Evidence That a Phosphatidylinositol 3,4,5-Trisphosphate-binding Protein Can Function in Nucleus*

PIP 3 BP is a phosphatidylinositol 3,4,5-trisphosphate-binding protein (PIP 3 BP) abundant in brain, containing a zinc finger motif and two pleckstrin homology (PH) domains. Staining of rat brain cells with anti-PIP 3 BP antibody and determination of localization of PIP 3 BP fused to the green fluorescent protein (GFP-PIP 3 BP) revealed that PIP 3 BP was targeted to the nucleus. Targeting was dependent on a putative nuclear localization signal in PIP 3 BP. Generation of PIP 3 in the nucleus was detected in H 2 O 2 -treated 293T cells, nerve growth factor (NGF)-treated PC12 cells, and platelet-derived growth factor (PDGF)-treated NIH 3T3 cells. Translocation of phosphatidylinositol 3-kinase (PI 3-kinase) to the nucleus

Phosphatidylinositol 3-kinase (PI 3-kinase) 1 is an enzyme that is activated immediately after growth factor or differentiation factor stimulation of the cells (1) and that generates second messengers, phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) and phosphatidylinositol 3,4-bisphosphate (PI 3,4-P 2 ) (2)(3)(4)(5). These 3Ј-phosphorylated phosphoinositides can activate serine, threonine kinases such as PKB/Akt, PKCs, and PDKs (6 -9). They are also suggested to be involved in other events such as rearrangement of cytoskeleton and vesicle transport because these phenomena are sensitive to the PI 3-kinase inhibitors and dominant negative mutants of PI 3-kinase (10). Recently, it was reported that the 3Ј-phosphorylated phosphoinositides can activate guanine nucleotide exchanging factors of Rac and Arf, small G proteins involved in actin rearrangement and vesicle transport, respectively (11,12). Therefore, G proteins as well as kinases are downstream of PI 3-kinase.
We have identified PIP 3 BP as a PIP 3 -binding protein, using a PIP 3 analogue column (13). It is abundant in brain, implying that it may be involved in the function of nerve systems. PIP 3 BP binds to PIP 3 but not to PI 3,4-P 2 or phosphatidylinositol 4,5-bisphosphate (PI 4,5-P 2 ). It has a zinc finger motif homologous to that of Arf-GTPase activating protein (GAP) and two PH domains. Both PH domains are shown to be involved in binding to PIP 3 . Another PIP 3 -binding protein, centaurin ␣, is highly homologous to PIP 3 BP (14). No GAP activity to Arf has been detected in either protein. Although the binding of centaurin ␣ and PIP 3 BP to PIP 3 was specific, the role of the protein is unclear. To address this question, we determined the intracellular localization by immunological techniques, using monoclonal antibody to PIP 3 BP as well as localization of green fluorescent protein (GFP) fusion proteins. Surprisingly, PIP 3 BP was found to localize in the nucleus, where the generation of PIP 3 was detected after stimulation, suggesting a new pathway of signal transduction through PI 3-kinase to PIP 3 BP in nucleus. PIP 3 BP was exported out of the nucleus by expression of a constitutively active PI 3-kinase. This suggests that PIP 3 BP can shuttle between nucleus and cytoplasm depending on the activity of PI 3-kinase.

EXPERIMENTAL PROCEDURES
Cell Lines and Transfection-COS-7 cells and 293T cells were cultured in Dulbecco's modified minimal essential medium (DMEM) supplemented with 10% calf serum. Transfection was done by the calcium phosphate method as described by Shirai et al. (15) except that pH of the buffer was 7.00 instead of 7.15.
Primary Culture of Rat Brain-Pregnant mice were sacrificed by cervical dislocation on the 18th-day of gestation. After isolation of the embryos from the uterus, by cutting the outer layer of the pelvis, the fetal meninges were removed and the cerebral cortices were placed in DMEM containing 10% fetal bovine serum (Life Technologies, Inc.). Following the mechanical dissociation, cells were passed through a #100 mesh, and were suspended in DMEM supplemented with 10% fetal bovine serum for glial cell culture. For neuronal cell culture, the * This work was supported by Grants-in-Aid from Ministry of Education, Science, Sports, and Culture of Japan (to Y. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § Present address: Division of Biochemistry and Cellular Biology, National Institute of Neuroscience, NCNP, 4-1-1 Ogawahigashi, Kodaira, Tokyo 187-8502, Japan.
