had been pretreated with either wortmannin or LY294002 for 1 h before NGF stimulation, no increase in nuclear activity was seen. The concentrations of the inhibitors we employed are well within the range of those reported to inhibit specifically PI 3-K activity (24 , 25) .



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  		Figure 2. Exposure of PC12 cells to NGF increases immunoprecipitatable nuclear PI 3-K activity. Filled circles, cells treated with NGF alone; filled triangles, cells treated with NGF plus wortmannin; filled squares, cells treated with NGF plus LY294002. Wortmannin was used at 100 nM, LY294002 at 50 µM. Basal levels measured were 432 (NGF alone), 213 (NGF plus wortmannin), and 135 dpm (NGF plus LY294002). Each point represents the mean from three different experiments ± SD 11%.

CLSM analysis of control PC12 cells immunostained with anti-PI 3-K antibody showed a predominantly cytoplasmic location of the enzyme. Nevertheless, nuclei, identified by means of phase-contrast microscopy (data not shown), showed also in control cells the presence of some immunostaining (Fig. 3A ). After 9 min of NGF stimulation, a marked intranuclear translocation of the enzyme became evident and was characterized by a fine punctate fluorescent pattern (Fig. 3B ).



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  		Figure 3. CLSM analysis of PI 3-K distribution in PC12 cells. A single optical section through the equatorial plane of the nucleus is shown. A) Unstimulated cells; B) cells exposed to NGF for 9 min; C) cells pretreated with wortmannin and then exposed to NGF for 9 min; D) cells pretreated with LY294002 and then exposed to NGF for 9 min. Wortmannin was used at 100 nM; LY294002 at 50 µM. Scale bar: 5 µm.

Cells were also pretreated as described above with either wortmannin or LY294002 and then exposed to NGF up to 9 min. Also in this case it was possible to observe that both the inhibitors did not block nuclear translocation of PI 3-K, which occurred on the same time scale and with comparable intensity (Fig. 3,C and D ).

In Table 2 we report the percentage of cells displaying a nuclear translocation of PI 3-K after NGF stimulation under the various conditions listed above, including either wortmannin or LY294002 exposure, which did not block NGF-dependent translocation of the enzyme.


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  		Table 2. Percentage of cells showing nuclear translocation of PI 3-Ka

Nuclear PtdIns(3,4,5)P3 changes after NGF stimulation
To further clarify a possible involvement of PI 3-K and PtdIns(3,4,5)P3 in the translocation of PKC-{zeta} to the nucleus, we decided that it would be necessary to determine the nuclear PtdIns(3,4,5)P3 level in vivo. Thus, polyphosphoinositides were labeled by means of carrier-free [32P]orthophosphate, nuclei were extracted, and inositol lipids were analyzed. A rapid increase in PtdIns(3,4,5)P3 levels could already be observed after 6 min of NGF exposure. The highest value was reached at 9 min, when a nearly 10-fold increase over the control was measured. Then the PtdIns(3,4,5)P3 level started to decrease, remaining ~3.5-fold higher after 30 min than in control nuclei. The administration of both PI 3-K inhibitors blocked the nuclear PtdIns(3,4,5)P3 rise at all the examined times (Fig. 4 ).



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  		Figure 4. Effects of NGF on the levels of nuclear PtdIns(3,4,5)P3 in PC12 cells labeled in vivo using [32P]orthophosphate. Filled circles, cells treated with NGF alone; filled triangles, cells treated with NGF plus wortmannin; filled squares, cells treated with NGF plus LY294002. Wortmannin was used at 100 nM, LY294002 at 50 µM. Basal levels measured were 169 dpm (NGF alone), 153 dpm (NGF plus wortmannin), 145 dpm (NGF plus LY294002). Each point represents the mean from three different experiments ± SD 10%.

NGF-induced nuclear translocation of PKC-{zeta} is dependent on PI 3-K activity
The increase in nuclear PtdIns(3,4,5)P3 described above might be the driving force that attracts PKC-{zeta} to the nucleus. Therefore, we first investigated by means of Western blot the behavior of PKC-{zeta} in NGF-stimulated PC12 cells. In nuclei prepared from unstimulated cells, a band with a Mr of ~75/78 kDa was seen (Fig. 5A , lane 1). After treatment with NGF there was a progressive increase in the amount of nuclear PKC-{zeta}, which peaked at 12 min (Fig. 5A , lane 3). Then the intranuclear amount of the isoform declined starting from 15 min of stimulation, but did not completely return to control level after 30 min (Fig. 5A , lanes 4–5). This translocation was not evident when either wortmannin or LY294002 were administered to the cells before NGF stimulation (Fig. 5B ).



