Ablation of Go alpha -Subunit Results in a Transformed Phenotype and Constitutively Active Phosphatidylcholine-specific Phospholipase C*

(Received for publication, February 4, 1997, and in revised form, April 30, 1997)

Jie Cheng Dagger , Jason D. Weber §, Joseph J. Baldassare and Daniel M. Raben Dagger par

From the Dagger  Department of Physiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 and the Departments of § Cell and  Pharmacological and Physiological Sciences, St. Louis University School of Medicine, St. Louis, Missouri 63104

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

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 alpha -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 Goalpha as a novel tumor suppressor. Interestingly, PC-PLC activity was constitutively active in the Goalpha -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 Go in 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.


INTRODUCTION

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)1 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 alpha , beta , and gamma  subunits, which, in response to receptor activation, dissociate into free alpha  subunits and beta gamma dimers. Both the GTP-bound alpha  subunits and beta gamma dimers have been shown to play roles in a variety mitogenic signal transduction cascades (1), including those involving induced lipid metabolism (2, 3).

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 alpha  subunits and beta gamma dimers, mutations that reduce the intrinsic GTPase activity in specific alpha -subunits transform these G proteins into oncoproteins. For example, mutations in the Gsalpha gene result in an oncogene (gsp), the protein product of which is a Gsalpha with substitutions at amino acids 201 (R201C/H) and 227 (Q227R/H/L) which have been found in growth hormone-secreting pituitary tumors (4). Similarly, mutations in the Gi2alpha gene yield another oncogene (gip2) characterized by a substitution of amino acid 179 in Gi2alpha (R179C/H) which has been found in ovarian sex cord stromal tumors and adrenal cortical tumors (5). 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 (6), it is now well recognized that induced PC metabolism is often just as, if not more, important (7). 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 (8).

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 (9-15), 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 (16), which are known to couple to G proteins (17) lead to an increase in PC-PLC activity (15, 18) 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-PLCbeta is regulated by a pertussis toxin-sensitive G protein (19), involving both alpha q and beta gamma dimers (20-22). In a similar manner, heterotrimeric G proteins have been implicated in the regulation of a high molecular weight PLA2 (23), while PC-PLD has been shown to be regulated by small molecular weight GTP-binding proteins (24). 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 Goalpha results in a transformed phenotype. Furthermore, in these Goalpha -ablated cells, PC-PLC is significantly elevated providing definitive evidence for a PC-PLC and implicating Go, Goalpha in particular, in the regulation of this enzyme in vivo. The relationship between Goalpha , the transformed phenotype and the constitutive activation of PC-PLC is discussed.


EXPERIMENTAL PROCEDURES

Materials

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 (approx 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. [gamma -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 Goalpha cDNA plasmid (pGEM-2/Goalpha ) was generously provided by Dr. Randy Reed (Howard Hughes Medical Institute, Johns Hopkins Medical Institutes, Baltimore MD).

Cells and Cell Culture

IIC9 cells, a subclone of Chinese hamster embryo fibroblasts (25), were grown, maintained, and serum-deprived as described previously (8). Briefly, cultures were grown and maintained in minimal essential medium-alpha /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 mM L-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 mM L-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.

Generation of Goalpha -ablated Cells

EcoRI fragment of Goalpha cDNA from pGEM-2/Goalpha was subcloned into a vector plasmid, pcDNAI, in a antisense orientation, i.e. the 3' end of the Goalpha 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 (8, 15, 26-29) as indicated in the figure legends.


RESULTS

Goalpha -ablated Cells Acquire a Transformed Phenotype

To investigate the physiological role of Goalpha , we stably transfected IIC9 cells with a Goalpha antisense construct (Fig. 1A). Western blot analysis demonstrated that Goalpha was absent in the transfected cells while other G protein alpha  subunits, Gi1alpha , Gi2alpha , Gsalpha , and Gqalpha , were present (Fig. 1B). This has been observed in at least three independently isolated Goalpha -ablated clones (data not shown).


