Shuttling of CTP:Phosphocholine Cytidylyltransferase between the Nucleus and Endoplasmic Reticulum Accompanies the Wave of Phosphatidylcholine Synthesis during the G0 → G1 Transition
1999; Elsevier BV; Volume: 274; Issue: 37 Linguagem: Inglês
10.1074/jbc.274.37.26240
ISSN1083-351X
AutoresI C Northwood, Amy H.Y. Tong, Bryan D. Crawford, Adrienne E. Drobnies, Rosemary B. Cornell,
Tópico(s)Mitochondrial Function and Pathology
ResumoThe transition from quiescence (G0) into the cell division cycle is marked by accelerated phospholipid turnover. We examined the rates of phosphatidylcholine (PC) synthesis and the activity, membrane affinity, and intracellular localization of the rate-limiting enzyme in the synthesis of PC, CTP:phosphocholine cytidylyltransferase (CT) during this transition. The addition of serum to quiescent IIC9 fibroblasts resulted in a wave of PC synthesis beginning at ∼10 min, peaking at ∼3 h with a >10-fold increase in rate, and declining to near basal rates by 10 h. CT activity, monitored in situ, was elevated ∼3-fold between 1 and 2 h postserum. Neither CT mass nor its phosphorylation state changed during the surge in PC synthesis and CT activity. On the other hand, the ratio of particulate/soluble CT surged and then receded in concert with the wave of PC synthesis. During quiescence, CT was confined to the nucleus, as assessed by indirect immunofluorescence. Within 10 min after serum stimulation, a portion of the CT fluorescence appeared in the cytoplasm, where it intensified until ∼4 h postserum. Thereafter, the cytoplasmic CT signal waned, while the nuclear signal increased, and by 8 h CT was once again predominantly nuclear. The dynamics of CT's apparent translocation in and out of the nucleus paralleled the wave of PC synthesis and the solubility changes of CT. Cytoplasmic CT co-localized with BiP, a resident endoplasmic reticulum protein, in a double labeling experiment. These data suggest that the wave of PC synthesis that accompanies the G0 → G1 transition is regulated by the coordinated changes in CT activity, membrane affinity, and intracellular distribution. We describe for the first time a redistribution of CT from the nucleus to the ER that correlates with an activation of the enzyme. We propose that this movement is required for the stimulation of PC synthesis during entry into the cell cycle. The transition from quiescence (G0) into the cell division cycle is marked by accelerated phospholipid turnover. We examined the rates of phosphatidylcholine (PC) synthesis and the activity, membrane affinity, and intracellular localization of the rate-limiting enzyme in the synthesis of PC, CTP:phosphocholine cytidylyltransferase (CT) during this transition. The addition of serum to quiescent IIC9 fibroblasts resulted in a wave of PC synthesis beginning at ∼10 min, peaking at ∼3 h with a >10-fold increase in rate, and declining to near basal rates by 10 h. CT activity, monitored in situ, was elevated ∼3-fold between 1 and 2 h postserum. Neither CT mass nor its phosphorylation state changed during the surge in PC synthesis and CT activity. On the other hand, the ratio of particulate/soluble CT surged and then receded in concert with the wave of PC synthesis. During quiescence, CT was confined to the nucleus, as assessed by indirect immunofluorescence. Within 10 min after serum stimulation, a portion of the CT fluorescence appeared in the cytoplasm, where it intensified until ∼4 h postserum. Thereafter, the cytoplasmic CT signal waned, while the nuclear signal increased, and by 8 h CT was once again predominantly nuclear. The dynamics of CT's apparent translocation in and out of the nucleus paralleled the wave of PC synthesis and the solubility changes of CT. Cytoplasmic CT co-localized with BiP, a