Plasmids Used in This Study-A cDNA fragment encoding the fulllength PIP 3 BP or mutant PIP 3 BPs was subcloned into pEGFP c-1, an expression vector for GFP fusion protein (CLONTECH), to produce pEGFP-PIP 3 BP, pEGFP-PIP 3 BP(ϪNLS), pEGFP(ϩNLS), and pEGFP-PIP 3 BP(ϪPH). PIP 3 BP-NLS was constructed by deletion of amino acid 1-9 residues using the restriction site, XhoI, in the cDNA. Point mutants in the PH domains in PIP 3 BP (PIP 3 BP-PH) were introduced as described previously (13) by substituting Cys for Arg (residues 149 and 272) of PIP 3 BP by the Kunkel method (16). To obtain Myc-tagged PIP 3 BP, an Myc-tag sequence with an initiation codon, ATGGAACA-GAAGCTGATCTCAGAAGAAGATCT, was attached at the 5Ј end of the cDNA of the PIP 3 BP. The resulting gene was expressed under the control of SR␣ promoter by an expression vector pMIKNeo (17). The expression vectors for constitutively active PI 3-kinase (BD110) and a kinase negative mutant of PI 3-kinase (BDKN) were described previously (18). The BD110 protein has a structure similar to that of p110* reported by Hu et al. (19). The protein has an inter-SH2 domain of p85, which binds to the p110 amino terminus. BDKN protein is a kinase negative counterpart of the BD110 protein with a point mutation in the kinase domain.
In Situ Hybridization-In situ hybridization was carried out as described previously (20). A cDNA fragment encoding the full-length PIP 3 BP was subcloned into pBluescript SK(ϩ), and the transcripts of T7 or T3 RNA polymerase labeled with digoxigenin were used as antisense or sense probes.
Production of the Monoclonal Antibody and Histocytochemistry-A monoclonal antibody, mAb 13-14, was produced. GST fusion protein of PIP 3 BP (GST-PIP 3 BP) was expressed in Escherichia coli and purified with a glutathione-Sepharose column. Eight-week-old male mice were injected subcutaneously with the purified protein mixed with complete Freund's adjuvant. Booster injections were given subcutaneously with the antigen mixed with incomplete Freund's adjuvant two times with an interval of two weeks. After the final booster injection, which was given intravenously, spleen cells of the mouse were taken and fused with the SP2/O cells by a polyethylene glycol method (21). Ten days after fusion, culture supernatant of the hybridomas were examined for the reactivity to purified GST-PIP 3 BP protein by enzyme-linked immunosorbent assay. After several cycles of cloning, a hybridoma clone producing mAb 13-14 was established. The epitope for mAb 13-14 was determined to be the region between amino acid position 42-109, which is within the zinc finger motif. Immunostaining was carried out as described previously (22) Fractionation of the Cells-The cells were collected by centrifugation and resuspended in a buffer containing 20 mM Tris-Cl (pH 7.5), 10 mM CaCl 2 . After homogenization in a Dounce homogenizer, they were centrifuged at 1,000 ϫ g for 5 min. After removal of the supernatant, two cycles of the same procedure were done to remove any non-nuclear membranes from the nucleus. The resulting pellet was used as a nuclear fraction. The supernatant was further ultracentrifugated at 100,000 ϫ g for 30 min. The supernatant and the pellet were used as cytosolic and membrane fractions, respectively.
In Vivo Labeling of the Cells and Analysis of Lipids-Cells were labeled with [ 32 P]orthophosphate (1 mCi/ml) for 4 h in a phosphate-free MEM supplemented with 25 mM Hepes-NaOH and treated with various stimuli. After fractionation of the cells, the lipids were extracted as described previously (23) and analyzed by TLC as described previously (24). High performance liquid chromatography analysis using SAX5 column (Whatman) was done to confirm the result of TLC (25).

RESULTS AND DISCUSSION
Localization of PIP 3 BP in Brain-In situ hybridization and immunostaining was carried out to determine the expression of PIP 3 BP in rat brain with mAb 13-14, anti-PIP 3 BP monoclonal antibody. In the rat brain section, roughly two types of the cells are clearly seen: large and round-shaped neuronal cells, and small and sharp-shaped glial cells (Fig. 1A). In situ hybridization revealed that only the neuronal cells were stained by the antisense probe in cerebral cortex, whereas the sense probe did not give clear signals (Fig. 1A, a and b). Consistent with this, immunostaining analysis suggested the same expression pattern (Fig. 1A, c and d). Interestingly, the staining of mAb 13-14 appeared to be restricted within the hematoxylin-stained areas, suggesting that PIP 3 BP might be located in the nucleus. Similar results were obtained in the hippocampus and the cerebellum (data not shown).