Fig. 1. Schematic representation of the Goalpha antisense construct and its effect on the expression of Goalpha protein in IIC9 cells. A, pGoas: pcDNAI containing Goalpha 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 (64) 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 (65) followed by washing and incubation for 1 h at room temperature with antibodies specifically directed against the indicated Galpha 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.
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In contrast to wild type IIC9 cells which are flat and extended (Fig. 2A), Goalpha -ablated cells appear round, retracted, and form multiple foci in confluent monolayer cultures (Fig. 2B). This morphology, observed in three independently isolated clones, suggest that the Goalpha -ablated cells have lost contact inhibition and acquired a transformed phenotype.


Fig. 2. Morphology of Go-ablated cells and wild type IIC9 cells in serum-containing and serum-free medium. Wild type IIC9 cells (A and C) and Goalpha -ablated cells (B and D) were grown in complete medium containing 5% FCS in 100-mm culture dishes. Cells were grown for 1 week (A and B) or shifted to serum-free medium after 3 days and incubated in serum-free media for an additional 9 days (C and D). Serum-free media was replaced with fresh serum-free media every 3 days. Magnification, × 100.
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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 Goalpha -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 and C). Cells transfected with control vectors (vectors without inserts) formed colonies similar to those seen with wild type cells (data not shown).

Table I. Information of colonies in soft agar

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.
Cell type Number of colonies

WT 10  ± 4
Goal 365  ± 39


Fig. 3. Colony formation of Go-ablated cells and wild type IIC9 cells in soft agar. Wild type IIC9 cells (A and C) and Goalpha -ablated cells (B and D) were grown in soft agar as described by Johansen et al. (29). Briefly, 103 cells were mixed into 1 ml of top agarose (0.35% SeaPlaque-agarose) in alpha -minimal essential medium/F-12 (1:1, v/v) supplemented with 10% FCS and seeded onto 2 ml of solidified bottom agarose (0.7% SeaPlaque-agarose) in alpha -minimal essential medium/F-12 (1:1, v/v) supplemented with 10% FCS in 35-mm diameter wells. The top agar was replenished every 4 days and colony formation was quantified after 14 days. Magnification, × 20 (A and B) and 200 (C and D).
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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 Goalpha -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 Goalpha -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 Goalpha -ablated cells are not growth arrested in serum-free medium and are consistent with the transformed phenotype of these cells.


Fig. 4. [3H]Thymidine incorporation in Goalpha -ablated cells. The incorporation of [3H]thymidine into cells was determined by radiolabeling culture cells with [3H]thymidine as described previously (26, 27). Briefly, subconfluent (3.5 × 105 cells/35-mm dish), serum-deprived cells were incubated in fresh serum-free media incubated either alone (open bars) or supplemented with thrombin (2 NIH units/ml) (filled bars) or FCS (10%) (stripped bars) for 16 h. Cultures were pulsed for 2 h with 5 µCi/ml [3H]thymidine. Incorporation of radioactivity into trichloroacetic acid-insoluble material was quantified by liquid scintillation counting as described previously (8, 26). The data presented is representative of at least three experiments each performed in duplicate. Each value represents the average of duplicate samples. Error bars represent the range of values and are present on all bars.
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DAG Level Is Chronically Elevated in Goalpha -ablated 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 (30-32). 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 (1, 6, 7). 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 Goalpha -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 Goalpha -ablated cells was twice that of quiescent wild type cells (Fig. 5A). Furthermore, while the addition of alpha -thrombin to the wild type cells resulted in a 2-fold increase in DAG mass level, addition of alpha -thrombin to the ablated cells did not induce a significant further increase in DAG levels (Fig. 5A). These results, observed in two independently isolated clones, indicate that the DAG level in Goalpha -ablated cells was constitutively elevated even in the absence of any added mitogens.