resident endoplasmic reticulum protein, in a double labeling experiment. These data suggest that the wave of PC synthesis that accompanies the G0 → G1 transition is regulated by the coordinated changes in CT activity, membrane affinity, and intracellular distribution. We describe for the first time a redistribution of CT from the nucleus to the ER that correlates with an activation of the enzyme. We propose that this movement is required for the stimulation of PC synthesis during entry into the cell cycle. phosphatidylcholine choline phosphotransferase CTP:phosphocholine cytidylyltransferase diacylglycerol fetal bovine serum endoplasmic reticulum minimal essential medium Dulbecco's modified Eagle's medium bovine serum albumin phosphate-buffered saline The regulation of phospholipid synthesis during the cell cycle is an important, yet understudied, problem. Phospholipid synthesis is required, not only to double the membrane mass, but also to replace phospholipid degraded by phospholipases. There are data suggesting regulated and periodic fluctuation of phospholipid synthesis and turnover rates during the cell cycle (1Jackowski S. J. Biol. Chem. 1996; 271: 20219-20222Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). For example, in BAC1.2F5 macrophage cells, phosphatidylcholine (PC)1 turnover rates were high during G1 and low during S (2Jackowski S. J. Biol. Chem. 1994; 269: 3858-3867Abstract Full Text PDF PubMed Google Scholar). PC is typically the major phospholipid of animal cells, and is a precursor to the synthesis of three other phospholipids: phosphatidylethanolamine, sphingomyelin, and phosphatidylserine. There is evidence that cell cycle progression may be sensitive to membrane PC content. Choline deprivation of WI-38 fibroblasts, L6 myoblasts, or C3H/10T½ fibroblasts led to decreased PC synthesis and mass and arrest in G1 (3Cornell R. Grove G.L. Rothblat G.H. Horwitz A.F. Exp. Cell Res. 1977; 109: 299-307Crossref PubMed Scopus (68) Google Scholar, 4Terce F. Brun H. Vance D.E. J Lipid Res. 1994; 35: 2130-2142Abstract Full Text PDF PubMed Google Scholar). The addition of choline (or lyso-PC, which is rapidly acylated to form PC) restored PC content and progression into S phase. Delipidated serum on its own did not effectively restore cell cycling (4Terce F. Brun H. Vance D.E. J Lipid Res. 1994; 35: 2130-2142Abstract Full Text PDF PubMed Google Scholar). These results suggest that serum growth factors cannot substitute for choline in the re-establishment of the cell cycle following choline starvation and that PC rather than another choline metabolite is the regulating molecule. The timing of the addition of choline during G1 to allow normal entry into S phase suggested a requirement for PC late in G1 (4Terce F. Brun H. Vance D.E. J Lipid Res. 1994; 35: 2130-2142Abstract Full Text PDF PubMed Google Scholar). Chinese hamster ovary cells harboring a temperature-sensitive CT do not synthesize PC at 40 °C, and the cells accumulate in G1. If not rescued by the addition of PC or lyso-PC, the cells undergo apoptosis rather than allowing DNA synthesis to occur (5Cui Z. Houweling M. Chen M.H. Record M. Chap H. Vance D.E. Terce F. J. Biol. Chem. 1996; 271: 14668-14671Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). Induction of apoptosis in HeLa cells by the CT inhibitor, edelfosine, was prevented by overexpression of CT or by lyso-PC (6Baburina I. Jackowski S. J. Biol. Chem. 1998; 273: 2169-2173Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). G1 arrest and induction of apoptosis in A549 cells by farnesol and geranylgeraniol, which act as competitive inhibitors of the final enzyme in the PC synthesis pathway, can be prevented by PC (7Miquel K. Pradines A. Terce F. Selmi S. Favre G. J. Biol. Chem. 1998; 273: 