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  		Figure 5. NGF induces nuclear translocation of PKC-{zeta}. A) Immunoblotting analysis for PKC-{zeta} in nuclei isolated from unstimulated cells (lane 1), and from cells exposed to NGF for 9 (lane 2), 12 (lane 3), 15 (lane 4), and 30 min (lane 5). B) Western blotting analysis for PKC-{zeta} in nuclei isolated from cells pretreated for 1 h with inhibitors. Lane 1, 100 nM wortmannin only; lane 2, 100 nM wortmannin plus NGF for 12 min; lane 3, 50 µM LY294002 only; lane 4, 50 µM LY294002 plus 12 min of NGF exposure. Eighty micrograms of nuclear protein were blotted to each lane.

The PKC-{zeta} activity present in isolated nuclei was immunoprecipitated and assayed using a synthetic peptide (corresponding to amino acids 113–125 of PKC-{zeta} regulatory subunit) with a serine substitution at amino acid 119. Low levels of activity were detected in nuclei from unstimulated cells (Fig. 6 ), in agreement with the results of immunochemical experiments. However, in nuclei prepared from cells treated for 12 min with NGF, a more than 12-fold increase in PKC activity was measured. This increase was completely abolished by either wortmannin or LY294002.



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  		Figure 6. Exposure of PC12 cells to NGF increases immunoprecipitatable nuclear PKC-{zeta} activity. Filled circles, cells treated with NGF alone; filled triangles, cells treated with NGF plus wortmannin; filled squares, cells treated with NGF plus LY294002. Wortmannin was used at 100 nM, LY294002 at 50 µM. Basal levels measured were 425 dpm (NGF alone), 368 dpm (NGF plus wortmannin), 240 dpm (NGF plus LY294002). Each point represents the mean from three different experiments ± SD 11%.

CLSM analysis showed PKC-{zeta} to be mainly localized in the cytoplasm in untreated cells (Fig. 7A ). Nuclei were immunolabeled to a much lower extent (Fig. 7A ). Incubation of cells for 12 min with NGF resulted in a striking increase in PKC-{zeta} immunolabeling in the nuclear interior (Fig. 7B ). This staining was characterized by the presence of several large brilliant dots and by fine punctate spots. When PC12 cells were pre-incubated with either wortmannin or LY294002, no NGF-elicited nuclear translocation of PKC-{zeta} was observed (Fig. 7,C and D ).



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  		Figure 7. CLSM analysis of PKC-{zeta} distribution in PC12 cells. A single optical section through the equatorial plane of the nucleus is shown. A) unstimulated cells; B) cells exposed to NGF for 12 min; C, cells pretreated with wortmannin and then exposed to NGF for 12 min; D, cells pretreated with LY294002 and then exposed to NGF for 12 min. Wortmannin was used at 100 nM, LY294002 at 50 µM. Scale bar: 5 µm.

In Table 3 we report the percentage of cells displaying a nuclear translocation of PKC-{zeta} after NGF stimulation under the various conditions listed above. It is evident that exposure to either wortmannin or LY294002 completely blocked nuclear translocation of the kinase.


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  		Table 3. Percentage of cells showing nuclear translocation of PKC-{zeta}a

NGF-induced nuclear translocation of PKC-{alpha} is independent of PI 3-K activity
Previous results have indicated, by means of Western blot, that NGF also induces a rapid nuclear translocation of PKC-{alpha} in PC12 cells (15 , 19) . To determine whether nuclear PI 3-K activity was involved in this phenomenon, the PKC-{alpha} activity present in isolated nuclei was immunoprecipitated and assayed using a synthetic peptide (corresponding to amino acids 19–36 of PKC-{alpha} regulatory subunit) with a serine substitution at amino acid 25. As shown in Figure 8 , a dramatic increase in PKC-{alpha} activity was induced by NGF after 9 min of stimulation. In this case, however, pretreatment with either wortmannin or LY294002 did not affect at all the increase of kinase activity in isolated nuclei. To examine this issue further, we carried out an immunochemical analysis for PKC-{alpha} in isolated nuclei. As shown in Figure 9 , nuclei from unstimulated cells retained a small amount of this isoform. Exposure for 9 min to NGF caused a marked increase in nuclear PKC-{alpha}. Neither wortmannin nor LY294002 were capable of blocking the intranuclear translocation of this isoform caused by NGF. In contrast, ET-18-OCH3, a specific inhibitor of phosphoinositide-specific phospholipase C (17) completely prevented the intranuclear increase in PKC-{alpha}.