Fig. 5. DAG and PC metabolism in Goalpha -ablated cells. A, DAG level in Goalpha -ablated cells: serum-deprived Goalpha -ablated and wild type cells in 35-mm dishes were incubated in the presence (open bars) or absence (closed bars) of 2 NIH units/ml (1.4 nM) thrombin for the indicated times. DAG mass level was then quantified as described previously (8). Each value represents the average of duplicate samples and is representative of at least three experiments. Error bars represent the range of values and are present on all bars. B, basal choline and phosphorylcholine levels: Goalpha -ablated (filled bars) and wild type (open bars) cells (3.5 × 105 cells/35-mm dish) were serum deprived and labeled with [3H]choline as described (15, 26, 34). Choline and phosphorylcholine, identified by comparison to known standards, were isolated by TLC and quantified as described previously (15, 26, 34). Each value represents the average of duplicate samples and is representative of at least three experiments. Error bars represent the range of values and are present on all bars. C, basal and thrombin-induced PLD activity: PLD activity was quantified as the production of phosphatidylethanol (PEt) (15). Briefly, wild type (open bars) and Go-ablated (filled bars) cells (3.5 × 105 cells/35-mm dish) incubated in serum-free medium supplemented with 5 µCi/[3H]myristate for 2 h at 37 °C. The cells were then incubated in fresh serum-free medium alone (Control) or serum-free medium supplemented with 100 mM ethanol in the presence or absence of 2 NIH units/ml (1.4 nM) thrombin for 30 min at 37 °C. Reactions were terminated by aspirating the medium and immediately adding ice-cold methanol. Radiolabeled phosphatidylethanol was then isolated and quantified as described previously (15). Each value represents the average of duplicate samples and is representative of at least three experiments. Error bars represent the range of values and are present on all bars. D, assessment of choline kinase activity in wild type and Goalpha -ablated cells: serum-deprived Go-ablated and wild type cells (3.5 × 105 cells/35-mm dish) were washed once and incubated in the serum-free medium supplemented with [3H]choline (5 µCi/ml). At the indicated times, cultures were washed three times in serum-free medium supplemented with 20 mM choline chloride and incubated for an additional 5 min at 37 °C. Total choline metabolites, [3H]phosphorylcholine ([3H]PCho), [3H]choline ([3H]Cho), and [3H]glycerolphosphorylcholine ([3H]GPC) were then isolated by TLC and quantified as described previously (28). Choline kinase activity was measured as the amount of choline converted to phosphorylcholine at the indicated times. Data are presented as the ratio of [3H]PCho relative to the total amount of water-soluble choline metabolites ([3H]PCho/[3H]PCho + [3H]Cho + [3H]glycerolphosphocholine). Each ratio represents the average of duplicate samples and is representative of at least three experiments. Error bars represent the range of values and are present on all bars.
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The Increased DAG Is Due to a Constitutively Activated PC-PLC

We have shown that PC hydrolysis is the major, if not exclusive, source of mitogen-induced DAGs in IIC9 cells (8, 15, 26, 33-35). In view of these data, we examined the possibility that an increase in PC hydrolysis contributed to the elevated DAG level in the Goalpha -ablated cells.

To determine if PC hydrolysis was affected in the Goalpha -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 (15). TLC analysis of water-soluble head groups indicated that the phosphorylcholine level in the Goalpha -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. 5B). 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 Goalpha -ablated cells. These results have been observed in three independently isolated clones.

The Increased PC Metabolism Is Not Due to PLD/PAPH/CK Activity

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 (36-38).

In view of the above, PLD activity was quantified in Goalpha -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 (15). 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. 5C, 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 Goalpha -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 Goalpha -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. 5D, the conversion of choline to phosphorylcholine was essentially identical in both cell types demonstrating that the CK activity was not elevated in the Goalpha -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 Goalpha -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 Goalpha -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 Goalpha -ablated cells. In addition, they indicate that Goalpha ablation-induced transformation is different from v-ras-induced transformation, since the later involves PLD/PAPH/CK activities (36-38).

The Increased PC Metabolism Is Not Due to PLA2 Activity

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 (27), both of which have been implicated in mitogenic signaling cascades (7). Basal and alpha -thrombin-induced PLA2 activities in Goalpha -ablated and wild type cells was assessed by quantifying the release of arachidonic acid and its metabolites. As observed for PLD, basal and alpha -thrombin-activated PLA2 activity was not affected by the ablation of Goalpha (Fig. 6A). 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 Goalpha -ablated cells.