26179-26186Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Together, these studies suggest the possibility that the membrane PC content could relay to a cell cycle check point late in G1. What regulates the synthesis of PC during the cell cycle? Under most conditions, the rate-limiting and regulated step in PC synthesis is the formation of CDP-choline, catalyzed by CTP:phosphocholine cytidylyltransferase (CT). CT is regulated post-translationally by reversible association with membrane lipids, which are required for its activity (8Tronchere H. Record M. Terce F. Chap H. Biochim. Biophys. Acta. 1994; 1212: 137-151Crossref PubMed Scopus (99) Google Scholar, 9Cornell R.B. Gross R. Advances in Lipobiology. 1. JAI Press, London1996: 1-38Google Scholar, 10Cornell R.B. Biochem. Soc. Trans. 1998; 26: 539-544Crossref PubMed Scopus (27) Google Scholar, 11Kent C. Biochim. Biophys. Acta. 1997; 1348: 79-90Crossref PubMed Scopus (192) Google Scholar). The equilibrium between soluble and membrane-bound forms is influenced by the lipid composition of the target membrane and by the phosphorylation state of the enzyme. Phosphorylation on multiple C-terminal sites stabilizes the soluble form of the enzyme (12Wang Y. Kent C. J. Biol. Chem. 1995; 270: 17843-17849Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 13Yang W. Jackowski S. J. Biol. Chem. 1995; 270: 16503-16506Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 14Arnold R.S. DePaoli-Roach A.A. Cornell R.B. Biochemistry. 1997; 36: 6149-6156Crossref PubMed Scopus (61) Google Scholar). The lipid second messengers phosphatidic acid and diacylglycerol promote CT-membrane binding (14Arnold R.S. DePaoli-Roach A.A. Cornell R.B. Biochemistry. 1997; 36: 6149-6156Crossref PubMed Scopus (61) Google Scholar, 15Arnold R.S. Cornell R.B. Biochemistry. 1996; 35: 9917-9924Crossref PubMed Scopus (85) Google Scholar). Pretranslational regulation of CT has also been observed in response to growth factors (16Tessner T.G. Rock C.O. Kalmar G.B. Cornell R.B. Jackowski S. J. Biol. Chem. 1991; 266: 16261-16264Abstract Full Text PDF PubMed Google Scholar, 17Houweling M. Tijburg L.B. Vaartjes W.J. Batenburg J.J. Kalmar G.B. Cornell R.B. Van Golde L.M. Eur. J. Biochem. 1993; 214: 927-933Crossref PubMed Scopus (29) Google Scholar) and during lung development (18Hogan M. Kuliszewski M. Lee W. Post M. Biochem. J. 1996; 314: 799-803Crossref PubMed Scopus (28) Google Scholar). In Bac1.2F5 cells, CT activity, assayed in cell extracts, was high in middle to late G1, declined during S, and reached a minimum in G2 (2Jackowski S. J. Biol. Chem. 1994; 269: 3858-3867Abstract Full Text PDF PubMed Google Scholar). This pattern in activity was paralleled by changes in the enzyme's phosphorylation state. Activity was lowest when the level of phosphorylation was highest (2Jackowski S. J. Biol. Chem. 1994; 269: 3858-3867Abstract Full Text PDF PubMed Google Scholar). The membrane interactions of the enzyme were not examined in this study. Using in situ methods, CT has been localized to the nucleus in some cells, the cytoplasm in others, and to both sites in still others (19Wang Y. Sweitzer T.D. Weinhold P.A. Kent C. J. Biol. Chem. 1993; 268: 5899-5904Abstract Full Text PDF PubMed Google Scholar, 20Houweling M. Cui Z. Anfuso C.D. Bussiere M. Chen M.H. Vance D.E. Eur J. Cell Biol. 1996; 69: 55-63PubMed Google Scholar). Biochemical fractionations of cells have yielded CT in the cytosol, nuclear, ER, and even Golgi fractions (21Vance J.E. Vance D.E. J. Biol. Chem. 1988; 263: 5898-5909Abstract Full Text PDF PubMed Google Scholar, 22Morand J.N. Kent C. J. Biol. Chem. 1989; 264: 13785-13792Abstract Full Text PDF PubMed Google Scholar, 23Terce F. Record M. Tronchere H. Ribbes G. Chap H. Biochim. Biophys. Acta. 1991; 1084: 