Nuclear Translocation of Phospholipase C-δ1 Is Linked to the Cell Cycle and Nuclear Phosphatidylinositol 4,5-Bisphosphate
2005; Elsevier BV; Volume: 280; Issue: 23 Linguagem: Inglês
10.1074/jbc.m413813200
ISSN1083-351X
AutoresJonathan D. Stallings, Edward G. Tall, Srinivas Pentyala, Mario J. Rebecchi,
Tópico(s)Nuclear Structure and Function
ResumoNuclear phosphoinositides, especially phosphatidylinositol 4,5-bisphosphate, fluctuate throughout the cell cycle and are linked to proliferation and differentiation. Here we report that phospholipase C-δ1 accumulates in the nucleus at the G1/S boundary and in G0 phases of the cell cycle. Furthermore, as wild-type protein accumulated in the nucleus, nuclear phosphatidylinositol 4,5-bisphosphate levels were elevated 3–5-fold, whereas total levels were decreased compared with asynchronous cultures. To test whether phosphatidylinositol 4,5-bisphosphate binding is important during this process, we introduced a R40D point mutation within the pleckstrin homology domain of phospholipase C-δ1, which disables high affinity phosphatidylinositol 4,5-bisphosphate binding, and found that nuclear translocation was significantly reduced at G1/S and in G0. These results demonstrate a cell cycle-dependent compartmentalization of phospholipase C-δ1 and support the idea that relative levels of phosphoinositides modulate the portioning of phosphoinositide-binding proteins between the nucleus and other compartments. Nuclear phosphoinositides, especially phosphatidylinositol 4,5-bisphosphate, fluctuate throughout the cell cycle and are linked to proliferation and differentiation. Here we report that phospholipase C-δ1 accumulates in the nucleus at the G1/S boundary and in G0 phases of the cell cycle. Furthermore, as wild-type protein accumulated in the nucleus, nuclear phosphatidylinositol 4,5-bisphosphate levels were elevated 3–5-fold, whereas total levels were decreased compared with asynchronous cultures. To test whether phosphatidylinositol 4,5-bisphosphate binding is important during this process, we introduced a R40D point mutation within the pleckstrin homology domain of phospholipase C-δ1, which disables high affinity phosphatidylinositol 4,5-bisphosphate binding, and found that nuclear translocation was significantly reduced at G1/S and in G0. These results demonstrate a cell cycle-dependent compartmentalization of phospholipase C-δ1 and support the idea that relative levels of phosphoinositides modulate the portioning of phosphoinositide-binding proteins between the nucleus and other compartments. A distinct phosphoinositide cycle is present in nucleus, and growing evidence suggests its metabolism is important for DNA repair, mRNA export, and gene transcription (1Irvine R.F. Nat. Rev. Mol. Cell. Biol. 2003; 4: 349-360Crossref PubMed Scopus (312) Google Scholar, 2Divecha N. Banfic H. Treagus J.E. Vann L. Irvine R.F. D'Santos C. Biochem. Soc. 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Because phospholipase C (PLC) 1The abbreviations used are: PLC, phospholipase C; LMB, leptomycin B; PH, pleckstrin homology; EGFP, enhanced green fluorescence protein; GFP, green fluorescence protein; DAPI, 4′,6-diamidino-2-phenylindole; PBS, phosphate-buffered saline; BSA, bovine serum albumin; PI, phosphatidylinositol; PI(4)P, phosphatidylinositol 4-phosphate; PI(5)P, phosphatidylinositol 5-phosphate; PIP2, phosphatidylinositol 4,5-bisphosphate. is a key regulator of phosphoinositide metabolism at the plasma membrane, understanding what controls the localization of this enzyme to the nucleus and how its activity there affects nuclear phosphoinositide metabolism is important. In mammals, PLC is a 13-member family of phosphodiesterases (β, γ, δ, ϵ, and ζ subtypes) essential to a wide range of cellular responses, including exocytosis, endocytosis, gene transcription, cytoskeletal remodeling, and membrane trafficking (8Payrastre B. Missy K. Giuriato S. Bodin S. Plantavid M. Gratacap M. 