Nuclear localization of phosphatidylinositol 4-kinase ß

Petra de Graaf, Elsa E. Klapisz, Thomas K. F. Schulz, Alfons F. M. Cremers, Arie J. Verkleij and Paul M. P. van Bergen en Henegouwen*

Molecular Cell Biology, Institute of Biomembranes, Universiteit Utrecht, The Netherlands

* Author for correspondence (e-mail: bergenp{at}bio.uu.nl )

Accepted 18 January 2002


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Whereas most phosphatidylinositol 4-kinase (PtdIns 4-kinase) activity is localized in the cytoplasm, PtdIns 4-kinase activity has also been detected in membranedepleted nuclei of rat liver and mouse NIH 3T3 cells. Here we have characterized the PtdIns 4-kinase that is present in nuclei from NIH 3T3 cells. Both type II and type III PtdIns 4-kinase activity were observed in the detergent-insoluble fraction of NIH 3T3 cells. Dissection of this fraction into cytoplasmic actin filaments and nuclear lamina-pore complexes revealed that the actin filament fraction contains solely type II PtdIns 4-kinase, whereas lamina-pore complexes contain type III PtdIns 4-kinase activity. Using specific antibodies, the nuclear PtdIns 4-kinase was identified as PtdIns 4-kinase ß. Inhibition of nuclear export by leptomycin B resulted in an accumulation of PtdIns 4-kinase ß in the nucleus. These data demonstrate that PtdIns 4-kinase ß is present in the nuclei of NIH 3T3 fibroblasts, suggesting a specific function for this kinase in nuclear processes.

Key words: Phosphatidylinositol 4-kinase, Detergent insoluble fraction, Actin filaments, Nucleus, NIH 3T3 fibroblasts


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The metabolism of phosphoinositides is known to play a crucial role in the transduction of signals triggered by a variety of hormones and growth factors. In particular, phosphatidylinositol(4,5)bisphosphate (PtdIns(4,5)P2) has been shown to be involved in the formation of stress fibers and focal adhesions via interactions with several actin-binding proteins, including profilin, gelsolin and N-WASP (Isenberg and Niggli, 1998Go). In addition, products of phosphoinositide 3-kinase (PI 3-kinase), PtdIns(3)P, PtdIns(3,4)P2 and PtdIns(3,4,5)P3 have been shown to act as lipid second messengers by binding to specific protein domains such as the Pleckstrin Homology, Phox- and FYVE domains and have been implicated in many intracellular processes such as intracellular trafficking, mitogenesis and actin rearrangements (Fruman et al., 1999Go; Kanai et al., 2001Go; Lemmon and Ferguson, 1998Go). An important precursor in both signaling pathways is PtdIns(4)P, which is synthesized by phosphatidylinositol 4-kinase (PtdIns 4-kinase).

PtdIns kinases from mammalian cells have been divided into three types (Endemann et al., 1987Go; Whitman et al., 1987Go). Type I is a PI 3-kinase, which is strongly inhibited by non-ionic detergents such as Triton X-100 (Whitman et al., 1988Go). Type II and type III PtdIns kinases are both PtdIns 4-kinases and are activated by non-ionic detergents. Different inhibitors can be used to discriminate between type II and III PtdIns 4-kinases. Adenosine inhibits only type II PtdIns 4-kinase, whereas both the PI 3-kinase inhibitors wortmannin and LY294002 inhibit only type III PtdIns 4-kinase. The latter requires a much higher concentration than for inhibition of PI 3-kinase (Downing et al., 1996Go).

We have previously reported that PtdIns 4-kinase activity is present in the detergent-insoluble fraction and membranedepleted nuclei of NIH 3T3 cells (Payrastre et al., 1992Go; Payrastre et al., 1991Go). Further fractionation of the nuclei showed that PtdIns 4-kinase activity was specifically associated with the lamina-pore complex, whereas PtdIns(4)P 5-kinase activity was detected in the internal matrix fraction (Payrastre et al., 1991Go). The importance of a nuclear inositol lipid metabolism is stressed by the finding that nuclear phosphoinositide levels were found to decrease during the S-phase of the cell cycle (York and Majerus, 1994Go). Furthermore, in Friend erythroleukaemia cells it was shown that insulin growth factor-1 (IGF-1) stimulates the hydrolysis of nuclear but not cytoplasmic phosphoinositides (Divecha et al., 1991Go). Recently, a large increase in nuclear PtdIns(5)P mass was observed during the G1 phase of murine erythro leukaemia cells, which suggests a role for this lipid in cell proliferation (Clarke et al., 2001Go).