69-77Crossref PubMed Scopus (18) Google Scholar). CT contains a functional nuclear localization signal near its N terminus (24Wang Y. MacDonald J.I. Kent C. J. Biol. Chem. 1995; 270: 354-360Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Deletion of this signal resulted in mostly cytoplasmic rather than nuclear localization when expressed in the Chinese hamster ovary cells lacking endogenous CT, but disruption of the nuclear localization did not perturb cell growth or PC synthesis (24Wang Y. MacDonald J.I. Kent C. J. Biol. Chem. 1995; 270: 354-360Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). These results suggest that nuclear localization of CT is not required for PC synthesis. Since other enzymes of phospholipid synthesis are ER residents, including cholinephosphotransferase, the enzyme catalyzing the step subsequent to the CT step, the ER localization makes functional sense. However, the role of the enzyme in the nucleus remains a mystery. The focus of the present work is on the changes in PC synthesis and CT activity and intracellular localization during progression from the quiescent G0 stage into the cell division cycle. Growth factors that release cells from G0 stimulate PC turnover and the production of lipid second messengers at early steps (1Jackowski S. J. Biol. Chem. 1996; 271: 20219-20222Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 25Wright T.M. Shin H.S. Raben D.M. Biochem. J. 1990; 267: 501-507Crossref PubMed Scopus (48) Google Scholar,26Exton J.H. Biochim. Biophys. Acta. 1994; 1212: 26-42Crossref PubMed Scopus (924) Google Scholar). Growth factors also stimulate PC synthesis (16Tessner T.G. Rock C.O. Kalmar G.B. Cornell R.B. Jackowski S. J. Biol. Chem. 1991; 266: 16261-16264Abstract Full Text PDF PubMed Google Scholar, 27Paddon H.B. Vance D.E. Biochim. Biophys. Acta. 1980; 620: 636-640Crossref PubMed Scopus (51) Google Scholar, 28Warden C.H. Friedkin M. J. Biol. Chem. 1985; 260: 6006-6011Abstract Full Text PDF PubMed Google Scholar, 29Wieprecht M. Wieder T. Geilen C.C. Orfanos C.E. FEBS Lett. 1994; 353: 221-224Crossref PubMed Scopus (16) Google Scholar, 30MacDonald J.I. Possmayer F. Biochem. J. 1995; 312: 425-431Crossref PubMed Scopus (12) Google Scholar, 31Tran K. Man R.Y. Choy P.C. Biochim. Biophys. Acta. 1995; 1259: 283-290Crossref PubMed Scopus (15) Google Scholar), perhaps as a homeostatic response to the rapid acceleration of PC degradation (8Tronchere H. Record M. Terce F. Chap H. Biochim. Biophys. Acta. 1994; 1212: 137-151Crossref PubMed Scopus (99) Google Scholar, 32Pelech S.L. Vance D.E. Trends Biochem. Sci. 1989; 14: 28-30Abstract Full Text PDF Scopus (274) Google Scholar, 33Morash S.C. Rose S.D. Byers D.M. Ridgway N.D. Cook H.W. Biochem. J. 1998; 332: 321-327Crossref PubMed Scopus (22) Google Scholar). The stimulation of PC synthesis by serum in 3T3 cells involved an acceleration at both the choline kinase and CT steps (28Warden C.H. Friedkin M. J. Biol. Chem. 1985; 260: 6006-6011Abstract Full Text PDF PubMed Google Scholar), whereas angiotensin (31Tran K. Man R.Y. Choy P.C. Biochim. Biophys. Acta. 1995; 1259: 283-290Crossref PubMed Scopus (15) Google Scholar) and fetal pneumocyte factor (30MacDonald J.I. Possmayer F. Biochem. J. 1995; 312: 425-431Crossref PubMed Scopus (12) Google Scholar) stimulated only CT. Our studies have employed IIC9 fibroblasts. The kinetics of lipid second messenger production in response to growth factors have been carefully dissected in IIC9 cells (25Wright T.M. Shin H.S. Raben D.M. Biochem. J. 1990; 267: 501-507Crossref PubMed Scopus (48) Google Scholar, 34Wright T.M. Rangan L.A. Shin H.S. Raben D.M. J. Biol. Chem. 1988; 263: 9374-9380Abstract Full Text PDF PubMed Google Scholar, 35Wright T.M. Willenberger S. Raben D.M. Biochem. J. 1992; 285: 