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Rudge S.A. Scarlata S. Petrova V. McLaughlin S. Rebecchi M.J. Biochemistry. 1995; 34: 16228-16234Crossref PubMed Scopus (260) Google Scholar, 18Yagisawa H. Sakuma K. Paterson H.F. Cheung R. Allen V. Hirata H. Watanabe Y. Hirata M. Williams R.L. Katan M. J. Biol. Chem. 1998; 273: 417-424Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 19Williams R.L. Katan M. Structure (Lond.). 1996; 4: 1387-1394Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). This interaction facilitates association with the plasma membrane, particularly with ruffles, where PIP2 is enriched (20Tall E.G. Spector I. Pentyala S.N. Bitter I. Rebecchi M.J. Curr. Biol. 2000; 10: 743-746Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). When a point mutation in the PH domain (R40D) is introduced, however, the ability of PLCδ1 to target the plasma membrane is greatly reduced, resulting in a higher cytosolic concentration (18Yagisawa H. Sakuma K. Paterson H.F. Cheung R. Allen V. Hirata H. Watanabe Y. Hirata M. Williams R.L. Katan M. J. Biol. Chem. 1998; 273: 417-424Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Several PLC isoforms are detected in the nuclear compartment, primarily due to alternative spliced variation (21D'Santos C.S. Clarke J.H. Divecha N. Biochim. Biophys. Acta. 1998; 1436: 201-232Crossref PubMed Scopus (159) Google Scholar). PLCδ1, however, is the only isoform that has been demonstrated to continuously cycle between the nucleus and cytoplasm (22Yamaga M. Fujii M. Kamata H. Hirata H. Yagisawa H. J. Biol. Chem. 1999; 274: 28537-28541Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 23Okada M. Fujii M. Yamaga M. Sugimoto H. Sadano H. Osumi T. Kamata H. Hirata H. Yagisawa H. Genes Cells. 2002; 7: 985-996Crossref PubMed Scopus (22) Google Scholar). When Madin-Darby canine kidney cells are treated with leptomycin B (LMB), which inhibits CRM1-dependent nuclear export, transiently expressed GFP-PLCδ1 accumulates in the nucleus, consistent with nucleocytoplasmic shuttling (22Yamaga M. Fujii M. Kamata H. Hirata H. Yagisawa H. J. Biol. Chem. 1999; 274: 28537-28541Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). PLCδ1 is also detected in nuclei of rat hepatoma cells (24Asano M. Tamiya-Koizumi K. Homma Y. Takenawa T. Nimura Y. Kojima K. Yoshida S. J. Biol. Chem. 1994; 269: 12360-12366Abstract Full Text PDF PubMed Google Scholar) and astrocytes (25Shimohama S. Sumida Y. Fujimoto S. Matsuoka Y. Taniguchi T. Takenawa T. Kimura J. Biochem. Biophys. Res. Commun. 1998; 243: 210-216Crossref PubMed Scopus (14) Google Scholar). It is unclear, however, what regulates the steady-state distribution of this enzyme between the nuclear and extranuclear compartments. Yamaga et al. (22Yamaga M. Fujii M. Kamata H. Hirata H. Yagisawa H. J. Biol. Chem. 1999; 274: 28537-28541Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) have indicated that the R40A point mutation within the pleckstrin homology domain of PLCδ1 results in faster and greater nuclear accumulation under conditions in which nuclear export is blocked, possibly due to a greater fraction available to engage the import apparatus. Although mechanisms that regulate nuclear import, such as dimerization and phosphorylation, have not been ruled out, it is likely that a PIP2-mediated sequestration at the plasma membrane influences the steady-state distribution of PLCδ1 between the nuclear and cytoplasmic compartments. The idea that phospholipids influence compartmentalization of PI-binding proteins is growing and evident in the recent work with Tub, which binds PIP2 through its tubby domain (26Santagata S. Boggon T.J. Baird C.L. Gomez C.A. Zhao J. Shan W.S. Myszka D.G. Shapiro L. Science. 