Fig. 6. Basal and thrombin-induced arachidonic acid release and inositol trisphosphate. A, basal and thrombin-induced arachidonic acid release: the release of arachidonic acid and its metabolites was quantified essentially as described previously (27). Briefly, serum-deprived wild type and Goalpha -ablated cells (3.5 × 105 cells/35-mm dish) were labeled with [3H]arachidonic acid for 48 h and then washed and incubated in serum-free medium supplemented with fatty acid-free bovine serum albumin for at least 30 min at 37 °C. Cells were then incubated in fresh serum free medium alone (open bars) or serum-free medium supplemented with 2 NIH units/ml (1.4 nM) thrombin (filled bars). The release of [3H]arachidonic acid and its metabolites into the media was then quantified (27). Each value represents the average of duplicate samples and is representative of at least three experiments. Error bars represent the range of values and are present on all points. B, basal and thrombin-induced inositol trisphosphate: inositol trisphosphate levels (IP3), were quantified in myo-[3H]inositol-labeled cultures essentially as described previously (27), except the medium was supplemented with 20 mM LiCl 1 min prior to the addition of growth factors and was present for the remainder of the experiment. Briefly, Goalpha -ablated and wild type cells (3.5 × 105 cells/35-mm dish) were labeled with myo-[3H]inositol (1 µCi/ml) for 48 h and then incubated in serum-free medium supplemented with 20 mM LiCl in the presence or absence of 2 NIH units/ml thrombin for 15 min at 37 °C. IP3 was then quantified using anion exchange columns as described (27). Each value represents the average of duplicate samples and is representative of at least three experiments. Error bars represent the range of values and are present on all bars.
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PI Hydrolysis Is Suppressed in Goalpha -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 cellular transformation. alpha -Thrombin induces the hydrolysis of PIs and contributes to some of the thrombin-induced increase in DAG in IIC9s at early times (39). We therefore examined PI metabolism in Goalpha -ablated cells to determine if this hydrolysis contributed to the increased level of DAGs in the ablated cells. PI metabolism was quantified in cells radiolabeled with myo-[3H]inositol as described previously (27). As shown in Fig. 6B, basal PI metabolism remains unaffected in the ablated cells. As in wild type cells, thrombin induced PI hydrolysis in the Goalpha -ablated cells. Interestingly, however, the level of PI metabolism, assessed as IP3 production, is attenuated in the ablated cells (11-fold increase in wild type cells and 2.5-fold in Goalpha -ablated cells). Similar results were obtained for the production of IP1 and IP2 and have been observed in two independently isolated clones (data not shown).

alpha -Transducin Reverses the Increase in PC-PLC in Goalpha -ablated Cells

The above data demonstrate that PC-PLC is constitutively active in Goalpha -ablated cells. To determine if this activation results from an increase in beta gamma dimers, Goa1 cells were transfected with the alpha  subunit of transducin (Gtalpha ) (see Ref. 66) which has been used to sequester these dimers (40, 41). As shown in Fig. 7, transfecting Goa1 cells with Gtalpha reduces the PC-PLC activity, indicated by the phosphorylcholine level, to that observed in wild type IIC9s. These data suggest that an increase in beta gamma dimers is involved in the increased PC-PLC activity observed in Goalpha -ablated cells and support the notion that these dimers are involved in the receptor-mediated regulation of this enzyme.


Fig. 7. Gtalpha reduces the elevated PC-PLC activity in Gao1 cells to wild type basal levels. Goa1 cells were transiently transfected with the alpha  subunit of transducin as using LipofectamineTM as described in the accompanying article (66). Gao1 and Gao1/Gtalpha cells (3.5 × 105 cells/35-mm dish) were then serum deprived and radiolabeled with [3H]choline as described (15, 26, 34). Choline and phosphorylcholine levels were then quantified in these cultures as described in the legend to Fig. 5B. Data represent the mean ± S.E. from two experiments each performed in duplicate.
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DISCUSSION

The data in this paper demonstrate for the first time that the alpha  subunit of the Go GTP-binding protein is involved in regulating cell growth and PC-PLC activity. Absence of this protein in IIC9 cells results in: (a) a transformed phenotype, and (b) a 10-fold increase in basal PC-PLC activity without increasing PLD, PLA2, CK, sphingomyelinase, and PI-PLC activities. These results indicate that Go selectively regulates PC-PLC and is an important component involved in the regulation of cell growth.