Primary neuronal and glial cultures were prepared separately from embryonic day 18 rat brains. Cell fractionation was

A Role of PI 3-Kinase in Nucleus 3920
correctly done because neuron-specific enolase (NSE) was specifically found in neuronal fraction, and the glial fiber acidic protein (GFAP) was found in the glial fraction (Fig. 1B, bottom  part). Expression of PIP 3 BP was examined by immunoblotting using mAb 13-14. As shown in Fig. 1B, PIP 3 BP was detected exclusively in neuronal cells. No detectable amounts of PIP 3 BP were observed in glial cells. These results suggest that PIP 3 BP is localized in nucleus of the neuronal cells in rat brain. Immunostaining using mAb 13-14 showed that native PIP 3 BP was also detected in nucleus of neuroblastoma, Neuro2A cells (Fig. 1C).
PIP 3 BP Is Targeted to the Nucleus-To confirm the nuclear localization of PIP 3 BP, COS-7 cells were transfected with a construct coding for PIP 3 BP fused to the green fluorescent protein (GFP-PIP 3 BP), and the localization of the protein in the intact cells was analyzed. The GFP-PIP 3 BP fusion protein was almost exclusively detected in the nucleus, supporting the immunostaining data (Fig. 1D, a). Similar results were obtained using PC12 cells and neuroblastoma Neuro 2A cells (26,27) (data not shown). Nuclear localization signal-like motif, KERRK, was found in the amino terminus part of PIP 3 BP. We tested whether or not this sequence directs the protein to the nucleus. GFP fused to amino-terminal 14 amino acids of PIP 3 BP, MAKERRKAVLELLQ, localized exclusively in the nucleus (Fig. 1D, c). A deletion mutant lacking amino acid 1-9 (GFP-PIP 3 BP(ϪNLS)) was diffusely distributed all over the cells (Fig. 1D, b), suggesting that the targeting mechanism of the protein to the nucleus was absent. GFP alone was detected in all parts of the cells (Fig. 1D, d). These results suggest that the amino acid 1-14 of PIP 3 BP targets the proteins to the nucleus. Fractionation of COS-7 cells transfected with an expression vector for Myc-PIP 3 BP by homogenizing and centrifugation revealed that PIP 3 BP free from GFP was also located in the nucleus (see below).
PIP 3 Is Generated in the Nucleus-The above results suggest that PIP 3 BP may play a role in the nucleus. We therefore determined whether or not PIP 3 was generated in the nucleus. Various cells were stimulated by agonists and fractionated, and the lipids were analyzed by TLC. PI 3-kinase is strongly activated to give a strong signal of PIP 3 in 293T cells treated with 10 mM H 2 O 2 . 2 We first used this system. As shown in Fig. 2A, generation of PIP 3 in the nucleus was detected in those cells as well as in the membrane. The fractionation was confirmed by Western blotting of Src and Myc, which are membrane and nuclear proteins, respectively (Fig. 2C). When the nuclear fraction of the H 2 O 2 -treated 293T cells was prepared and incubated with [ 32 P]ATP-MgCl 2 , generation of PIP 3 was clearly detected; it was not seen in that of the untreated cells (Fig. 2B). The presence of PIP 3 in the samples was confirmed by high performance liquid chromatography analysis of the lipids, using a SAX5 column (data not shown).
In the H 2 O 2 -treated 293T cells, tyrosine phosphorylation of the proteins was extremely elevated, suggesting the activation of many signaling pathways (28). Fractionation of the cells revealed that the level of p85 in the nuclear fraction was markedly elevated after H 2 O 2 treatment and considerable tyrosine phosphorylation on nuclear p85 was detected (Fig. 2C), suggesting that the activation of PI 3-kinase activity in the nucleus may be because of relocalization of the enzyme. PIP 3 was also detected in the nucleus in 293T cells transiently expressed, constitutively active PI 3-kinase, NGF-treated PC12 cells, and PDGF-treated NIH 3T3 cells (Fig. 2A). Recently, several groups have used PIP 3 -binding proteins, such as ARNO and GRP1, fused to GFP, as a means to visualize changes in cellular PIP 3 levels (29,30). However, they failed to detect the nuclear PIP 3 . This may be because of failure of nuclear localization of these proteins.

