Ablation of Go α-Subunit Results in a Transformed Phenotype and Constitutively Active Phosphatidylcholine-specific Phospholipase C
1997; Elsevier BV; Volume: 272; Issue: 28 Linguagem: Inglês
10.1074/jbc.272.28.17312
ISSN1083-351X
AutoresJie Cheng, Jason D. Weber, Joseph J. Baldassare, Daniel M. Raben,
Tópico(s)Nuclear Structure and Function
ResumoModulation of the components involved in mitogenic signaling cascades is critical to the regulation of cell growth. GTP-binding proteins and the stimulation of phosphatidylcholine (PC) hydrolysis have been shown to play major roles in these cascades. One of the enzymes involved in PC hydrolysis, a PC-specific phospholipase C (PC-PLC) has received relatively little attention. In this paper we examined the role of a particular heterotrimeric GTP-binding protein, Go, in the regulation of cell growth and PC-PLC-mediated hydrolysis of PC in IIC9 fibroblasts. The Go α-subunit was ablated in IIC9 cells by stable expression of antisense RNA. These stably transfected cells acquired a transformed phenotype as indicated by: (a) the formation of multiple foci in monolayer cultures, (b) the acquisition of anchorage-independent growth in soft agar; and (c) an increased level of thymidine incorporation in the absence of added mitogens. These data implicate Goα as a novel tumor suppressor. Interestingly, PC-PLC activity was constitutively active in the Goα-ablated cells as evidenced by the chronically elevated levels of diacylglycerol and phosphorylcholine in the absence of growth factors. In contrast, basal activities of PC-phospholipase D, phospholipase A2, or phosphoinositol-PLC were not affected. These data demonstrate, for the first time, a role for Goin regulating cell growth and provide definitive evidence for the existence of a PC-PLC in eukaryotic cells. The data further indicate that a subunit of Go, is involved in regulating this enzyme. Modulation of the components involved in mitogenic signaling cascades is critical to the regulation of cell growth. GTP-binding proteins and the stimulation of phosphatidylcholine (PC) hydrolysis have been shown to play major roles in these cascades. One of the enzymes involved in PC hydrolysis, a PC-specific phospholipase C (PC-PLC) has received relatively little attention. In this paper we examined the role of a particular heterotrimeric GTP-binding protein, Go, in the regulation of cell growth and PC-PLC-mediated hydrolysis of PC in IIC9 fibroblasts. The Go α-subunit was ablated in IIC9 cells by stable expression of antisense RNA. These stably transfected cells acquired a transformed phenotype as indicated by: (a) the formation of multiple foci in monolayer cultures, (b) the acquisition of anchorage-independent growth in soft agar; and (c) an increased level of thymidine incorporation in the absence of added mitogens. These data implicate Goα as a novel tumor suppressor. Interestingly, PC-PLC activity was constitutively active in the Goα-ablated cells as evidenced by the chronically elevated levels of diacylglycerol and phosphorylcholine in the absence of growth factors. In contrast, basal activities of PC-phospholipase D, phospholipase A2, or phosphoinositol-PLC were not affected. These data demonstrate, for the first time, a role for Goin regulating cell growth and provide definitive evidence for the existence of a PC-PLC in eukaryotic cells. The data further indicate that a subunit of Go, is involved in regulating this enzyme. Defects in signal transduction cascades involved in the regulation of cell growth often lead to pathological conditions, including the development of neoplasms. Heterotrimeric GTP-binding proteins (G proteins) 1The abbreviations used are: G protein, heterotrimeric GTP-binding