In this paper we have characterized the PtdIns 4-kinase activity that is present in the detergent-insoluble fraction and membrane-depleted nuclei of NIH 3T3 fibroblasts. We show that a mixture of type II and type III PtdIns 4-kinase activity is bound to the detergent-insoluble fraction of NIH 3T3 cells. Further dissection of this fraction revealed that the PtdIns 4-kinase activity in the actin filament fraction is predominately type II whereas the PtdIns 4-kinase activity present in isolated lamina-pore complexes is type III. Fractionation studies indicate that the nuclear type III kinase is PtdIns 4-kinase ß (PI4Kß). The localization of PtdIns 4-kinase ß in the nucleus suggests a specific role for this PtdIns 4-kinase in the nuclear inositol cycle.


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Materials
The antibody directed against lamin A/C (41CC4) was a generous gift of R. van Driel (E.C. Slater Institute, University of Amsterdam, The Netherlands), and the antibody against PI4Kß was a generous gift of Rachel Meyers (MIT, Boston, USA) (Meyers and Cantley, 1997Go). The monoclonal antibody against actin was from Amersham (Amersham International, UK), the antibodies against caveolin-1 and against GM 130 were from R&D Systems (Minneapolis, USA), the antibody against myc-epitope 9E10 was from Roche Molecular Biochemicals (Germany) and the monoclonal antibody against tubulin was from Calbiochem (San Diego, USA). The secondary antibodies donkey anti-mouse-horse radish peroxidase and donkey anti-rabbit-horse radish peroxidase were from Jackson ImmunoResearch Laboratories Inc. (West Grove, USA). Leptomycin B and lipids were obtained from Sigma Co. (St. Louis, USA), and [{gamma}-32P]-ATP was from Amersham. Restriction enzymes were obtained from Roche Molecular Biochemicals.

Construction of eukaryotic expression vector for epitopetagged PI4K230 and PI4Kß
The human PI4K230 cDNA was a kind gift from Thor Gehrmann, and details of it have been published previously (Gehrmann et al., 1999Go). The cDNA was cut out of the pFastBac HTa vector (GibcoBRL, Paisley, UK) using EcoRI and XhoI and cloned into the pcDNA3.1 zeo+ vector (Invitrogen, USA). The cDNA was tagged with the myc-epitope (MEQKLISEEDL) using an oligo-linker with restriction sites HindIII and EcoRI. The human PI4Kß cDNA was obtained by PCR from the human brain QUICK-Clone TM cDNA library (Clontech, USA) using the Expand High-Fidelity System (Roche Molecular Biochemicals, Germany). Primers were designed on the basis of the sequence of PI4Kß (Meyers and Cantley, 1997Go), with the restriction sites XhoI (forward primer) and XbaI (reverse primer). The cDNA was cloned into the pcDNA3.1 zeo+ vector (Invitrogen, USA) and tagged with the coding sequence of the myc epitope (MEQKLISEEDL) using an oligo-linker with restriction sites KpnI and XhoI.

Cell culture
Mouse NIH 3T3 fibroblasts (ATCC # CRL-1658), Chinese hamster ovary cells (CHO-K1, ATCC # CCL-61) and COS-1 cells (ATCC # CRL-1650) were grown in Dulbecco's modified Eagle's medium supplemented with 7.5% fetal calf serum (GibcoBRL, Paisley, UK) at 37°C in a humidified atmosphere containing 5% CO2. Cells were continuously grown to near confluency in 150 cm2 culture dishes (GibcoBRL, Paisley, UK). CHO and COS-1 cells were transfected using FuGENE 6 transfection reagent (Roche Molecular Biochemicals, Germany) according to the manufacturer.

Production of monoclonal antibody against PI4K230
The DNA fragment coding for amino acids 1691-1779 of PI4K230 was obtained by PCR from the human brain QUICK-CloneTM cDNA library (Clontech, USA) using the Expand High-Fidelity System (Roche Molecular Biochemicals, Germany). This fragment was cloned into the pGEX-2T vector (Amersham, UK). After transformation of this vector into DH5{alpha}, a GST fusion protein was isolated and cut with thrombin as described previously (Whitehead et al., 1999Go). Mice were injected with 50 µg of purified protein mixed (1:1) with Freund's complete adjuvants; the GST fusion protein was used for further screening. Production and isolation of the monoclonal antibody against PI4K230 was performed according to standard procedures (Schulz et al., 1987Go).