395-400Crossref PubMed Scopus (27) Google Scholar), making them ideal for analysis of the coupling between phospholipid degradation and synthesis during exit from G0. We show that exit from G0 is accompanied by a wave of PC synthesis that is coordinated with CT activation, CT translocation to membranes, and redistribution from the nucleus to the ER. The data give rise to the novel hypothesis that it is the ER-associated and not the nuclear form of CT that is enzymatically active with respect to PC synthesis. Monoclonal anti-Grp78 (BiP) was purchased from StressGen. Anti-cyclin D-1 (H-295) and purified cyclin D-1 protein (amino acids 1–295) were purchased from Santa Cruz Biotechnologies, Inc. (Santa Cruz, CA). Generation of anti-M (rabbit polyclonal antibody directed against a peptide corresponding to amino acids 256–288 of rat liver CT) has been previously described (36Johnson J.E. Aebersold R. Cornell R.B. Biochim. Biophys. Acta. 1997; 1324: 273-284Crossref PubMed Scopus (39) Google Scholar). Antibody to CT-β was a generous gift from Dr. Suzanne Jackowski (St. Judes Children's Research Hospital, Memphis, TN). Oregon Green and Texas Red-conjugated antibodies, rhodamine hexyl ester, and rhodamine phalloidin were all purchased from Molecular Probes, Inc. (Eugene, OR). Horseradish peroxidase-conjugated goat anti-rabbit antibody was from Sigma. IIC9 cells (a generous gift from Dr. D. Raben, Johns Hopkins, Baltimore, MD) were maintained in α-MEM/F-12 (1:1), 10% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin (Life Technologies, Inc.) as described (34Wright T.M. Rangan L.A. Shin H.S. Raben D.M. J. Biol. Chem. 1988; 263: 9374-9380Abstract Full Text PDF PubMed Google Scholar) and were used at passage numbers 30–40. To generate quiescent cells, cultures (80% confluent) were washed twice with serum-free α-MEM/F12 and incubated for 48 h in starvation medium containing DMEM, 100 units/ml penicillin, 100 μg/ml streptomycin, 1 mml-glutamine (Life Technologies, Inc.), 1 mg/ml radioimmunoassay grade BSA (Sigma), 20 mm Hepes, pH 7.4 (Sigma), and 5 μg/ml human transferrin (Calbiochem). Nonadherent cells were removed at ∼30 h by aspiration, and fresh serum starvation medium was replaced. For each analysis described below, following serum starvation for 48 h, FBS was added to one set of cultures to a final concentration of 10%. The control set was maintained on serum starvation medium. To measure DNA synthesis rates, [3H]thymidine (NEN Life Science Products) was added to duplicate 30-mm dishes to a concentration of 2 μCi/ml in a volume of 2 ml. After 2 h at 37 °C, the medium was removed, and the dishes were washed three times with ice-cold PBS, twice with ice-cold 5% trichloroacetic acid, and once with ice-cold ethanol. The acid-insoluble material was solubilized with 0.2% SDS in 0.1 n NaOH, and the label was quantitated by liquid scintillation counting. Duplicate 60-mm dishes were labeled for 10 min at 37 °C with 10 μCi of [methyl-3H]choline (Amersham Pharmacia Biotech) in 3 ml of medium. Incorporation of label was quenched by removing the medium, washing three times with ice-cold buffer A (50 mm Tris-HCl, pH 7.4, 0.15 m NaCl, 1 mm EDTA), and extracting into methanol. The 3H incorporated into PC was analyzed in the CHCl3 fraction of a Bligh-Dyer extraction (37Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 62: 983-988Google Scholar). To measure choline transport into the cells, triplicate dishes were pulsed for 5 min with 10 μCi of [3H]choline in 3 ml of medium. Incorporation of label was quenched as above. The cells were scraped from the dish into methanol and sonicated. Radioactivity was quantitated by liquid scintillation counting. Duplicate 100-mm dishes were washed three times with buffer A, and cells were released from