protein; PC, phosphatidylcholine; PLC, phospholipase C; PI, phosphoinositide; PLD, phospholipase D; PA, phosphatidic acid; DAG, diacylglycerol; PAPH, phosphatidic acid phosphohydrolase; TLC, thin layer chromatography; PLA2, phospholipase A2; FCS, fetal calf serum; CK, choline kinase; IP, inositol phosphate. and induced lipid metabolism are important components in growth factor-coupled cellular signal transduction pathways. Heterotrimeric G proteins are a family of membrane-bound proteins composed of α, β, and γ subunits, which, in response to receptor activation, dissociate into free α subunits and βγ dimers. Both the GTP-bound α subunits and βγ dimers have been shown to play roles in a variety mitogenic signal transduction cascades (1Seuwen K. Pouyssegur J. Adv. Cancer Res. 1992; 58: 75-94Crossref PubMed Scopus (42) Google Scholar), including those involving induced lipid metabolism (2Sternweis P.C. Smrcka A.V. Trends Biochem. Sci. 1992; 17: 502-506Abstract Full Text PDF PubMed Scopus (175) Google Scholar, 3Wakelam M.J. Briscoe C.P. Stewart A. Pettitt T.R. Cross M.J. Paul A. Yule J.M. Gardner S.D. Hodgkin M. Biochem. Soc. Trans. 1993; 21: 874-877Crossref PubMed Scopus (14) Google Scholar). There is now strong evidence indicating that G proteins play crucial roles in the regulation of mitogenic signals and that specific defects in these proteins lead to the development of transformed phenotypes. In addition to the observation that the activation of certain growth factor receptors stimulates the dissociation of G proteins into GTP-bound α subunits and βγ dimers, mutations that reduce the intrinsic GTPase activity in specific α-subunits transform these G proteins into oncoproteins. For example, mutations in the Gsα gene result in an oncogene (gsp), the protein product of which is a Gsα with substitutions at amino acids 201 (R201C/H) and 227 (Q227R/H/L) which have been found in growth hormone-secreting pituitary tumors (4Landis C.A. Masters S.B. Spada A. Pace A.M. Bourne H.R. Vallar L. Nature. 1989; 340: 692-696Crossref PubMed Scopus (1225) Google Scholar). Similarly, mutations in the Gi2α gene yield another oncogene (gip2) characterized by a substitution of amino acid 179 in Gi2α (R179C/H) which has been found in ovarian sex cord stromal tumors and adrenal cortical tumors (5Lyons J. Landis C.A. Harsh G. Vallar L. Grunewald K. Feichtinger H. Duh Q.Y. Clark O.H. Kawasaki E. Bourne H.R. McCormick F. Science. 1990; 249: 655-659Crossref PubMed Scopus (927) Google Scholar). These data provide strong support for the notion that these G proteins are important components involved in the regulation of mitogenic signal transduction cascades and represent potential targets for oncogenic mutations in human tumors. In addition to G proteins, agonist-induced lipid metabolism also plays a central role in mitogenic signaling cascades. While induced hydrolysis of phosphoinositides (PIs) has long been recognized as playing such a role (6Noh D.Y. Shin S.H. Rhee S.G. Biochim. Biophys. Acta. 1995; 1242: 99-113Crossref PubMed Scopus (254) Google Scholar), it is now well recognized that induced PC metabolism is often just as, if not more, important (7Exton J.H. Biochim. Biophys. Acta. 1994; 1212: 26-42Crossref PubMed Scopus (921) Google Scholar). Although PI and PC hydrolysis are induced by a variety of mitogens, PI hydrolysis is often transient while PC hydrolysis is usually sustained in the continuous presence of growth factors. In this regard, PC hydrolysis correlates with the requirement for the prolonged presence of growth factors for full mitogenic responses (8Wright T.M. Rangan L.A. Shin H.S. Raben D.M. J. Biol. Chem. 1988; 263: 9374-9380Abstract Full Text PDF PubMed Google Scholar). Depending on the cell type and specific