2001; 292: 2041-2050Crossref PubMed Scopus (311) Google Scholar), and ING2, which binds PI(5)P through its plant homeodomain finger (27Gozani O. Karuman P. Jones D.R. Ivanov D. Cha J. Lugovskoy A.A. Baird C.L. Zhu H. Field S.J. Lessnick S.L. Villasenor J. Mehrotra B. Chen J. Rao V.R. Brugge J.S. Ferguson C.G. Payrastre B. Myszka D.G. Cantley L.C. Wagner G. Divecha N. Prestwich G.D. Yuan J. Cell. 2003; 114: 99-111Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar). Upon G-protein-linked hydrolysis of PIP2, Tub translocates to the nucleus (26Santagata S. Boggon T.J. Baird C.L. Gomez C.A. Zhao J. Shan W.S. Myszka D.G. Shapiro L. Science. 2001; 292: 2041-2050Crossref PubMed Scopus (311) Google Scholar), whereas ING2 accumulates in the nucleus as the levels of nuclear PI(5)P increase (27Gozani O. Karuman P. Jones D.R. Ivanov D. Cha J. Lugovskoy A.A. Baird C.L. Zhu H. Field S.J. Lessnick S.L. Villasenor J. Mehrotra B. Chen J. Rao V.R. Brugge J.S. Ferguson C.G. Payrastre B. Myszka D.G. Cantley L.C. Wagner G. Divecha N. Prestwich G.D. Yuan J. Cell. 2003; 114: 99-111Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar). We set out to test whether high affinity PIP2 binding plays a role in targeting PLCδ1 to the nuclear compartment and whether this could be related to the cell cycle. Here we establish that PLCδ1 accumulates in the nucleus in G0 and at the G1/S-phase boundary and that its accumulation is significantly decreased by the R40D point mutation in its PH domain, which abrogates high affinity PIP2-binding. Furthermore, nuclear accumulation of PLCδ1 correlates with increased total nuclear levels of PIP2, consistent with a general mechanism by which phosphoinositides can influence the nuclear accumulation of proteins that bind these lipids. Synchrony, Flow Cytometry, and Cell Cycle Analysis—NIH-3T3 fibroblasts (American Type Culture Collection) were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum, 1 mm penicillin and streptomycin, 1 mm non-essential amino acids, and 1 mm sodium pyruvate (all supplements were from Invitrogen) at 37 °C in a 10% CO2 humidified incubator. To synchronize cells to G0, adherent cells were washed three times and maintained in a medium with reduced serum (0.5% fetal bovine serum) for 30 h, a technique that causes many cell types to exit the cell cycle and become quiescent (28Percival J.M. Thomas G. Cock T.A. Gardiner E.M. Jeffrey P.L. Lin J.J. Weinberger R.P. Gunning P. Cell Motil. Cytoskeleton. 2000; 47: 189-208Crossref PubMed Scopus (59) Google Scholar, 29Pagon Z. Volker J. Cooper G.M. Hansen U. J. Cell. Biochem. 2003; 89: 733-746Crossref PubMed Scopus (24) Google Scholar, 30Shaw R.J. McClatchey A.I. Jacks T. J. Biol. Chem. 1998; 273: 7757-7764Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). To synchronize cells to the G1/S boundary, adherent fibroblasts were twice blocked with 2 mm thymidine (6York J.D. Majerus P.W. J. Biol. Chem. 1994; 269: 7847-7850Abstract Full Text PDF PubMed Google Scholar, 31Flygare J. Falt S. Ottervald J. Castro J. Dackland A.L. Hellgren D. Wennborg A. Exp. Cell Res. 2001; 268: 61-69Crossref PubMed Scopus (51) Google Scholar). Briefly, cultures growing at an exponential rate were washed with Dulbecco's modified Eagle's medium and incubated in growth medium supplemented with 2 mm thymidine for 12–15 h, washed three times with growth medium, and incubated for an additional 8–10 h without thymidine. Cells were then treated again with growth medium supplemented with 2 mm thymidine for an additional 12–15 h. To release them from thymidine block, cells were washed three times in growth medium. To analyze DNA content, cells were washed with phosphate-buffered saline (PBS) (Invitrogen), harvested with trypsin/EDTA, washed again, and fixed in 3.7% formaldehyde with 0.1% BSA in PBS at 4 °C overnight. Cells were permeabilized in PBS containing 0.1% BSA and 0.1% Triton X-100 for 5 min, washed with PBS, and incubated in 50 mm citrate buffer with 50 μg/ml propidium iodide and 10 μg of RNase A for 30 min at 37 °C. After washing the cells