the dishes with buffer A containing 2.5 mm EDTA and counted with a hemocytometer. The lipids were extracted into CHCl3 by the method of Bligh-Dyer (37Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 62: 983-988Google Scholar). The CHCl3 fraction was dried under N2 and dissolved in 30 μl of CHCl3. PC was separated from the other lipids by TLC on Silica Gel G (Analtech) using CHCl3/MeOH/NH4OH (65:35:5). The PC was visualized by I2 staining and quantitated using the Bartlett assay (38Bartlett G.R. J. Biol. Chem. 1959; 234: 466-468Abstract Full Text PDF PubMed Google Scholar). Egg PC (Avanti) was used as a standard. IIC9 cells were labeled with 2 μCi/ml [3H]choline in DMEM (28 μm choline) per 60-mm dish during the final hour of serum starvation. This labeling time was sufficient to saturate the aqueous choline metabolites in the serum-starved cells. Cultures were chased with fresh, unlabeled DMEM containing 250 μm choline with or without 10% FBS. At times after chase, the medium was aspirated, and dishes were washed three times with ice-cold PBS and quenched with 1.4 ml of MeOH. The cells were scraped into the methanol, and the aqueous phase of a Bligh-Dyer extraction was obtained. Choline (3.75 μmol), phosphocholine (4.2 μmol), and CDP-choline (0.46 μmol) were added as carriers to the samples, followed by evaporation to dryness in a Speed Vac. The residue was redissolved in 130 μl of water, and the choline metabolites were separated as described (39Walkey C.J. Kalmar G.B. Cornell R.B. J. Biol. Chem. 1994; 269: 5742-5749Abstract Full Text PDF PubMed Google Scholar). Digitonin permeabilization was carried out in a 3 °C room. Duplicate 100-mm dishes were rinsed with ice-cold PBS and placed on a Bellco rocker platform. 1.3 ml of permeabilization buffer (10 mm Hepes, pH 7.4, 0.1m KCl, 2 mm dithiothreitol, 0.5 mmphenylmethylsulfonyl fluoride, 0.2 mg/ml digitonin (Calbiochem)) was added to each dish. At intervals thereafter, the medium was collected, and the cell ghosts were scraped in 1.2 ml of permeabilization buffer. Both fractions were transferred to tubes containing 25 μl of 5 mm PC/oleic acid (1:1) vesicles to stabilize the CT. The ghost fraction was sonicated for 20 s on ice, and aliquots were removed immediately from both fractions for assay of CT activity (40Cornell R. J. Biol. Chem. 1989; 264: 9077-9082Abstract Full Text PDF PubMed Google Scholar). 100-mm dishes were grown to subconfluence, washed twice in cysteine- and methionine-free DMEM (Life Technologies, Inc.), and starved for 30 min in the same medium. The cells were then incubated with cysteine- and methionine-free DMEM supplemented with 50 μCi/ml [35S]methionine and cysteine (Amersham Pharmacia Biotech) and 5% FBS. After 4 h, the cells were washed twice with ice-cold PBS and 1 ml of immunoprecipitation buffer was added to each plate. Immunoprecipitation of cell extracts was carried out as described below. 150-mm dishes were washed twice with PBS, and the cells were scraped into 1 ml of immunoprecipitation buffer (PBS containing 50 mm Tris pH 8.0, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 1% Triton X-100, 1 mm dithiothreitol). The cells were homogenized by freeze-thaw followed by five passages through a 27-gauge needle. Insoluble material was removed by centrifugation at 14,000 × g for 15 min, and the supernatant was incubated with 2 μl/ml of rabbit polyclonal anti-M overnight at 4 °C. 40 μl of protein A beads (Amersham Pharmacia Biotech) were added, and the sample was incubated for 1 h at 4 °C. The protein A beads were washed four times with immunoprecipitation buffer, 40 μl of Laemmli buffer was added to the washed beads, and the samples were boiled for 5 min. Immunoprecipitated CT was resolved on a 12% SDS-polyacrylamide gel. For Western