mitogen, three enzymes have been implicated in mitogen-induced PC metabolism: PLA2, PC-PLD, and PC-PLC. PLA2 removes the fatty acid esterified at sn-2 of the glycerol backbone in PC resulting in the liberation of a free fatty acid, often arachidonic acid, and a lysophospholipid. PLD-mediated hydrolysis of PC results in the production of phosphatidic acid (PA) and free choline. This generated PA is often, but not always, hydrolyzed by phosphatidic acid phosphohydrolase (PAPH) leading to the production of diacylglycerol (DAG). Alternatively, in some systems (9Baldi E. Musial A. Kester M. Am. J. Physiol. 1994; 266: F957-FF65Crossref PubMed Google Scholar, 10Cifone M.G. Roncaioli P. De Maria R. Camarda G. Santoni A. Ruberti G. Testi R. EMBO J. 1995; 14: 5859-5868Crossref PubMed Scopus (277) Google Scholar, 11Larrodera P. Cornet M.E. Diaz-Meco M.T. Lopez-Barahona M. Diaz-Laviada I. Guddal P.H. Johansen T. Moscat J. Cell. 1990; 61: 1113-1120Abstract Full Text PDF PubMed Scopus (119) Google Scholar, 12Laviada I.D. Baudet C. Galve-Roperh I. Naveilhan P. Brachet P. FEBS Lett. 1995; 364: 301-304Crossref PubMed Scopus (18) Google Scholar, 13Randell E. Mulye H. Mookerjea S. Nagpurkar A. Biochim. Biophys. Acta. 1992; 1124: 273-278Crossref PubMed Scopus (8) Google Scholar, 14Sands W.A. Clark J.S. Liew F.Y. Biochem. Biophys. Res. Commun. 1994; 199: 461-466Crossref PubMed Scopus (43) Google Scholar, 15Wright T.M. Willenberger S. Raben D.M. Biochem. J. 1992; 285: 395-400Crossref PubMed Scopus (27) Google Scholar), PC-derived DAGs, in addition to phosphorylcholine, are produced from a PC-PLC-mediated hydrolysis of PC. Most studies have focused on PLA2 and PLD while very little attention has been given to PC-PLC. Indeed, the existence of a eukaryotic PC-PLC remains somewhat controversial. Given this lack of attention to eukaryotic PC-PLC, it is not surprising that the cellular components involved in its regulation have not been identified. The observation that activation of receptors, such as the thrombin receptor (16Vu T.K. Hung D.T. Wheaton V.I. Coughlin S.R. Cell. 1991; 64: 1057-1068Abstract Full Text PDF PubMed Scopus (2675) Google Scholar), which are known to couple to G proteins (17Hung D.T. Wong Y.H. Vu T.-K.H. Coughlin S.R. J. Biol. Chem. 1992; 267: 20831-20834Abstract Full Text PDF PubMed Google Scholar) lead to an increase in PC-PLC activity (15Wright T.M. Willenberger S. Raben D.M. Biochem. J. 1992; 285: 395-400Crossref PubMed Scopus (27) Google Scholar, 18Murthy K.S. Makhlouf G.M. Mol. Pharmacol. 1995; 48: 293-304PubMed Google Scholar) suggest that this enzyme is regulated by a G protein. That a G protein couples to PC-PLC is consistent with the fact that these proteins are known to regulate other specific phospholipases. PI-PLCβ is regulated by a pertussis toxin-sensitive G protein (19Exton J.H. Adv. Second Messenger Phosphoprotein Res. 1993; 28: 65-72PubMed Google Scholar), involving both αq and βγ dimers (20Blank J.L. Brattain K.A. Exton J.H. J. Biol. Chem. 1992; 267: 23069-23075Abstract Full Text PDF PubMed Google Scholar, 21Lee S.B. Shin S.H. Hepler J.R. Gilman A.G. Rhee S.G. J. Biol. Chem. 1993; 268: 25952-25957Abstract Full Text PDF PubMed Google Scholar, 22Rhee S.G. Choi K.D. Adv. Second Messenger Phosphoprotein Res. 1992; 26: 35-61PubMed Google Scholar). In a similar manner, heterotrimeric G proteins have been implicated in the regulation of a high molecular weight PLA2 (23Axelrod J. Trends. Neurosci. 1995; 18: 64-65Abstract Full Text PDF PubMed Scopus (42) Google Scholar), while PC-PLD has been shown to be regulated by small molecular weight GTP-binding proteins (24Cockcroft S. Thomas G.M. Fensome A. Geny B. Cunningham E. Gout I. Hiles I. Totty N.F. Truong O. Hsuan J.J. Science. 