Isolation of the detergent insoluble fraction
Cells were grown to near confluency in 150 cm2 culture dishes and washed twice with ice-cold phosphate-buffered saline pH 7.4 (PBS). DIF was prepared as described by Van Bergen en Henegouwen et al. (Van Bergen en Henegouwen et al., 1989Go) with minor modifications. Cells were lysed for 5 minutes in 2 ml cytoskeleton-stabilizing (CSK) buffer (20 mM Hepes pH 7.4, 250 mM sucrose, 3 mM MgCl2, 150 mM NaCl, 1 mM Na3VO4, 1 mM benzamidine, 1 mM PMSF and 0.5% Triton X-100). After removal of the lysate the remaining DIF in the dish was briefly washed with CSK-buffer without detergent and scraped in 50 mM Tris-HCl (pH 7.4, with 1 mM Na3VO4, 1 mM benzamidine and 1 mM PMSF) and immediately used for assaying enzymatic activities.

Isolation of actin filaments
Cells were washed twice with ice cold PBS, and DIF was prepared as described above. After washing the DIF, the dish was incubated on ice with actin depolymerization buffer (20 mM Hepes pH 7.4, 250 mM sucrose, 3 mM MgCl2, 150 mM NaCl, 0.15% Triton X-100, 0.3 M KI, 1 mM EGTA, 1 mM Na3VO4, 1 mM benzamidine and 1 mM PMSF). After 10 minutes the lysate was centrifuged at 10,000 g for 20 minutes, and the supernatant was dialyzed overnight against CSK-buffer (20 mM Hepes pH 7.4, 250 mM sucrose, 3 mM MgCl2, 150 mM NaCl) without KI and Triton X-100. The polymerized actin was pelleted at 12,000 g for 5 minutes. The pellet was washed twice with ice cold CSK-buffer, resuspended in 50 mM Tris-HCl (pH 7.4 with 1 mM Na3VO4, 1 mM benzamidine and 1 mM PMSF) and immediately used.

Isolation of lamina-pore complexes
Membrane-depleted nuclei were isolated from NIH 3T3 fibroblasts according to a slightly modified procedure of Payrastre et al. (Payrastre et al., 1992Go). To isolate lamina-pore complexes, the membrane-depleted nuclei were incubated with DNaseI (200 units/107 nuclei) for 30 minutes at 37°C in hypotonic buffer (20 mM Hepes, pH 7.4, 10 mM KCl, 3 mM MgCl2, 1 mM Na3VO4, 1 mM benzamidine, 1 mM PMSF). (NH4)2SO4 was added to a final concentration of 0.25 M. After 15 minutes on ice the nuclear matrices were centrifuged at 1000 g for 5 minutes and washed once with hypotonic buffer without detergent. In order to remove the internal matrix, the nuclear matrices were incubated with 0.25 M (NH4)2SO4 and 40 mM dithiothreitol (DTT) for 20 minutes at 37°C. The peripheral matrices were centrifuged at 10,000 g for 5 minutes, washed with ice cold hypotonic buffer without detergent, resuspended in the same buffer and used immediately.

Lipid kinase assay
Lipid kinase activities were measured as previously described by Payrastre et al. (Payrastre et al., 1991Go) in a final volume of 200 µl containing 50 mM Tris-HCl (pH 7.4), 10 mM MgCl2, 50 µM ATP, 10 µCi [{gamma}-32P] ATP, 0.3% Triton X-100, 0.2 mg/ml PtdIns/PtdChol vesicles (2:1) and 30-50 µg protein. Proteins were sonicated three times for 10 seconds on ice prior to use. The inhibitors adenosine (200 µM), wortmannin (1 µM) or LY294002 (10 µM) were added to the reaction mixture when indicated. The reaction was started by adding the PtdIns/PtdChol vesicles and carried out at 37°C for 20 minutes under constant shaking. Under these conditions, lipid synthesis was linear with protein concentration and with time. The reaction was stopped by adding 400 µl chloroform/methanol (1:1) and the lipids were immediately extracted following the modified method of Bligh and Dyer (Bligh and Dyer, 1959Go; Payrastre et al., 1991Go). Phosphoinositides were separated by thin layer chromatography (TLC) on silica-gel-coated glass plates using chloroform/methanol/4.3 M NH4OH (90/70/20) as a solvent (Gonzales-Sastre and Floch-Pi, 1968Go). TLC plates were analyzed using a phosphoimager (Phosphoimager SITM, Molecular Dynamics Inc.), and the spots were quantified using ImageQuaNT software.