While some G proteins (e.g. Ras, Gi, and Gs) are known to be involved in regulating cell growth (1, 42, 43), the data in this work are the first to demonstrate that Go plays a role in mitogenesis. Ablation of Goalpha results in a transformed phenotype. This is indicated by anchorage-independent growth (Table I and Fig. 3), formation of foci in confluent cultures (Fig. 2), and increased level of thymidine incorporation when cells are serum-deprived (Fig. 4). We should note that the Goalpha is selectively ablated in this cells. Other homologous G proteins, including Gialpha whose coding sequence shows the greatest homology to Goalpha , are not affected (Fig. 1). This is likely due to the fact that the antisense construct included the 5'- and 3'-untranslated region of Go which does not posses significant homology to other G proteins, including Gialpha (Basic Local Alignment Search Tool (BLAST) data base, NCBI, Dec. 1996).

The above data suggest that the presence of Goalpha in quiescent cells is involved in suppressing cell growth and supports the notion that this protein acts as a tumor suppressor (44). Consistent with this hypothesis, Goalpha was selectively deficient in two pituitary tumors (7315a, MtTW15) (45). In this regard, it is interesting to note that the human Goalpha gene maps to the chromosomal region (16q13) (Online Mendelian Inheritance in Man, OMIMTM), associated with the loss of heterozygosity identified in a variety of tumors (46-51). Furthermore, because dominant negative variants of tumor suppressor genes often result in the overexpression of their proteins, our data suggest that the increased levels of Goalpha protein observed in many neuroendocrine tumors (52, 53) and in Merkel cell carcinoma (54) may result from a dominant negative defect in this G protein, consistent with its role in suppressing cell proliferation. Taken together, these data demonstrate that Goalpha is an important component of a mechanism involved in the regulation of cell growth. The suppression of mitogenesis by Goalpha is a novel observation in that most defects in G proteins resulting in cell transformation involve expression of a constitutively active form (GTPase deficient) of G proteins (4, 5).

In addition to a transformed phenotype, a phospholipase associated with the regulation of cell growth (11, 15, 55-58), PC-PLC, is constitutively active in the Goalpha -ablated cells. The basal levels of other phospholipases involved in signal transduction cascades, PLA2 (Fig. 6A), PLD (Fig. 5C), and PI-PLC (Fig. 6B) are not affected in the ablated cells. Thrombin-induced levels of PLA2 and PLD are also unaffected. Interestingly, induced levels of PI-PLC appears to be blunted (Fig. 6B), which may be due to a protein kinase C-mediated inhibition of this enzyme resulting from the elevated levels of DAG as a result of constitutive PC-PLC activation. Importantly, however, the suppression of PI hydrolysis cannot account for the increase in DAG levels observed in the Goalpha -ablated cells.

The DAG elevation is significant in that the mass amount of this lipid is strictly regulated in wild type cells reflecting its importance in initiating cell proliferation. It is noteworthy that the amount of DAG present in serum-deprived Goalpha -ablated cells is similar to that observed in wild type cells stimulated with a maximal concentration of mitogen (2 NIH units/ml thrombin or 10% FCS). It is conceivable, therefore, that the elevated level of DAG in the ablated cells is at least partly involved in generating the transformed phenotype. Consistent with these data, comparable DAG levels were observed in NIH 3T3 cells stably transfected with Bacillus cereus PC-PLC which, interestingly, also displayed a transformed phenotype (29).

In the accompanying article (66), we present data demonstrating that Ras and ERK are constitutively active, as well as elevated expression of cyclin D1 and constitutively active cyclin D1-CDK complexes, in the Goalpha -ablated cells. In this regard, it is interesting to note that PC-PLC has been suggested to play a role in this pathway. Transfection of 3T3 with bacterial PC-PLC reversed the inhibition of cell growth mediated by a dominant negative Ras, but not dominant negative Raf, constructs (55, 59). Consistent with this notion, transfection of cells with bacterial PC-PLC results in a transformed phenotype (60). Although Ras also activates choline kinase that confuses the identification of a PC-PLC (30, 61), there is direct evidence for a Ras-mediated activation of PC-PLC (57). Homogenates of Ras-transformed cells contain a higher PC-PLC activity than their non-transformed counterparts, that appears to be accompanied by an increase in membrane-associated PC-PLC protein (57). Together, these data support the notion that the increased PC-PLC activity observed in the Goalpha -ablated cells reflects the constitutive activation of the Ras/Raf/ERK signaling pathway.