1994; 263: 523-526Crossref PubMed Scopus (585) Google Scholar). In view of these data, it is reasonable to hypothesize that PC-PLC may also be regulated by a G protein. In this report, we demonstrate that the ablation of Goα results in a transformed phenotype. Furthermore, in these Goα-ablated cells, PC-PLC is significantly elevated providing definitive evidence for a PC-PLC and implicating Go, Goα in particular, in the regulation of this enzyme in vivo. The relationship between Goα, the transformed phenotype and the constitutive activation of PC-PLC is discussed. Tissue culture media components, Lipofectin reagents, Geneticin (G418), and calf alkaline phosphatase (1000 units/μg) were purchased from Boehringer Mannheim. Plastic culture dishes were purchased from Falcon Labware. Highly purified human thrombin (≈4000 NIH units/ml) and bovine serum albumin (radioimmunoassay grade, fraction V) were purchased from Sigma.Escherichia coli diacylglycerol kinase was obtained from Lipidex or CalBiochem. AG1X8 Resin (200–400 mesh, formate form) was from Bio-Rad. TLC plates were purchased from EM Diagnostics, Analabs, and Analtech. CytoScint scintillation counting fluid was obtained from ICN. Radioactive materials were purchased from Amersham. Molecular biology enzymes were purchased from Stratagene, Life Technologies, Inc., New England Biolabs, and Boehringer Mannheim. [methyl-3H]Choline (83 Ci/mmol) and [9,10-3H]myristic acid (53 Ci/mol) were purchased from Amersham. [γ-32P]ATP (3000 Ci/mmol), [methyl-3H]thymidine (6.7 Ci/mmol), and [5,6,8,9,11,12,14,15-3H]arachidonic acid (180–240 Ci/mmol) were purchased from NEN Life Sciences Products. Rat Goα cDNA plasmid (pGEM-2/Goα) was generously provided by Dr. Randy Reed (Howard Hughes Medical Institute, Johns Hopkins Medical Institutes, Baltimore MD). IIC9 cells, a subclone of Chinese hamster embryo fibroblasts (25Low D.A. Scott R.W. Baker J.B. Cunningham D.D. Nature. 1982; 298: 476-478Crossref PubMed Scopus (51) Google Scholar), were grown, maintained, and serum-deprived as described previously (8Wright T.M. Rangan L.A. Shin H.S. Raben D.M. J. Biol. Chem. 1988; 263: 9374-9380Abstract Full Text PDF PubMed Google Scholar). Briefly, cultures were grown and maintained in minimal essential medium-α/Ham's F-12 medium (1:1, v/v) containing 5% (v/v) fetal calf serum, 100 units of penicillin/ml of 100 mg of streptomycin/ml, and 2 mml-glutamine (complete media). Subconfluent cultures were serum-deprived by washing three times with Dulbecco's modified Eagle's medium containing 1 mg/ml bovine serum albumin (radioimmunoassay grade), 100 units of penicillin, 100 mg of streptomycin/ml, 2 mml-glutamine, and 20 mm NaHepes, pH 7.4. The cultures were then incubated in this media supplemented with 5 mg/ml human transferrin (serum-free medium) and incubated for 2 days at 37 °C. Cultures were washed twice and equilibrated in fresh serum-free media for at least 30 min prior to addition of each experiment. EcoRI fragment of Goα cDNA from pGEM-2/Goα was subcloned into a vector plasmid, pcDNAI, in a antisense orientation, i.e. the 3′ end of the Goα cDNA was immediately adjacent to cytomegalovirus promoter under which control the antisense sequence is transcribed. Plasmids were sequenced and transfected into IIC9 cells using a Lipofectamine protocol (Life Technologies, Inc.). pcDNAI without inserts were transfected into IIC9s as a control. Briefly, subconfluent cells in 60-mm culture dishes were washed with Opti-MEM (Life Technologies, Inc.) and transfected by incubation with 5-μg plasmids and Lipofectamine for 24 h at 37 °C. The Opti-MEM media was then replaced with complete media and the cells were grown for 48 h at 37 °C to allow expression of the neomycin resistance gene products. The transfected