Gel electrophoresis and western blotting
Protein concentrations were determined according to Peterson (Peterson, 1977Go). Protein samples were resuspended in sample buffer (60 mM Tris-HCl, 10% glycerol (v/v), 45 mM dithiothreitol, 80 mM sodium dodecyl sulfate, pH 6.8), separated by SDS-PAGE and blotted onto PVDF membrane. The membrane was blocked using 3% milk powder (Protifar, Nutricia, Zoetermeer, The Netherlands) in TBST (20 mM Tris-HCl pH 7.4, 150 mM NaCl and 0.1% Tween-20) for 45 minutes and incubated with the primary antibody diluted 1:5,000 (or 1:40,000 for PI4Kß) in 0.3% milk powder in TBST for 1 hour. The membrane was washed four times in the same buffer for 5 minutes each and subsequently incubated with the secondary antibody (donkey anti-mouse-horse radish peroxidase, or donkey anti-rabbit-horse radish peroxidase for PI4Kß) diluted 1:10,000 in 0.3% milk powder in TBST for 45 minutes. The blot was washed three times with the same buffer for 5 minutes and once for 5 minutes in TBST. Proteins were detected using the chemiluminescence procedure as described by the manufacturer (Lumilight Plus, Roche Diagnostics), and visualized using a lumino-imager (FluorS, BioRad)

Immunofluorescence
COS-1 cells were seeded on 15 mm cover slips and, when indicated, transiently transfected with myc-PI4Kß using FuGENE 6 transfection reagent (Roche Molecular Biochemicals, Germany) according to the manufacturer. Cells were fixed for 20 minutes in 4% formaldehyde, washed twice with PBS, followed by a 5 minutes incubation with 0.2% Triton X-100 in PBS. Cover slips were washed twice with PBS followed by a 10 minutes quenching of formaldehyde using 50 mM Glycine in PBS. After washing twice with 0.2% Gelatin in PBS (PBG) cells were incubated for 60 minutes with anti-myc antibody (9E10, Roche Molecular Biochemicals), at a concentration of 800 ng/ml in PBG. After washing four times for 5 minutes, cells were incubated with GAM-Cy3 (Jackson ImmunoResearch) at a concentration of 1.3 µg/ml in PBG for 45 minutes. After washing four times with PBG and once with PBS, cells were imbedded in mowiol/PPD and analyzed by confocal microscopy.


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PtdIns 4-kinase activity in the detergent insoluble fraction of NIH 3T3 fibroblasts is a mixture of type II and type III activity
To discriminate between type II and III PtdIns 4-kinases we used the PtdIns kinase inhibitors adenosine and wortmannin. Adenosine specifically inhibits type II PtdIns 4-kinase activity, whereas wortmannin can be used to inhibit type III PtdIns 4-kinase activity. In total cell lysates, PtdIns 4-kinase activity was significantly inhibited by 200 µM adenosine (Fig. 1A). In contrast, no effect of wortmannin was observed, which indicates that the majority of the PtdIns 4-kinase activity in total cell lysates is a type II PtdIns 4-kinase. To exclude any contribution of PI 3-kinase activity, the non-ionic detergent Triton X-100 was added to the lipid kinase assay, which inhibits PI 3-kinase activity. This was controlled by adding a low concentration of LY294002, and this inhibitor did not reduce PtdIns kinase activity any further.



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Fig. 1. Lipid kinase activity in total cell lysate and detergent insoluble fraction. (A) Total cell lysate (10 µg) and detergent insoluble fraction (DIF) (50 µg) from NIH 3T3 cells were applied to an in vitro lipid kinase assay using PtdIns as substrate. After an incubation of 20 minutes at 37°C, the lipids were extracted, separated on TLC and the PtdIns(4)P spots were quantified (total cell lysates, s.e.m., n=3; DIF, s.e.m., n=4) (*P<0.01). Adenosine, 200 µM; wortmannin, 1 µM; LY294002, 10 µM. (B) Total cell lysates (10 µg) from NIH 3T3 cells were incubated with increasing amounts of adenosine and applied to an in vitro kinase assay. Results are expressed as the percentage of control activity without adenosine.