PIP 3 BP Is Exported Out of the Nucleus by the Expression of Constitutively Active PI 3-Kinase-
To test the effect of PI 3-kinase on the localization of PIP 3 BP, COS-7 cells were transfected with the constructs for expression of PIP 3 BP and constitutively active PI 3-kinase (BD110), and the cells were fractionated and distribution of PIP 3 BP was determined. As shown in Fig. 3A, both PIP 3 BPs fused to GFP and a Myc tag were fractionated in nucleus in the absence of the activated PI 3-kinase. In contrast, they were localized in the membrane or cytosolic fractions when the constitutively active PI 3-kinase was co-expressed. The cells were observed under the microscope to detect the relocalization of GFP-PIP 3 BP. Co-expression of BD110 resulted in exportation out of the protein from the nucleus in more than 75% of the transfected cells (Fig. 3B) 1, 2, 5 , 6, 9, 10, 13, and 14) and nuclear (N) (lanes 3, 4, 7, 8, 11, 12, 15, and 16) fractions of NIH 3T3 cells treated with (lanes 2 and 4) or without (lanes 1 and 3) PDGF for 3 min, PC12 cells treated with (lanes 6 and 8) or without (lanes 5 and 7) NGF for 3 min, 293T cells treated with (lanes 10 and 12) or without (lanes 9 and 11) 10 mM H 2 O 2 for 3 min, or 293T cells transfected with (lanes 14 and 16) or without (lanes 13 and 15) the expression vector for constitutively active PI 3-kinase (BD110) were analyzed on TLC. B, generation of PIP 3 in membrane (M) and nuclear (N) fractions. Membrane and nuclear fractions were prepared from 293T cells treated with or without 10 mM H 2 O 2 for 3 min. They were then incubated with [ 32 P]ATP and MgCl 2 for 5 min at 25°C. The resulting lipids were analyzed on TLC (top). Distribution of Src in each fraction was analyzed by Western blotting with anti-Src antibody, 327 (bottom). C, distribution of PI 3-kinase. 293T cells treated with or without 10 mM H 2 O 2 were fractionated into membrane and nuclear fractions. The distribution of p85␣, Src, Myc, was analyzed by Western blotting. In one experiment, levels of p85␣ in anti-phosphotyrosine immunoprecipitates were analyzed. Cell fractionation was correctly done because Src was specifically found in the membrane fraction and Myc in the nuclear fraction.
A Role of PI 3-Kinase in Nucleus 3921 ( Fig. 3B). Treatment of the cells with wortmannin resulted in relocation of PIP 3 BP to the nucleus within 30 min, suggesting that cells were not damaged by the expression of the constitutively active PI 3-kinase (Fig. 3B). Point mutations of PH domains in PIP 3 BP were suggested to abolish the binding to PIP 3 , previously (13). This mutant PIP 3 BP was not exported out of the nucleus (Fig. 3B). These results suggest that interaction of the PH domains and PIP 3 is responsible for the relocation of PIP 3 BP. These results suggest that PIP 3 BP can shuttle between the nucleus and the cytoplasm depending on the activity of PI 3-kinase. It is well known that PI 4,5-P 2 is present in the nucleus, probably in the nuclear membrane. It is possible that PI 3-kinase which is present in the cytosol can approach the nuclear membrane at least from the cytosolic side to produce PIP 3 . We found that PI 3-kinase can be targeted after H 2 O 2 treatment of 293T cells. The condition used here was artificial; however, PI 3-kinase may be a specific protein that is targeted to the nucleus because Coomassie Blue staining patterns of the proteins in the nuclear fractions from H 2 O 2 -treated and -untreated cells were almost identical. Although a drastic condition may be required for nuclear localization of a considerable amount of PI 3-kinase, this finding implicates that a small amount of PI 3-kinase, which is undetectable by the present methods, can be targeted to the nucleus after appropriate stimulation of the cells. The results in this paper clearly indicate that PIP 3 BP can function as a PIP 3 -binding protein. Therefore, the fact that PIP 3 BP is targeted to the nucleus suggests that there may be an unknown function of PIP 3 BP in the nucleus. The exportation of PIP 3 BP out of the nucleus was resistant to leptomycin B, an inhibitor of nuclear exportation signal (NES)-dependent exportation. We are currently examining how PIP 3 BP is ex-ported out of the nucleus to understand the role of the protein.
FIG. 3. Exportation of GFP-PIP 3 BP from nucleus by the constitutively active PI 3-kinase. A, Western blotting of the PIP 3 BPs. COS-7 cells were transfected with the constructs coding for GFP-PIP 3 BP or Myc-PIP 3 BP with or without the construct for the BD110 expression. Distribution of PIP 3 BPs was analyzed by Western blotting with anti-GFP polyclonal antibody or with anti-Myc monoclonal antibody, 9E10 (15). B, distribution of GFP-PIP 3 BPs in various conditions. GFP fusion proteins of wild type PIP 3 BP or PIP 3 BP with point mutations in the PH domains were co-expressed with the BD110 protein or the kinase negative version of it in COS-7 cells. The cells whose fluorescence is clearly found in the nucleus were scored. More than 1000 cells were counted, and the percentages to the total cells expressing PIP 3 BP are shown. Control, wild type PIP 3 BP ϩ BD110; WT, wild type PIP 3 BP ϩ BD110, treated with 10 Ϫ7 M wortmannin for 30 min; PH(Ϫ), PIP 3 BP with point mutations of the PH domains ϩ BD110; BDKN, wild type PIP 3 BP ϩ kinase negative BD110.
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