cells were subcultured and grown for several weeks in selection medium (complete medium supplemented with 500 μg/ml G418). G418-resistant clones were isolated with cloning cylinders and the transfected clones were maintained in complete medium supplemented with 250 μg/ml G418. All other assays, including growth in soft agar, [3H]thymidine incorporation, Western blot analysis, and quantification of DAG mass, choline metabolites, and PLD activation were performed as described previously (8Wright T.M. Rangan L.A. Shin H.S. Raben D.M. J. Biol. Chem. 1988; 263: 9374-9380Abstract Full Text PDF PubMed Google Scholar, 15Wright T.M. Willenberger S. Raben D.M. Biochem. J. 1992; 285: 395-400Crossref PubMed Scopus (27) Google Scholar, 26Rangan L.A. Wright T.M. Raben D.M. Cell Regul. 1991; 2: 311-316Crossref PubMed Scopus (12) Google Scholar, 27Raben D.M. Yasuda K.M. Cunningham D.D. J. Cell. Physiol. 1987; 130: 466-473Crossref PubMed Scopus (13) Google Scholar, 28Wright T.M. Shin H.S. Raben D.M. Biochem. J. 1990; 267: 501-507Crossref PubMed Scopus (48) Google Scholar, 29Johansen T. Bjorkoy G. Overvatn A. Diaz-Meco M.T. Traavik T. Moscat J. Mol. Cell Biol. 1994; 14: 646-654Crossref PubMed Scopus (124) Google Scholar) as indicated in the figure legends. To investigate the physiological role of Goα, we stably transfected IIC9 cells with a Goα antisense construct (Fig.1 A). Western blot analysis demonstrated that Goα was absent in the transfected cells while other G protein α subunits, Gi1α, Gi2α, Gsα, and Gqα, were present (Fig.1 B). This has been observed in at least three independently isolated Goα-ablated clones (data not shown).Figure 1Schematic representation of the Goα antisense construct and its effect on the expression of Goα protein in IIC9 cells. A, pGoas: pcDNAI containing Goα cDNA in a antisense orientation as described under “Experimental Procedures.”B, cell lysates (50 μg/lane) were subjected to Western blot analysis. For this analysis, 50 μg of protein in sample buffer was separated by electrophoresis in 9% polyacrylamide gels (64Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207218) Google Scholar) and transferred to Immobilon-P by electroblotting. The blot was incubated overnight in wash buffer (20 mm Tris, pH 8.0, 150 mm NaCl, 0.01% Tween 20) containing 5% dry milk as described (65Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44923) Google Scholar) followed by washing and incubation for 1 h at room temperature with antibodies specifically directed against the indicated Gα subunits. After washing and incubation one-half hour at room temperature with anti-IgG horseradish peroxidase conjugate, the blot was then developed using chemiluminescence detection (Amersham). The figure was constructed by photocopying autoradiograms of Western blots onto a transparency. The appropriate lanes were mounted onto white paper and then photographed. WT, wild type IIC9 cells;Goa1 and Goa2, two cell lines stably transfected with pGoas/pNeo; Gv, cell line stably transfected with pcDNAI without an insert. The data are representative of at least three experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In contrast to wild type IIC9 cells which are flat and extended (Fig.2 A), Goα-ablated cells appear round, retracted, and form multiple foci in confluent monolayer cultures (Fig. 2 B). This morphology, observed in three independently isolated clones, suggest that the Goα-ablated cells have lost contact inhibition and acquired a transformed phenotype. An important characteristic of transformed fibroblasts is their ability to grow in an anchorage independent manner. In view of this and the above data, we assessed the ability of the Goα-ablated cells to grow in soft agar. As shown in Table I, Goa1 cells