again, cell cycle analysis was immediately performed using a fluorescence-activated cell sorter (FAC-Scan; BD Biosciences), and the data were analyzed using SyncWizard. myo-[3H]Inositol Labeling, Lipid Extraction, and Thin Layer Chromatography—Cells were cultured in growth media supplemented with 2–8 μCi/ml myo-[3H]inositol (PerkinElmer Life Sciences) in 60-mm dishes for 48–72 h to reach isotopic steady state. Cultures were then washed with Dulbecco's modified Eagle's medium and taken through the above synchronization procedures in the presence of radiolabel. Whole cell levels of phosphoinositides were measured essentially as described previously (32Auger K.R. Serunian L.A. Cantley L.C. Irvine R. Methods in Inositide Research. Raven Press, New York1990: 159-166Google Scholar). Briefly, synchronized cells were rinsed twice in ice-cold PBS with 1 mm calcium chloride and 0.5 mm magnesium chloride (PBS-Ca), followed by ice-cold PBS. Monolayers were scraped into 750 μl of methanol:0.1 m HCl (v/v, 1:1), placed in silianized borosilicate glass tubes, and mixed vigorously for 30 s. 500 μl of chloroform was then added to each sample, which was mixed for 30 s, and then placed on a rocker at room temperature for 15 min. The aqueous and organic phases were separated by centrifugation for 5 min at 1000 × g. The upper aqueous phase was removed and saved for inositol polyphosphate analysis. The lower phase was back extracted twice with 500 μl each of methanol:0.1 m EDTA (v/v, 1:0.9) and then dried under nitrogen and dissolved in chloroform:methanol (v/v, 1:1). Lipid extracts were applied to pre-scored Linear KD silica plates (Whatman) that were treated with 40% methanol, 1% potassium oxalate, and 1 mm EGTA in water and heat-activated. Purified PI, PI(4)P, and PIP2 (10 μg of each) were used as standards. The solvent system used to separate the lipids was chloroform:methanol:water:concentrated ammonium hydroxide (v/v/v/v, 60:47:11.3:2), similar to that described in Ref. 33Rebecchi M.J. Basic Medical Sciences Program (Pharmacology) of the Sackler Institute of Graduate Biomedical Sciences of the School of Medicine. New York University, New York1984: 168Google Scholar. Areas corresponding to migration of PI, PI(4)P, and PIP2 standards were scraped into vials containing 100 μl of methanol:10% Nonidet P-40 (v/v, 1:1). 4 ml of scintillation fluid (EcoLite) was added to each vial and counted the following day in a liquid scintillation spectrometer. The cpm values were normalized to total lipid phase-extractable phosphorus and specific activity of the radiolabeled medium. Subcellular Fractionation—Cells were lysed by osmotic swelling, similar to a previously described protocol (7Clarke J.H. Letcher A.J. D'Santos C.S. Halstead J.R. Irvine R.F. Divecha N. Biochem. J. 2001; 357: 905-910Crossref PubMed Scopus (126) Google Scholar). In 60-mm dishes, NIH-3T3 fibroblasts were briefly washed with ice-cold PBS-Ca. Cells were then treated briefly with PBS containing 1 mm EDTA, centrifuged at 600 × g for 5 min at 4 °C, and promptly resuspended in 500 μl of pre-chilled resuspension buffer (10 mm NaCl, 1.5 mm MgCl2, 10 mm Tris-HCl, pH 7.4) on ice for 7 min. The degree of cytoplasmic swelling was assessed by phase-contrast microscopy. The swollen cells were then transferred to a Dounce homogenizer and lysed by 20 strokes of the glass pestle. Separated nuclei were then layered onto a sucrose cushion (320 mm sucrose, 7.7 mm MgCl2, 2.1 mm EGTA, and 0.1 mm phenylmethylsulfonyl fluoride) and centrifuged at 300 × g for 3 min at 4 °C. The supernatant was removed, and nuclei were washed twice with 1 ml of resuspension buffer. 