blots, proteins were transferred from 12% gels to a polyvinylidene difluoride membrane (Bio-Rad) at 150 mA for 1 h. The transfer buffer consisted of 39 mm glycine, 48 mm Tris, 20% methanol, 0.0375% SDS. Immunoblots were blocked in PBS containing 6% powdered milk, 0.5% Tween 20 and probed with anti-M (1:1000) for 1 h at room temperature. Following three washes with PBS containing 0.5% Tween 20, blots were incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:2000) for 1 h. The blots were washed three times with PBS-Tween, and CT was visualized using enhanced chemiluminescence (Amersham Pharmacia Biotech). Detection of cyclin D1 followed a similar protocol, using an anti-cyclin D1 antibody at a dilution of 1:500 . IIC9 cells were grown to 80% confluence in 100-mm dishes and serum-starved as described above for 32 h. Dishes were then washed twice with phosphate-free DMEM and incubated for 16 h with the same medium containing [32P]orthophosphate (0.1 mCi/ml). This labeling time was sufficient to saturate the ATP pools and the 32P label in CT. FBS or BSA was added to the dishes to final concentrations of 10 or 5%, respectively, and the incubation was continued. At various times, the medium was removed, and the cells were washed twice with ice-cold PBS and lysed as described above using 1 ml of immunoprecipitation buffer containing 60 mm β-glycerophosphate and 50 mm Na3VO4. Lysates were immunoprecipitated and resolved as described above.32P-Labeled CT was detected by autoradiography. IIC9 cells were grown on coverslips to 80% confluence and serum-starved as described above. Cells were fixed in 2% paraformaldehyde-PBS for 20 min and permeabilized by incubation in PBS containing 0.5% Triton X-100, 5% BSA for 1 h at room temperature. The fixed, permeabilized cells were incubated with primary antibodies (1:100) in PBS containing 0.5% Triton X-100, 5% BSA for 2 h at room temperature or overnight at 4 °C. The cells were then washed three times with PBS and incubated with secondary antibody (1:200) for 2 h at room temperature, washed three times with PBS, and mounted in Prolong antifade reagent (Molecular Probes). For double labeling, the primary antibodies and their corresponding secondary antibodies were incubated sequentially. Cells were viewed with a Zeiss LSM-410 confocal microscope equipped with a krypton/argon laser (Omnichrome), using a 63 × 1.4 NA lens. To set the background fluorescence, the gain and slope settings for the photomultipliers were adjusted using cells treated with normal rabbit serum followed by secondary antibody. In the colocalization analysis, a yellow pixel represents equal intensities from the red and green channel. IIC9 fibroblasts were arrested in G0 by serum starvation for 48 h. The incorporation of [3H]thymidine into DNA was negligible in the serum-starved cells (Fig.1 A). The addition of 10% serum resulted in a wave of [3H]thymidine incorporation beginning ∼14 h poststimulation, peaking at ∼22 h, and declining thereafter. Since at the peak (22 h) the thymidine incorporation rate was ∼3 times that of nonsynchronized cells at the onset of serum starvation, this suggests that 3 times as many cells were in S phase. S phase typically occupies 30–40% of the cell cycle time. Thus, nearly all of the cells participated in the DNA synthesis wave, suggesting that they were synchronized initially in G0. To further assess the cell cycle status of the serum-starved cells, the level of cyclin D1 was examined by immunoblot analysis of cell lysates. The expression of cyclin D1 is low in G0 cells, increases during the period of entry into G1, and plummets during S phase (41Baldin V. Lukas J. Marcote M.J. Pagano M. Draetta G. Genes Dev. 1993; 7: 812-821Crossref PubMed Scopus (1438) Google Scholar). Serum-starved IIC9 cells