formed 20–30-fold more colonies in soft agar than wild type cells and this has been observed in a second, independently isolated, clone (data not shown). Furthermore, each of the colonies formed by the ablated cells were much larger and more dense (Fig. 3, B and D) than the colonies formed by the wild type cells (Fig. 3, A andC). Cells transfected with control vectors (vectors without inserts) formed colonies similar to those seen with wild type cells (data not shown).Table IInformation of colonies in soft agarCell typeNumber of coloniesWT10 ± 4Goal365 ± 39The ability of wild type IIC9 cells and Goa-ablated cells (Gao1) to grow in soft agar was assesed as described under “Experimental Prodecures.” The data represent the average number of colonies in three 35-mm dishes for each cell type and is representative of three independent experiments. Error values indicate the range. Open table in a new tab The ability of wild type IIC9 cells and Goa-ablated cells (Gao1) to grow in soft agar was assesed as described under “Experimental Prodecures.” The data represent the average number of colonies in three 35-mm dishes for each cell type and is representative of three independent experiments. Error values indicate the range. To further investigate the possibility that the ablated cells were transformed, we assessed the “basal” level of thymidine incorporation. As shown in Fig. 4, wild type IIC9 cells became quiescent after serum deprivation for 48 h and the level of [3H]thymidine incorporation was low. In contrast, serum-deprived Goα-ablated cells displayed a 10-fold higher level of [3H]thymidine incorporation than the quiescent wild type cells (Fig. 4). FCS (10%) or thrombin (2 NIH units/ml) stimulated only a modest increase in [3H]thymidine incorporation in the ablated cells while these treatments stimulated a 10-fold increase in [3H]thymidine incorporation in the quiescent wild type cells (Fig. 4). These data have been observed in three independently isolated clones. Consistent with these data, the Goα-ablated cells survive in serum-free media for an extended period of time while the wild type cells do not (Fig. 2,C and D). These data indicate that the Goα-ablated cells are not growth arrested in serum-free medium and are consistent with the transformed phenotype of these cells. Cells transformed as a consequence of a defect in a signal transduction component normally associated with the regulation of mitogenesis often show changes in the concentrations of second messengers under their control (30Macara I.G. Mol. Cell Biol. 1989; 9: 325-328Crossref PubMed Scopus (109) Google Scholar, 31Weinstein I.B. Adv. Second Messenger Phosphoprotein Res. 1990; 24: 307-316PubMed Google Scholar, 32Waterfield M.D. Br. Med. Bull. 1989; 45: 570-581Crossref PubMed Scopus (12) Google Scholar). Many of the signaling cascades known to be involved in mediating mitogenic signals involve the stimulation of lipid metabolism and G proteins are known to play a role in some of these cascades (1Seuwen K. Pouyssegur J. Adv. Cancer Res. 1992; 58: 75-94Crossref PubMed Scopus (42) Google Scholar, 6Noh D.Y. Shin S.H. Rhee S.G. Biochim. Biophys. Acta. 1995; 1242: 99-113Crossref PubMed Scopus (254) Google Scholar, 7Exton J.H. Biochim. Biophys. Acta. 1994; 1212: 26-42Crossref PubMed Scopus (921) Google Scholar). In addition, the elevation of DAG levels plays a central early role in transducing the mitogenic signal in these cascades. In view of this and the above observations regarding the transformed phenotype of the Goα-ablated cells, we measured the mass of DAG in the ablated cells. Subconfluent wild type and ablated cells were incubated in serum-free medium for 2 days and DAG levels were quantified. Interestingly, the basal DAG level