750 μl of methanol:0.1 m HCl was added to the nuclear pellet and taken through the lipid extraction procedure described above. The degree of contamination was assessed using standard compartmental markers for the cytoplasm (lactate dehydrogenase) and endoplasmic reticulum (cytochrome-c oxidase) according to the manufacturer's protocol (Sigma). DNA Constructs and Cell Transfection—Full-length human PLCδ1 cDNA was inserted into pEGFP-N1, a cytomegalovirus-driven mammalian expression vector that carries the gene for enhanced green fluorescent protein (EGFP; Clontech). This catalytically active chimera, designated PLC-δ1-EGFP, served as a template for the production of various mutant PLC-δ1-EGFP fusion proteins, as described previously (34Tall E.G. Physiology and Biophysics. Stony Brook University, Stony Brook, NY2002: 224Google Scholar). Briefly, a catalytically inactive (H356A) form and a double mutant (R40D/H356A) that fails to bind PIP2 with high affinity were constructed using a Pfu mutagenesis approach (Stratagene). The mutants were designated PLCδ1H356AEGFP and PLCδ1R40DH356AEGFP, respectively. We also utilized PH(δ1)EGFP (residues 1–126 of full-length PLCδ1 fused to EGFP) and its corresponding R40D point mutant (20Tall E.G. Spector I. Pentyala S.N. Bitter I. Rebecchi M.J. Curr. Biol. 2000; 10: 743-746Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Point mutations have been verified via DNA sequencing, in vitro binding, and enzymatic activity assays (35Tall E. Dorman G. Garcia P. Runnels L. Shah S. Chen J. Profit A. Gu Q.M. Chaudhary A. Prestwich G.D. Rebecchi M.J. Biochemistry. 1997; 36: 7239-7248Crossref PubMed Scopus (66) Google Scholar). For transient expression of all PLCδ1EGFP chimeric proteins, NIH-3T3 cells were plated on #1.0 borosilicate chambered glass coverslips (Nalge Nunc International) coated with fibronectin (50 μg/ml; Sigma) and allowed to adhere overnight. The following day, cells were transfected with FuGENE 6 reagent according to manufacturer's protocol (Roche Applied Science). Epifluorescence Microscopy and Image Deconvolution—Living cells transiently expressing fusion proteins were viewed in OptiMEM (Invitrogen) supplemented with 10% or 0.5% serum, 1 mm penicillin and streptomycin, 1 mm non-essential amino acids, 1 mm sodium pyruvate, and 2.25 mm CaCl2, with an Axiovert 200M inverted microscope (Zeiss). In some experiments, cells were fixed with 3.7% formaldehyde for 5 min and incubated in 4′,6-diamidino-2-phenylindole (DAPI; 5 μg/ml) for 5 min to clearly identify the nuclear compartment. Images were captured with an AxioCam 330mA 12-bit charge-coupled device camera (Zeiss) and viewed with Carl Zeiss Axovision 3.1 software. To avoid bias, samples were coded, and the investigator was blinded to the experimental conditions. Captured Z-stacks were deconvolved using a constrained iterative point spread function according to recommended Niquest criteria and analyzed. Images were further analyzed in Life Science Resources software. The degree of nuclear accumulation was determined by analysis of intensity profiles in both the nuclear and cytoplasmic compartments. The formula used to determine the average nuclear to cytoplasmic (N:Cavg) pixel intensity ratio is ∑[(Navg – Bavg)/(Cavg – Bavg)]/n, where Navg = average nuclear pixel intensity excluding nucleoli, Cavg = average cytoplasmic pixel intensity excluding perinuclear regions and vacuoles, Bavg = average background pixel intensity, and n = the number of cells sampled. A comparable method was used to determine the ratio of plasma membrane (Pavg) to cytoplasm pixel intensity for analysis of PH(δ1)EGFP compartmentalization in deconvolved images. The Pavg, however, was determined by sampling a stretch of plasma membrane no less than 10 μm in length. The method provides an average of plasma membrane intensities, which vary significantly along the membrane surface. Fixation and Indirect Immunofluorescence—Monolayers were rinsed once in PBS-Ca and then fixed with 3.7% formaldehyde solution (Fisher-Scientific) in PBS for 10 min at room temperature. Samples were then washed three times in PBS for 5 min and permeabilized with 0.2% Triton X-100 (Rohm & Haas Co.) in PBS