 

We next analyzed the PtdIns 4-kinase activity that is present in the detergent-insoluble fraction (DIF) of NIH 3T3 cells. This fraction was isolated by a treatment of the cells with the non-ionic detergent Triton X-100. The fraction of total PtdIns 4-kinase activity that is associated with the DIF of NIH 3T3 cells was approximately 11% of total PtdIns 4-kinase activity. Analysis of the PtdIns kinase activity that is associated with the detergent insoluble fraction showed no inhibition by adenosine, but this time a high concentration of wortmannin inhibited the lipid kinase activity by more than 60% (Fig. 1A). Again, no significant inhibition by a low concentration of LY294002 was observed. These observations demonstrate that the DIF of NIH 3T3 cells contains both type II and type III PtdIns 4-kinase activity.

An unexpected finding was that the type II PtdIns 4-kinase activity in total cell lysates could not be completely inhibited by adenosine. This observation suggests the existence of an adenosine-insensitive PtdIns 4-kinase activity. In order to investigate this possibility we analyzed the effect of higher concentrations of this inhibitor on PtdIns 4-kinase activity in total cell lysates. Adenosine at a concentration of 1 mM inhibited PtdIns 4-kinase activity by 90%, which indicates that the existence of an adenosine-insensitive pool is highly unlikely (Fig. 1B).

Type II and type III PtdIns 4-kinase activity is differentially localized in the DIF
The DIF is composed of cytoskeletal structures and membrane-depleted nuclei. In order to determine the location of the two types of PtdIns 4-kinases within the DIF, we dissected this fraction into cytoplasmic actin filaments and nuclear lamina-pore complexes. Actin filaments were isolated using an in vitro depolymerization/repolymerization reaction as described previously (Payrastre et al., 1991Go). Lamina-pore complexes were isolated from membrane-depleted nuclei according to Payrastre et al. (Payrastre et al., 1992Go). The purity of the nuclei was investigated enzymatically using 5'-nucleotidase, lactate dehydrogenase and antimycin A-insensitive NADH-cytochrome c reductase as markers for the plasma membrane, cytoplasm and endoplasmatic reticulum respectively, and their activity was found to be less than 1% in the nuclear fraction (Payrastre et al., 1992Go).

Both fractions were analyzed by western blotting using antibodies against different proteins that are characteristic for these fractions. Anti-caveolin-1 was used as a marker for the DIF, anti-GM130 as a marker for the Golgi apparatus, anti-actin as a marker for the cytoskeleton, and anti-lamin was used as a marker for the nuclear matrix. The DIF marker caveolin-1 is clearly present in the total cell lysate and in the DIF but strongly reduced in nuclear and actin filament fractions (Fig. 2). The Golgi marker GM130 is present in the total lysate but greatly reduced in the DIF. Furthermore, this marker is absent in the fractions containing actin filaments and nuclear matrix, indicating that these two fractions were not contaminated by Golgi proteins. The nuclear marker lamin is clearly present in the DIF and nuclear matrices and completely absent in the actin filament fraction. Lamin was also detected in the total cell lysate, but the intensity of signal for lamin was, for unknown reasons, less than that observed in the DIF. The cytoskeletal marker actin is present in the total cell lysate, the DIF and in the actin filaments. A small amount of actin is, however, also detectable in nuclear matrix fraction, suggesting the presence of nuclear actin or a small contamination of the nuclei by this cytoskeletal protein. By changing the isolation conditions we have tried to optimise the nuclear isolation procedure, but changes in the used detergent (NP40) or differences in salt conditions did not result in the extraction of actin (data not shown). This observation suggests that actin is a component of the nuclear matrix, an observation that is in agreement with previous studies (for a review, see Rando et al., 2000Go).



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Fig. 2. Western blot analysis of subfractions of the detergent insoluble fraction (DIF) of NIH 3T3 cells. Proteins from cell equivalents of cell lysate (CL), total DIF (DIF), nuclear matrix (NM) and actin-enriched fraction (AF) were separated on 10% SDS-PAGE and subsequently western blotted and incubated with antibodies directed against caveolin-1 (A), GM130 (B), actin (C) and lamin A/C (D).