The increase in PC-PLC is particularly interesting in that there has been some controversy regarding the existence and regulation of this enzyme (7). As mentioned, it is indeed possible that the "apparent" PC-PLC activity observed in some studies was due to the combined increase in PC-PLD, PAPH, and/or CK (30, 36, 37). The data in this work, however, provide definitive evidence that a PC-PLC is present and constitutively active in the Goalpha -ablated cells. Phosphorylcholine and DAG, two products for PC-PLC mediated hydrolysis, are elevated in the ablated cells (Fig. 5, A and B). The increase in phosphorylcholine is not due to an increase in PLD (Fig. 5C) and/or CK (Fig. 5D) activities or sphingomyelin hydrolysis (data not shown). DAG levels are not due to any other phospholipid hydrolysis as products (head groups) of other phospholipases are not elevated in the ablated cells (Fig. 6B and data not shown).

Three simplified working models for the mechanism by which Go modulates PC-PLC. In one scheme (referred to as Scheme I), the beta gamma activation model, PC-PLC is activated by beta gamma dimers dissociated from Go in response to agonist stimulation. A second model (referred to as Scheme II), the Go-bound inhibitor model, PC-PLC is bound to an inhibitor which is in a complex with the Go heterotrimer in quiescent cells. Receptor activation leads to a dissociation of this complex which relieves the inhibition of PC-PLC. In both Schemes I and II, the interaction of the G protein with the enzyme may be direct or may involve an auxiliary protein. This is particularly true for Scheme I as Ras has been implicated in mediating the beta gamma dimer-induced activation of PLD. The third model (referred to as Scheme III), the dual G protein model, PC-PLC is regulated by two G proteins, Go, and another as yet unidentified G protein, G?. The GTP-bound form of Goalpha suppresses PC-PLC activity while this form of G?alpha stimulates activity. In the basal state, a certain proportion of the Galpha subunits of each G protein is present in the free GTP-bound state (62), but the predominant influence is suppression of PC-PLC by Goalpha . Receptor stimulation results in an increase in GTP-G?alpha binding to PC-PLC to stimulate its activity.

These models can be used to explain the increase in PC-PLC activity observed in the Goalpha -ablated cells. In Scheme I, ablation of Goalpha results in an increased level of beta gamma dimers resulting in an increase in PC-PLC activity. In Scheme II, ablation of Goalpha results in an inability of the inhibitor to complex and inhibit PC-PLC. In Scheme III, the ablation of Goalpha removes the inhibitory G protein, resulting in the presence of the stimulatory G protein, G?alpha , only. We should note that Schemes II and III are consistent with the observation that Gialpha constitutively suppresses PI-PLC. Watkins et al. (63) demonstrated that ablation of Gi2alpha resulted in enhanced basal PI-PLC in a beta gamma dimer-independent manner. The data in Fig. 7, however, lend strong support to Scheme I. This is supported in the accompanying paper (66) demonstrating that the specific growth-associated activities that are constitutively elevated in Goa1 (Ras/ERK and cyclin D1) are reduced to basal levels in the presence of Gtalpha . We cannot, however, completely rule out the possibility that Gtalpha functionally complements Goalpha in Schemes II or III. Experiments to discriminate among these three models, and to completely define the role of this G protein in regulating PC-PLC and cell growth, are in progress.


FOOTNOTES

*   This work was supported by National Institutes of Health Grants GM51593 (to D. M. R.) and HL40901 (to J. J. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
par    To whom the correspondence should be addressed: Dept. of Physiology, The Johns Hopkins University, School of Medicine, 725 North Wolfe St., Baltimore, MD 21205. Tel.: 410-955-1289; Fax: 410-955-0461; E-mail: draben{at}wpo.bs.jhu.edu.
1   The 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.

ACKNOWLEDGEMENTS

We thank Drs. Jeremy Nathans and Yanshu Wang for critically reading this manuscript and helpful suggestions.