for 5 min at room temperature. The detergent was replaced with blocking solution (PBS, 5% BSA (Sigma), 5% goat serum (Pierce)) for 30 min at room temperature. The blocking solution was then replaced with primary antibody solution (1:200 dilution of anti-PLCδ1 clone S-11-2 (05-343; Upstate) or 1:200 dilution of anti-PIP2 (Assay Design) in PBS with 1% BSA and 1% goat serum) and placed in an incubator at 37 °C for 1 h. In some experiments, anti-PIP2 was pre-incubated with vesicles containing 20 μm PI(4)P or PIP2 (vesicles prepared as a 70% phosphatidylcholine, 20% phosphatidylserine, 10% phosphatidylinositol mix) for 1 h. Samples were washed three times in 1 ml of PBS for 7 min and then incubated at 37 °C for 1 h in goat anti-mouse IgG Alexa488 secondary antibody (Molecular Probes) or goat anti-mouse IgG (H+L) conjugate Texas Red (Molecular Probes) diluted 1:3000 in PBS with 1% BSA and 1% goat serum. Each well was then washed three times in PBS for 7 min. Fixed cells were visualized by epifluorescence microscopy (Olympus IMT-2 inverted microscope with 100-watt Mercury arc lamp), and images were taken with Nikon Plan Fluor ×40 oil objective (numerical aperture, 1.3) and Olympix AstroCam (Life Science Resources). Images were processed and analyzed with Esprit imaging software (Life Science Resources). Alternatively, Z-stacks were obtained and deconvolved on the Zeiss microscope as described above. Western Blot Analysis—C6 glioma cells were grown to 50% confluence and fed normal growth media or blocked to the G1/S boundary as described above. After synchronization was complete and verified by fluorescence-activated cell-sorting analysis, an equal number of cells were harvested and washed in PBS-Ca. Nuclei were isolated as described above, without the use of a sucrose cushion. After several washes, the nuclear pellets were lysed with a high-salt extraction buffer (50 mm Tris-HCl, pH 8.0, 450 mm NaCl, 1 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, 1% Nonidet P-40, 1% mammalian protease inhibitor) on ice for 15 min and centrifuged for 5 min at 14,000 × g; the supernatant was removed and designated the nuclear fraction. Total volume of nuclear fraction (∼1 × 106 cells) was loaded into each lane and compared with 10% of the cytoplasmic fraction. Lysates were transferred to a polyvinylidene difluoride membrane (Bio-Rad), probed with the antibody described above (1:200 in PBS with 3% dried, fat-free milk), and detected using enhanced chemiluminescence (Amersham Biosciences). Statistical Analysis—All statistical analysis was preformed in GraphPad Prism. Nuclear to cytoplasm ratios were pooled from duplicate wells from multiple experiments. To determine whether the distribution of pooled data passed normality, we used the Kolomogorov-Smirnov test. To determine significance of the differences among mean values, we used a one-way analysis of variance and Newman-Kuels post test. PLCδ1, fused to EGFP, was expressed in NIH-3T3 mouse fibroblasts. These large cells have a relatively flat morphology that facilitates localization studies of this type. To prevent hydrolysis of cellular phosphoinositides, the enzyme was inactivated by H356A mutation. In our initial work, we observed that 6% of cells transiently expressing PLCδ1H356AEGFP had a greater degree of fluorescence associated with the nuclear compartment than the cytoplasmic compartment. Because PLCδ1 is a nucleocytoplasmic shuttling protein, we reasoned that nuclear accumulation could be attributed to a brief stage of the cell cycle, in which the steady-state distribution has shifted toward the nuclear compartment. Because this protein is well known to bind strongly to PIP2, we also hypothesized that a change in nuclear or extranuclear PIP2 could influence this redistribution. We first set out to determine whether and how nuclear PLCδ1 levels fluctuate during the cell cycle of NIH-3T3 fibroblasts and to measure, in parallel, changes in total cellular and nuclear PIP2 concentrations. Cell Cycle Synchrony and NIH-3T3 Cells—Subconfluent NIH-3T3 fibroblasts were synchronized by serum withdrawal or double thymidine block. When asynchronous cultures growing at logarithmic rates are withdrawn from serum, they exit the cell cycle at G1 and become quiescent (G0) (28Percival J.M. Thomas G. Cock T.A. Gardiner E.M. Jeffrey P.L. Lin J.J. Weinberger R.P. Gunning P. Cell Motil. Cytoskeleton. 