 

Subsequently, we tested the effects of the different lipid kinase inhibitors on the PtdIns 4-kinase activity in these fractions. In contrast to wortmannin, adenosine significantly inhibited PtdIns 4-kinase activity present in the actin filament fraction (Fig. 3). In the nuclear fraction, however, a high concentration of wortmannin has a strong inhibitory effect on the formation of radioactive PtdIns(4)P, whereas adenosine had no significant effect on the PtdIns 4-kinase activity. These data demonstrate that isolated actin filaments contain type II PtdIns 4-kinase activity, whereas the nuclei contain PtdIns 4-kinase activity belonging to the type III family of PtdIns 4-kinases.



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Fig. 3. Lipid kinase activity in actin filaments and lamina-pore complexes. Proteins from actin filaments and lamina-pore complexes (both 50 µg) from NIH 3T3 cells were used in a lipid kinase assay as described in the legend to Fig. 1, and the PtdIns(4)P spots were quantified (actin filaments: s.e.m., n=8; lamina-pore complexes: s.e.m., n=7) (*P<0.01). Adenosine, 200 µM; wortmannin, 1 µM; LY294002, 10 µM.

 

PI4Kß is the nuclear type III PtdIns 4-kinase
At present, two type III PtdIns 4-kinases have been described: PI4K230 and PI4Kß (Gehrmann et al., 1999Go; Meyers and Cantley, 1997Go). In order to determine which of these PtdIns 4-kinases is present in NIH 3T3 cells we used specific antibodies against these two type III PtdIns 4-kinases. To perform these experiments we raised a monoclonal antibody against amino acids 1691-1779 of PI4K230 as described in the Materials and Methods. The polyclonal antibody against PI4Kß was obtained from R. Meyers (MIT, Boston, USA) (Meyers and Cantley, 1997Go). The monoclonal antibody reacted strongly with a myctagged PI4K230, which was expressed in COS-1 cells (Fig. 4). Remarkably, the anti-PI4K230 recognized several bands, whereas anti-myc showed a positive reaction with a single band. This is probably caused by N-terminal degradation, as an N-terminal myc-epitope was used. Furthermore, no positive reaction of the monoclonal anti-PI4K230 was found in lysates from mock-transfected COS-1 or NIH 3T3 cells. In addition, no signal was observed when this antibody was used with immunofluorescence microscopy on NIH 3T3, BHK or CHO cells (data not shown). In contrast, PI4Kß could easily be detected in lysates from both COS-1 and NIH 3T3 cells (Fig. 4). From these results we conclude that from the currently identified type III kinases only PI4Kß and not PI4K230 is present in COS-1 and NIH 3T3 cells.



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Fig. 4. PI4K230 is absent in NIH 3T3 and COS-1 cells. Cells were either mock transfected or transfected with cDNA encoding myctagged PI4K230. Proteins from cell lysates from equal amounts of cells were separated on 6% SDS-PAGE for PI4K230 and on 8% SDS-PAGE for PI4Kß and subsequently blotted and incubated with antibodies directed against myc epitope (9E10), PI4K230 and PI4Kß. Anti-PI4K230 was prepared as described in the Materials and Methods. Epitope-tagged PI4K230 was used as a control to demonstrate the expression of PI4K230 in COS-1 cells.

 

Subsequently, we analyzed whether PI4Kß is present in the nuclei of NIH 3T3 and CHO cells. Membrane-depleted nuclei were isolated, and proteins from total cell lysates, supernatant and nuclei were analyzed by western blotting. Anti-tubulin was used as a marker for the cytoplasm, and only a minor band was observed in the nuclear fraction (Fig. 5). PI4Kß is clearly present in the nuclear fraction of both NIH 3T3 and CHO cells. In addition, CHO cells transiently transfected with epitope (myc)-tagged PI4Kß, leading to a higher expression level of PI4Kß, shows a clear band representing PI4Kß in the nuclear fraction (Fig. 5).



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Fig. 5. The presence of PI4Kß in the nuclear fraction. Proteins from cell equivalents of cell lysate (CL), post nuclear supernatant (PNS) and five times cell equivalents of membrane-depleted nuclei (N) were separated on 8% SDS-PAGE and subsequently western blotted and incubated with antibodies directed against tubulin, PI4Kß and the myc-epitope (9E10). 3T3, proteins from mock-transfected NIH 3T3 cells; CHO, proteins from mock-transfected CHO cells, and CHO + mPI4Kß, CHO cells transfected with epitope-tagged PI4Kß (myc).