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  		Figure 8. Exposure of PC12 cells to NGF increases immunoprecipitatable nuclear PKC-{alpha} activity. Filled circles, cells treated with NGF alone; filled triangles, cells treated with NGF plus wortmannin; filled squares, cells treated with NGF plus LY294002. Wortmannin was used at 100 nM, LY294002 at 50 µM, and ET-18-OCH3 at 100 µM. Basal levels measured were 513 (NGF alone), 340 (NGF plus wortmannin), and 628 (NGF plus LY294002). Wortmannin was used at 100 nM, LY294002 at 50 µM. Each point represents the mean from three different experiments ± SD 12%.



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  		Figure 9. NGF induces nuclear translocation of PKC-{alpha}. Immunoblotting analysis for PKC-{alpha} in nuclei isolated from unstimulated cells (lane 1), and from cells exposed for 9 min to NGF alone (lane 2), or to NGF plus wortmannin (lane 3), or to NGF plus LY294002 (lane 4), or to NGF plus ET-18-OCH3 (lane 5). Wortmannin was used at 100 nM, LY294002 at 50 µM, and ET-18-OCH3 at 100 µM. Eighty micrograms of nuclear protein were blotted to each lane.

CLSM analysis showed PKC-{alpha} to be mainly localized in the cytoplasm in unstimulated cells, whereas nuclei showed only a scarce immunoreactivity (Fig. 10A ). Incubation of cells with NGF for 9 min resulted in a dramatic increase in PKC-{alpha} labeling in the nuclear interior, constituted of a diffuse punctate immunofluorescence (Fig. 10B ). Pre-incubation of PC12 cells with either wortmannin or LY294002 did not affect nuclear translocation of PKC-{alpha} (Fig. 10C and D , respectively). On the other hand, pre-incubation of cells for 5 min with 100 µM ET-18-OCH3 blocked the migration of PKC-{alpha} to the nucleus (Fig. 10E ), in agreement with our own previous results obtained in Swiss 3T3 cells (17) .



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  		Figure 10. CLSM analysis of PKC-{alpha} distribution in PC12 cells. A single optical section through the equatorial plane of the nucleus is shown. A) unstimulated cells; B) cells exposed to NGF for 9 min; C) cells pretreated with wortmannin and then exposed to NGF for 9 min; D) cells pretreated with LY294002 and then exposed to NGF for 9 min; E) cells pretreated with ET-18-OCH3 and then exposed to NGF for 9 min. Wortmannin was used at 100 nM, LY294002 at 50 µM, and ET-18-OCH3 at 100 µM. Scale bar: 5 µm.

In Table 4 we report the percentage of cells displaying nuclear translocation of PKC-{alpha} after NGF stimulation plus or minus various inhibitors.


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  		Table 4. Percentage of cells showing nuclear translocation of PKC-{alpha}a

Nuclear DAG levels in PC12 cell nuclei after NGF stimulation
As a further clue to the existence of separate mechanisms promoting the nuclear translocation of PKC isozymes belonging to different subfamilies, we evaluated the effect of either wortmannin or LY294002 on the generation of DAG at the nuclear level. As shown in Table 5 , at 6 min after NGF treatment, there was a marked increase in the mass of nuclear DAG that rose more than fourfold above control levels. Incubation of cells with the PI 3-K inhibitors did not modify nuclear DAG production. On the other hand, ET-18-OCH3, which effectively blocks nuclear DAG production (17 , 26) , was able to fully inhibit this increase.

In Table 6 we demonstrate that there was no ET-18-OCH3-dependent inhibition of nuclear PI 3-K activity, PtdIns(3,4,5)P3 generation, and PKC-{zeta} activity elicited by NGF, that were all similar to the samples from cells not exposed to the chemical. On the contrary, when PKC-{alpha} activity was assayed under the same conditions, it became evident that ET-18-OCH3, but not wortmannin or LY294002, inhibited the increase in nuclear activity elicited by NGF.


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  		Table 6. Effect of different inhibitors on nuclear PI 3-K activity, PtdIns(3,4,5)P3 generation, PKC-{zeta}, and PKC-{alpha} activities after stimulation with NGFa


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
At present, increasing interest surrounds phospholipid signaling in the nucleus and particularly the generation of second messengers capable of attracting to this cell compartment different PKC isozymes (1 , 27) .