2000; 47: 189-208Crossref PubMed Scopus (59) Google Scholar, 29Pagon Z. Volker J. Cooper G.M. Hansen U. J. Cell. Biochem. 2003; 89: 733-746Crossref PubMed Scopus (24) Google Scholar, 30Shaw R.J. McClatchey A.I. Jacks T. J. Biol. Chem. 1998; 273: 7757-7764Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). NIH-3T3 fibroblasts have asynchronous distributions of DNA content (Fig. 1A) that indicate distinct G1 (54.2%), S (34.7%), and G2/M (11.2%) populations. Reducing the serum to 0.5% for 30 h (Fig. 1B) resulted in cells with more G1 DNA content (81.7%) and fewer cells in S (14.8%) or G2/M (3.6%). Because G0 and G1 DNA content are equivalent with fluorescence-activated cell-sorting analysis, we assayed cell proliferation under logarithmic growth conditions (doubling time of 17 h; Fig. 1E) and compared that to growth in low-serum medium (doubling time of 29 h). These data show that reduced serum (0.5%) for 30 h enriched the number of quiescent NIH-3T3 cells in these cultures. 2 mm double thymidine block (Fig. 1C) was used to synchronize cultures to the G1/S checkpoint (6York J.D. Majerus P.W. J. Biol. Chem. 1994; 269: 7847-7850Abstract Full Text PDF PubMed Google Scholar). 97.2% of cells had S-phase DNA content after a 2 mm double thymidine block (G1, 1.7%; G2/M, 1.1%). As others have shown, washing out the thymidine releases cells from G1/S block (31Flygare J. Falt S. Ottervald J. Castro J. Dackland A.L. Hellgren D. Wennborg A. Exp. Cell Res. 2001; 268: 61-69Crossref PubMed Scopus (51) Google Scholar); as early as 3 h after release, 93.2% of cells were in G2/M as indicated by increased DNA content (Fig. 1D). These data demonstrate that these treatments synchronize NIH-3T3 cultures to a predicted DNA content, blocking them at well-defined points in the cell cycle. Nuclear Accumulation of PLCδ1 is Cell Cycle-dependent, Occurring at the G0 and the G1/S Checkpoint in NIH-3T3 Fibroblasts and at the G1/S Checkpoint in C6 Glioma Cells—To explain the degree of variance in nuclear PLCδ1 levels in asynchronous cell populations, we hypothesized that the enzyme accumulated in the nucleus in a cell cycle-dependent manner. We found that when cells were blocked at G0 or G1/S, the ratios of nuclear to cytoplasmic PLCδ1H356AEGFP intensity were enhanced by 1.4- and 2.0-fold, respectively, indicating that PLCδ1 accumulated in the nucleus during these stages of the cell cycle (Fig. 2, B and C) as a result of translocation, when compared with asynchronous cultures. Treatment with leptomycin B, a toxin used to block CRM1-driven nuclear export (Fig. 2, A and D), showed similar results (36Kudo N. Wolff B. Sekimoto T. Schreiner E.P. Yoneda Y. Yanagida M. Horinouchi S. Yoshida M. Exp. Cell Res. 1998; 242: 540-547Crossref PubMed Scopus (715) Google Scholar, 37Yashiroda Y. Yoshida M. Curr. Med. Chem. 2003; 10: 741-748Crossref PubMed Scopus (88) Google Scholar). To determine whether transient expression of PLCδ1H356AEGFP led to cell cycle synchrony, we analyzed DNA content by fluorescence-activated cell-sorting analysis. We found that cell cycle partitioning was similar to that of non-transfected cells, suggesting that the catalytically inactive mutant did not disrupt cell cycle progression in these fibroblasts (data not shown). Furthermore, synchronized NIH-3T3 cells expressing PLCδ1H356AEGFP were also released from thymidine block and observed over time; 4 h later, PL
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