 

PI4Kß accumulates in the nucleus upon inhibition of nuclear export
To obtain further proof of the nuclear localization of PI4Kß, we performed immunofluorescence microscopy on normal COS-1 cells, and COS-1 cells transfected with epitope-tagged PI4Kß. A similar distribution of endogenous and transiently expressed PI4Kß was found. PI4Kß is mainly located in the cytoplasm and is concentrated at the Golgi complex. Only a weak staining of the nuclei was observed (Fig. 6A). A similar distribution of epitope-tagged PI4Kß was observed in transiently transfected COS-1 cells (Fig. 6C). Sequence analysis of PI4Kß showed that this kinase also contains two nuclear localization signals and a leucine-rich sequence resembling a nuclear export signal (NES). This type of NES is involved in the nuclear export of proteins regulated by Crm1, which can be inhibited by leptomycin B (Fornerod et al., 1997Go; Ossareh-Nazari et al., 1997Go). Control and transfected COS-1 cells were treated for 16 hours with 10 ng/ml leptomycin B, fixed and immunostained for PI4Kß. As shown in Figure 6B, leptomycin B induced an accumulation of endogenous PI4Kß in the nucleus. No staining is observed in nucleoli. Similar results were obtained with COS-1 cells that were transiently transfected with epitope-tagged PI4Kß (Fig. 6D). These data demonstrate that PI4Kß is exported from the nucleus in a Crm1-dependent way. Moreover, this indicates that in vivo PI4Kß translocates to the nucleus, supporting our biochemical data that PI4Kß is present in the nucleus.



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Fig. 6. PI4Kß accumulates in the nucleus upon leptomycin B treatment. COS-1 cells were mock transfected (A,B) or transfected with myc-PI4Kß (C,D). Cells were left untreated (control, A,C) or treated for 16 hours with 10 ng/ml leptomycin B (+LB, B,D). After incubation, cells were fixed and stained with anti-PI4Kß followed by GAR-Cy3 (A,B) or with an anti-myc antibody (9E10) followed by GAM-Cy3 (C,D).

 


    Discussion
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 Materials and Methods
 Results
 Discussion
 References
 
In this paper we have characterized the PtdIns 4-kinases that are present in the DIF and membrane-depleted nuclei of mouse NIH 3T3 cells. Although total cellular PtdIns 4-kinase activity could not be inhibited by wortmannin, the DIF contained a PtdIns 4-kinase activity that was significantly inhibited by wortmannin. This indicates that the majority of total PtdIns 4-kinase activity belongs to the type II kinases and, furthermore, that the DIF contains type III PtdIns 4-kinase activity. Further dissection of the DIF into a cytoskeletal and nuclear fraction revealed that the actin filament fraction predominantly contains type II PtdIns 4-kinase activity, whereas the nuclear PtdIns 4-kinase activity almost completely consists of type III PtdIns 4-kinase activity.

An important question is which of the known PtdIns 4-kinase isoforms is responsible for the observed kinase activities in the different fractions. Type II PtdIns 4-kinase is indicated as the PI4K55 and has recently been cloned by two independent groups (Barylko et al., 2001Go; Minogue et al., 2001Go). This type II PtdIns 4-kinase binds tightly to the membrane and bears little similarity to other known lipid or protein kinases. Two mammalian type III PtdIns 4-kinase isoforms have been described: PI4Kß, the orthologue of PtdIns 4-kinase from the budding yeast Saccharomyces cerevisiae Pik1p, and PI4K230, the mammalian orthologue for the yeast isoform Stt4p (Gehrmann and Heilmeyer Jr., 1998Go; Meyers and Cantley, 1997Go). Interestingly, Pik1p was found to bind to the nuclear pores of yeast nuclei, whereas Stt4p was strictly located in the cytoplasm (Flanagan et al., 1993Go; Yoshida et al., 1994Go). Both the human and bovine PI4K230 have been sequenced, and this isoform contains besides an SH3 and PH domain, two nuclear localization signals (NLS). Also PI4Kß contains two NLSs and one NES, which are all present in the N-terminal part of the protein.