This study provides the first evidence for a possible novel mechanism regulating nuclear translocation of PKC-{zeta} after a differentiating stimulus mediated by the activation of nuclear PI 3-K in NGF-treated PC12 cells. Because two phosphorylation products of PI 3-K, PtdIns(3,4)P2 and PtdIns(3,4,5)P3, have been described to activate PKC-{zeta}, implying that this atypical isoform may be a downstream target of PI 3-K (10) , we envisioned the hypothesis that PtdIns(3,4,5)P3 may be the driving force for the nuclear translocation of PKC-{zeta} in NGF-treated PC12 cells.

The involvement of cellular PI 3-K in the initiation of signal transduction by NGF in PC12 cells was previously demonstrated by Soltoff et al. (20) . They observed, by immunoprecipitation of PI 3-K activity with anti-phosphotyrosine antibody, a ninefold increase in whole-cell activity after 5 min of NGF exposure and we have confirmed their findings. Furthermore, we have detected a similar increase in immunoprecipitable nuclear PI 3-K activity. However, this increase was delayed in comparison with the one measured in whole cells. We feel it unlikely that the increase we measured in nuclear PI 3-K activity was due to a contamination from cytoplasm, for a number of reasons: first, our nuclear preparations were free from ß-tubulin, a well-established marker for cytoplasmic contamination in this cell line (15 , 16 , 19) ; second, our unpublished data revealed that nuclear PI 3-K activity represented ~10% of the activity found in the cytosol, i.e., a value that seems much higher than the possible contribution from cytoplasmic PI 3-K, even if there is a small contamination of the nuclear fraction not revealed by Western blotting; third and more convincing, we have demonstrated that translocation of PI 3-K to the nucleus is detectable in situ in intact cells and this argues against contamination by other organelles as recently underlined by Divecha (28) .

It is noteworthy that the nuclear translocation of PI 3-K may rapidly occur after agonist stimulation, as shown in the past by means of immunofluorescence for platelet-derived growth factor (PDGF)-treated 3T3-L1 cells (29) . This finding conforms to the reports that the main components of the canonical phosphoinositide pathway, including PtdIns(4,5)P2 and PLCß1, are activated in the nucleus in the early stages of cell responses (23 , 30 , 31) .

Our data demonstrate that specific inhibitors of PI 3-K did not block the translocation of the enzyme to the nucleus, but rather inhibited its activation in this organelle. Therefore, we could assume that migration of PI 3-K to the nucleus is related to the p85 regulatory subunit rather than to the activity of the p110 catalytic subunit. Very recently, we have obtained similar results employing vitamin D3-stimulated HL-60 leukemia cells (32) . In contrast, in Saos-2 cells treated with interleukin-1, intranuclear migration of PI 3-K was inhibited by either wortmannin or LY294002 (33) . It is conceivable that these differences are dependent on the cell types and/or agonist employed.

In agreement with our own observations, Tanaka et al. (34) have very recently demonstrated a considerable NGF-elicited tyrosine phosphorylation of nuclear p85 subunit of PI 3-K, suggesting that the activation of the enzyme in the nucleus may be due to its relocalization. The increase in nuclear PI 3-K activity that follows NGF treatment was paralleled by an increase in the levels of nuclear PtdIns(3,4,5)P3. In addition, the same authors identified in the nucleus of PC12 cells a specific PtdIns(3,4,5)P3-binding protein.

Originally, Divecha et al. (35) proposed that conventional PKC isoforms (such as PKC-{alpha}) may continuously cycle in and out of the nucleus and may become ‘fixed’ in this cell compartment by an increase in DAG. Our finding supports the hypothesis that nuclear PtdIns(3,4,5)P3, generated by PI 3-K activity, is the driving force for PKC-{zeta} translocation to the nucleus, similar to what has previously been demonstrated for DAG and PKC-{alpha} in IGF-I-stimulated Swiss 3T3 cells (17) . The nuclear translocation of PKC-{zeta} in NGF-treated PC12 cells was demonstrated by three different methods: Western blot analysis revealed a marked increase in PKC-{zeta} in purified nuclei after NGF treatment; biochemical assays for PKC-{zeta} indicated a low specific activity in nuclei from control cells, which increased more than 10-fold in nuclei obtained from NGF-treated cells; finally, in situ CLSM analysis of intact cells showed an increased staining for PKC-{zeta} within nuclei of NGF-stimulated cells. In any case, with all the aforementioned techniques, we saw that in cells exposed to either wortmannin or LY294002 nuclear translocation of PKC-{zeta} was inhibited. It should be emphasized that already after 9 min of NGF exposure a maximal nuclear PI 3-K activation could be observed. In contrast, maximal PKC-{zeta} activity was observed at 12 min of stimulation, thus showing an ordered sequence in PI 3-K nuclear translocation, PtdIns(3,4,5)P3 production, and PKC-{zeta} migration. It is interesting that both PKC-{zeta} and PI 3-K have been demonstrated to associate with the insoluble nuclear matrix (6 , 15) .