in the serum-starved Goα-ablated cells was twice that of quiescent wild type cells (Fig. 5 A). Furthermore, while the addition of α-thrombin to the wild type cells resulted in a 2-fold increase in DAG mass level, addition of α-thrombin to the ablated cells did not induce a significant further increase in DAG levels (Fig.5 A). These results, observed in two independently isolated clones, indicate that the DAG level in Goα-ablated cells was constitutively elevated even in the absence of any added mitogens. We have shown that PC hydrolysis is the major, if not exclusive, source of mitogen-induced DAGs in IIC9 cells (8Wright T.M. Rangan L.A. Shin H.S. Raben D.M. J. Biol. Chem. 1988; 263: 9374-9380Abstract Full Text PDF PubMed Google Scholar, 15Wright T.M. Willenberger S. Raben D.M. Biochem. J. 1992; 285: 395-400Crossref PubMed Scopus (27) Google Scholar, 26Rangan L.A. Wright T.M. Raben D.M. Cell Regul. 1991; 2: 311-316Crossref PubMed Scopus (12) Google Scholar,33Pessin M.S. Altin J.G. Jarpe M. Tansley F. Bradshaw R.A. Raben D.M. Cell Regul. 1991; 2: 383-390Crossref PubMed Scopus (19) Google Scholar, 34Pessin M.S. Baldassare J.J. Raben D.M. J. Biol. Chem. 1990; 265: 7959-7966Abstract Full Text PDF PubMed Google Scholar, 35Pessin M.S. Raben D.M. J. Biol. Chem. 1989; 264: 8729-8738Abstract Full Text PDF PubMed Google Scholar). In view of these data, we examined the possibility that an increase in PC hydrolysis contributed to the elevated DAG level in the Goα-ablated cells. To determine if PC hydrolysis was affected in the Goα-ablated cells, the cells were radiolabeled to isotopic equilibrium with [3H]choline chloride in serum-free medium for 48 h and the intracellular [3H]choline and [3H]phosphorylcholine level were quantified (15Wright T.M. Willenberger S. Raben D.M. Biochem. J. 1992; 285: 395-400Crossref PubMed Scopus (27) Google Scholar). TLC analysis of water-soluble head groups indicated that the phosphorylcholine level in the Goα-ablated cells was 5–10-fold higher than that found in wild type cells. The level of choline in the ablated and wild type cells, however, was identical (Fig. 5 B). To ensure that the increased level of radiolabeled phosphorylcholine was not due to contaminant which co-migrated with the phosphorylcholine, the radioactivity in the region of the TLC plate containing phosphorylcholine was recovered, subjected to alkaline phosphatase hydrolysis, and the products were identified by TLC. All of the radioactivity that migrated with phosphorylcholine was converted to choline indicating that co-migrating contaminants were not present (data not shown). These data indicate that both phosphorylcholine and DAG, the two products of PC-PLC, are elevated in ablated cells and strongly suggest that PC-PLC is constitutively activated in Goα-ablated cells. These results have been observed in three independently isolated clones. An alternative explanation for the above results is that PC is hydrolyzed via a PLD and the resulting PA is dephosphorylated to DAG, via PAPH, while the free choline is phosphorylated via CK. As a result of the combined action of all three enzymes, PLD, PAPH, and choline kinase (CK), an apparent PC-PLC activity would be detected similar to that observed in v-ras transformed cells (36Carnero A. Dolfi F. Lacal J.C. J. Cell. Biochem. 1994; 54: 478-486Crossref PubMed Scopus (36) Google Scholar, 37Carnero A. Cuadrado A. del Peso L. Lacal J.C. Oncogene. 