To identify the type III isoform present in the nucleus we first analyzed which of these PtdIns 4-kinases is present in NIH 3T3 and COS-1 cells. Using specific antibodies against both isoforms we could not detect PI4K230 in these cells. This observation is in accordance with the work of Gehrmann and colleagues who have shown that PI4K230 is mainly expressed in the brain (Gehrmann et al., 1999Go). In contrast, western blot analysis of NIH 3T3 and COS-1 cells clearly showed the presence of this kinase in these cells. Interestingly, endogenous PI4Kß was also present in membrane-depleted nuclei from NIH 3T3 cells. An increase in nuclear presence was found when epitope-tagged PI4Kß was expressed in CHO cells. The nuclear presence of PI4Kß is not caused by contamination of the nuclear fraction by cytosolic proteins, as the nuclear preparations were negative for tubulin and the cis-Golgi marker GM130.

Previously, PI4Kß has been localized by fluorescence microscopy, and it was predominantly found at the Golgi complex (Wong et al., 1997Go). In our experiments we also observed a very weak staining of the nucleus, which is in agreement with the low level of nuclear PI4Kß, as determined with the western blotting experiments. The nuclear PtdIns 4-kinase is, however, shown to bind to the lamina-pore complex, which may be difficult to discern in the fluorescent light microscope (Payrastre et al., 1992Go). PI4Kß also contains, next to the two NLSs, a leucine-rich sequence resembling a nuclear export signal (NES). Nuclear export of a leucine-rich NES is regulated by Crm1, which can be blocked by leptomycin B (Ossareh-Nazari et al., 1997Go). Inhibition of nuclear export by leptomycin B resulted in an accumulation of PI4Kß in the nucleus. PI4Kß remained excluded from nucleoli. This suggests that PI4Kß is shuttling between the cytoplasm and the nucleus in a Crm1-dependent process. Our previous results showed that PtdIns 4-kinase activity is associated with the lamina-pore complex (Payrastre et al., 1992Go). It is tempting to suggest that a small fraction of PI4Kß that is transported into the nucleus remains in the nucleus as a result of its binding to a component of the nuclear-pore complex. This intranuclear location of PI4Kß is to be expected, because at this location it is in proximity to its substrate PtdIns, which was recently shown to be present in the nuclear envelope (Vann et al., 1997Go). The component of the lamina-pore complex that is responsible for the binding of PI4Kß remains to be elucidated.

The nuclear fraction of PI4ß activity may fulfil a role in nuclear inositol signaling (for a review, see Irvine, 2000Go). Stimulation of the cell with growth factors such as EGF and PDGF did not stimulate this lipid kinase (P.d.G, unpublished). On the other hand, the translocation of PI4Kß into the nucleus may result in the stimulation of the nuclear phosphoinositide cycle by inducing the generation of PtdIns(4)P, which is the substrate of the nuclear type I PtdIns(4)P 5-kinase. We have previously shown that this PtdIns P kinase activity is located in the inner nuclear matrix, which is consistent with the localization of the type I PtdIns(4)P 5-kinase in nuclear speckles (Boronenkov et al., 1998Go). Components of the mRNA machinery are also located in these nuclear speckles, suggesting a function for the nuclear PtdIns metabolism in mRNA processing. On the other hand, the products of PtdIns 4-kinase, PtdIns(4)P and inositol(1,4)bisphosphate may have a function themselves, as they are known to activate a low specificity form of DNA polymerase {alpha} in vitro (Sylvia et al., 1988Go). Moreover, the level of PtdIns(5)P was recently shown to increase dramatically during the G1 phase of murine erythro leukemia cells (Clarke et al., 2001Go). These data all point to a function for the nuclear inositol cycle in the regulation of cell proliferation and cell differentiation.

In conclusion, the results shown in this paper reveal that type II PtdIns 4-kinase activity is associated to actin filaments, whereas type III PtdIns 4-kinase activity is present in nuclei. The type III PtdIns 4-kinase was identified as the PI4Kß. Studies using leptomycin B suggest that the nuclear levels of PI4Kß are controlled by processes regulating the nuclear shuttling of this kinase.


    Acknowledgments
 
The authors wish to thank J. C. Stam (Molecular Cell Biology, Universiteit Utrecht, The Netherlands) and B. Payrastre (INSERM unit 326, Toulouse, France) for discussions and critical reading of the manuscript, and S. C. A. van den Berg for his assistance during the production of monoclonal antibodies against PI4K230.


    References
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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