To further support the hypothesis of PtdIns(3,4,5)P3 being the driving force for PKC-{zeta} migration to the nucleus, we investigated in the same experimental model the behavior of a conventional PKC isozyme, i.e., the -{alpha} isozyme. NGF also induced the translocation to the nucleus of this isoform. However, this migration was independent of nuclear PI 3-K activation and PtdIns(3,4,5)P3 generation, as demonstrated by its insensitivity to PI 3-K inhibitors. In contrast, we showed that PKC-{alpha} nuclear translocation was dependent on the nuclear generation of a different driving force, such as DAG. To this end, we used a chemical, ET-18-OCH3, which is thought to be quite a specific inhibitor of phosphoinositide-specific PLC (17 , 26) . Therefore, it has been possible to assess the independence of the different mechanisms that regulate the nuclear translocation of specific PKC isozymes by PtdIns(3,4,5)P3 or DAG.

There are several studies that have shown translocation to the nucleus of PKC-{zeta} in different cell types subjected to various differentiative stimuli (15 , 16 , 36) . Moreover, a nuclear translocation of PKC-{zeta}, inhibited by wortmannin, was described to occur in ischemic rat hearts (37) .

Several recent reports hinted at the activation of PKC-{zeta} as an event dependent on PI 3-K activity. Herrera-Velit et al. (38) demonstrated that the response of human monocytes to bacterial lipopolysaccharide is consistent with a model in which PKC-{zeta} is activated downstream of PI 3-K. It is more interesting that, also in differentiation models, the two enzymes have been associated: in insulin-stimulated rat adipocytes, PKC-{zeta} may act as a downstream effector of PI 3-K and contribute to the activation of Glut-4 translocation (39) . In L6 myotubes, phorbol ester treatment did not elicit increases in glucose transport and translocation, whereas both wortmannin and LY294002 blocked PKC-{zeta}-mediated insulin-stimulated glucose transport (40) . These authors suggested that although PKC-{zeta} is a reasonable candidate downstream of PI 3-K to participate in insulin stimulation of glucose transport, DAG-sensitive PKCs are unlikely participants. IGF-I-inducible PI 3-K in vitamin D3-primed HL-60 cells induced activation of PKC-{zeta} and enhanced macrophage differentiation that was totally blocked with either wortmannin or LY294002 (41) . However, the exact role played by PtdIns(3,4,5)P3 in the control of PKC-{zeta} activity is still controversial. Indeed, two recent reports have shown that this isoform is phosphorylated and activated by the protein kinase PDK-1 and this is enhanced by the presence of PtdIns(3,4,5)P3 (42 , 43) . The association between PDK-1 and PKC-{zeta} reveals extensive cross-talk between enzymes in the PI 3-K signaling pathways.

PKC-{zeta} may not be the only target of 3-phosphoinositides in the nucleus, because it has been shown that these lipids could be involved in the modulation of different protein kinases (44) subsequently identified for example as Bruton’s tyrosine kinase (45) , PDK1 (42) , and Akt/PKB (13) .

Therefore, in the future it will be interesting to investigate the presence of PDK-1 in the nucleus, since the nuclear location of PI 3-K and phosphoinositide-activated kinases such as PKC-{zeta} and PKB/Akt (46) suggests the existence of an expanding family of downstream targets of nuclear polyphosphoinositide metabolism.


   ACKNOWLEDGMENTS
 
The authors wish to thank Giovanna Baldini for the illustrations. Luca M. Neri is grateful to Paola Ziccone for her continuous support, encouragement, and understanding. This work was supported by Associazione Italiana per la Ricerca sul Cancro grants to Alberto M. Martelli and Silvano Capitani, Progetto Finalizzato Biotecnologie to S. Capitani, 40% by Italian MURST and 60% by grants to the Universities of Trieste and Ferrara and grant for biomedical research Az. Osp. ‘Arcispedale S. Anna’, Ferrara University. M.L.C. is a recipient of a FIRC fellowship.


   FOOTNOTES
 
2 L. M. N. and A. M. M. contributed equally to this work.

Received for publication March 22, 1999. Revised for publication July 20, 1999.


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 DISCUSSION
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