1994; 9: 1387-1395PubMed Google Scholar, 38Preiss J. Loomis C.R. Bishop W.R. Stein R. Niedel J.E. Bell R.M. J. Biol. Chem. 1986; 261: 8597-8600Abstract Full Text PDF PubMed Google Scholar). In view of the above, PLD activity was quantified in Goα-ablated and wild type IIC9 cells by taking advantage of the unique transphosphatidylation activity of PLD and the ability to preferentially label PC by acute labeling with [3H]myristate (15Wright T.M. Willenberger S. Raben D.M. Biochem. J. 1992; 285: 395-400Crossref PubMed Scopus (27) Google Scholar). In the transphosphatidylation reaction, a small molecular weight alcohol such as ethanol is used as the nucleophile in lieu of water resulting in the generation of phosphatidylethanol instead of PA. As shown in Fig. 5 C, PLD activity is indistinguishable in the ablated and wild type cells in the absence of thrombin or FCS. Furthermore, the addition of thrombin to both cell types results in comparable increases in PLD activity. These data indicate that both basal and thrombin activated PLD activity are unaltered in Goα-ablated cells and have been observed in three independently isolated clones. To further examine the possible involvement of PLD/PAPH/CK activities, CK activity was quantified in wild type and Goα-ablated serum-deprived cells. These cells were incubated with [3H]choline for 15 and 30 min and the level of radiolabeled phosphorylcholine was quantified. As shown in Fig.5 D, the conversion of choline to phosphorylcholine was essentially identical in both cell types demonstrating that the CK activity was not elevated in the Goα-ablated cells. These results have been observed in two independently isolated clones. We should note that sphingomyelinase was also not contributing to the increased phosphorylcholine levels in Goα-ablated cells. If this choline metabolite was generated from a sphingomyelinase-mediated hydrolysis of sphingomyelin ceramide, in addition to phosphorylcholine, would be generated. Ceramide levels were quantified, therefore, in wild type and Goα-ablated cells and was found to be identical in both cell types (data not shown). Taken together, the above data eliminate the involvement of PLD/PAPH/CK as a mechanism for the chronic elevation of DAG and phosphorylcholine levels in Goα-ablated cells. In addition, they indicate that Goα ablation-induced transformation is different from v-ras-induced transformation, since the later involves PLD/PAPH/CK activities (36Carnero A. Dolfi F. Lacal J.C. J. Cell. Biochem. 1994; 54: 478-486Crossref PubMed Scopus (36) Google Scholar, 37Carnero A. Cuadrado A. del Peso L. Lacal J.C. Oncogene. 1994; 9: 1387-1395PubMed Google Scholar, 38Preiss J. Loomis C.R. Bishop W.R. Stein R. Niedel J.E. Bell R.M. J. Biol. Chem. 1986; 261: 8597-8600Abstract Full Text PDF PubMed Google Scholar). Another mitogen-activated PC hydrolyzing enzyme is PLA2. In IIC9 cells, thrombin and FCS stimulate PLA2 activity which hydrolyze PC to lysophosphocholine and arachidonic acid (27Raben D.M. Yasuda K.M. Cunningham D.D. J. Cell. Physiol. 1987; 130: 466-473Crossref PubMed Scopus (13) Google Scholar), both of which have been implicated in mitogenic signaling cascades (7Exton J.H. Biochim. Biophys. Acta. 1994; 1212: 26-42Crossref PubMed Scopus (921) Google Scholar). Basal and α-thrombin-induced PLA2activities in Goα-ablated and wild type cells was assessed by quantifying the release of arachidonic acid and its metabolites. As observed for PLD, basal and α-thrombin-activated PLA2 activity was not affected by the ablation of Goα (Fig. 6 A). Consistent with these data, glycerolphosphocholine, another metabolite produced by the hydrolysis of PLA2-generated lysophospholipid, was also at similar levels in both cell types (data not shown). These data, observed in three independently isolated clones, indicated that PLA2 activity is unaffected in the Goα-ablated cells. Induced PI hydrolysis has been observed in response to a wide variety of mitogens and defects in this metabolism have been implicated in
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