had low but detectable levels of cyclin D1. This low expression level persisted at 2, 4, 6, and 8 h postserum, rose markedly between 8 and 10 h, and remained high until at least 24 h postserum (Fig. 1 B). The incorporation of [3H]choline into PC during 10-min pulses was stimulated 3-fold as early as 10 min after the addition of serum compared with serum-starved cells (Fig.1 C). A dramatic elevation in PC synthesis rates continued for 3–4 h, at which time there was an 11 ± 3-fold (n = 6) increase over control rates. The rate of PC synthesis declined to near basal levels between 4 and 10 h postserum. The return to basal PC synthesis rates probably coincides with the G0/G1 boundary (42Zetterberg A. Thomas N.S.B. Apoptosis and Cell Cycle Control in Cancer. Bios Scientific, Oxford1996: 17-35Google Scholar). To determine whether the burst in the PC synthesis rate resulted in accumulation of PC, we quantified the mass of PC in cells following stimulation by serum. In two independent determinations, the PC content of quiescent cells was 23 ± 2 nmol of PC/106 cells. The PC content at 4 and 7 h postserum was 26 ± 3 and 24 ± 3 nmol of PC/106 cells, respectively. Thus, the synthesis wave was accompanied by a nearly equivalent wave of PC degradation, as has been observed previously, for example in macrophages re-entering the cell cycle in response to CSF1 (2Jackowski S. J. Biol. Chem. 1994; 269: 3858-3867Abstract Full Text PDF PubMed Google Scholar). The stimulation of the rate of [3H]choline incorporation into PC was not due to an acceleration of choline transport. Uptake of [3H]choline into the cells, monitored by 5-min pulses, was not significantly different for serum-starved and serum-stimulated cultures. The uptake rates were constant over a 4-h time course in both serum-starved and serum-stimulated cultures (not shown). The increase in choline incorporation into PC could be due to acceleration of the reactions catalyzed by choline kinase, cytidylyltransferase, cholinephosphotransferase (CPT), or a combination of these. The specific radioactivity of the pools of choline, phosphocholine, and CDP-choline reached equilibrium within 1 h of labeling with 2–3 μCi/ml [3H]choline (28 μm), and the relative pool sizes in serum-starved cells were as follows: choline, 3 ± 2%; phosphocholine, 83 ± 4%; and CDP-choline, 14 ± 2% (n = 3). These ratios suggest a rate-limiting step at the conversion of phosphocholine into CDP-choline, catalyzed by CT. The relative pool sizes were altered in cells treated with serum for 2–3 h: choline, 1%; phosphocholine, 73 ± 1%; and CDP-choline, 26.4 ± 0.6%. The phosphocholine:CDP-choline ratio decreased >2-fold, indicative of an acceleration of the CT-catalyzed step. The flux of [3H]choline through the CDP-choline pathway was monitored by a pulse-chase regime (43Vance D.E. Trip E.M. Paddon H.B. J. Biol. Chem. 1980; 255: 1064-1069Abstract Full Text PDF PubMed Google Scholar). The label associated with intracellular choline completely turned over within 20 min after the onset of the cold chase in both control and serum-stimulated cultures. The turnover of phosphocholine and CDP-choline is shown in Fig. 2. The turnover times determined from these plots reflect the rates of the CT and CPT reactions in situ. The t12values for both phosphocholine and CDP-choline were reduced ∼3-fold in the serum-stimulated cells (Fig. 2, A and B). These data suggest that the stimulation of PC synthesis during exit from G0 is accompanied by acceleration of the CT and CPT reactions. We further examined the regulation of CT by serum. The serum-stimulated increase in the CT reaction was not due to an increase in the levels of the enzyme in the cell. Endogenous CT was immunoprecipita
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