REFERENCES

  1. Seuwen, K., and Pouyssegur, J. (1992) Adv. Cancer Res. 58, 75-94 [Medline] [Order article via Infotrieve]
  2. Sternweis, P. C., and Smrcka, A. V. (1992) Trends Biochem. Sci. 17, 502-506 [CrossRef][Medline] [Order article via Infotrieve]
  3. Wakelam, M. J., Briscoe, C. P., Stewart, A., Pettitt, T. R., Cross, M. J., Paul, A., Yule, J. M., Gardner, S. D., and Hodgkin, M. (1993) Biochem. Soc. Trans. 21, 874-877 [Medline] [Order article via Infotrieve]
  4. Landis, C. A., Masters, S. B., Spada, A., Pace, A. M., Bourne, H. R., and Vallar, L. (1989) Nature 340, 692-696 [CrossRef][Medline] [Order article via Infotrieve]
  5. Lyons, J., Landis, C. A., Harsh, G., Vallar, L., Grunewald, K., Feichtinger, H., Duh, Q. Y., Clark, O. H., Kawasaki, E., Bourne, H. R., and McCormick, F. (1990) Science 249, 655-659 [Medline] [Order article via Infotrieve]
  6. Noh, D. Y., Shin, S. H., and Rhee, S. G. (1995) Biochim. Biophys. Acta 1242, 99-113 [CrossRef][Medline] [Order article via Infotrieve]
  7. Exton, J. H. (1994) Biochim. Biophys. Acta 1212, 26-42 [Medline] [Order article via Infotrieve]
  8. Wright, T. M., Rangan, L. A., Shin, H. S., and Raben, D. M. (1988) J. Biol. Chem. 263, 9374-9380 [Abstract/Free Full Text]
  9. Baldi, E., Musial, A., and Kester, M. (1994) Am. J. Physiol. 266, F957-F65 [Abstract/Free Full Text]
  10. Cifone, M. G., Roncaioli, P., De Maria, R., Camarda, G., Santoni, A., Ruberti, G., and Testi, R. (1995) EMBO J. 14, 5859-5868 [Abstract]
  11. Larrodera, P., Cornet, M. E., Diaz-Meco, M. T., Lopez-Barahona, M., Diaz-Laviada, I., Guddal, P. H., Johansen, T., and Moscat, J. (1990) Cell 61, 1113-1120 [Medline] [Order article via Infotrieve]
  12. Laviada, I. D., Baudet, C., Galve-Roperh, I., Naveilhan, P., and Brachet, P. (1995) FEBS Lett. 364, 301-304 [CrossRef][Medline] [Order article via Infotrieve]
  13. Randell, E., Mulye, H., Mookerjea, S., and Nagpurkar, A. (1992) Biochim. Biophys. Acta 1124, 273-278 [Medline] [Order article via Infotrieve]
  14. Sands, W. A., Clark, J. S., and Liew, F. Y. (1994) Biochem. Biophys. Res. Commun. 199, 461-466 [CrossRef][Medline] [Order article via Infotrieve]
  15. Wright, T. M., Willenberger, S., and Raben, D. M. (1992) Biochem. J. 285, 395-400 [Medline] [Order article via Infotrieve]
  16. Vu, T. K., Hung, D. T., Wheaton, V. I., and Coughlin, S. R. (1991) Cell 64, 1057-1068 [Medline] [Order article via Infotrieve]
  17. Hung, D. T., Wong, Y. H., Vu, T.-K. H., and Coughlin, S. R. (1992) J. Biol. Chem. 267, 20831-20834 [Abstract/Free Full Text]
  18. Murthy, K. S., and Makhlouf, G. M. (1995) Mol. Pharmacol. 48, 293-304 [Abstract]
  19. Exton, J. H. (1993) Adv. Second Messenger Phosphoprotein Res. 28, 65-72 [Medline] [Order article via Infotrieve]
  20. Blank, J. L., Brattain, K. A., and Exton, J. H. (1992) J. Biol. Chem. 267, 23069-23075 [Abstract/Free Full Text]
  21. Lee, S. B., Shin, S. H., Hepler, J. R., Gilman, A. G., and Rhee, S. G. (1993) J. Biol. Chem. 268, 25952-25957 [Abstract/Free Full Text]
  22. Rhee, S. G., and Choi, K. D. (1992) Adv. Second Messenger Phosphoprotein Res. 26, 35-61 [Medline] [Order article via Infotrieve]
  23. Axelrod, J. (1995) Trends. Neurosci. 18, 64-65 [CrossRef][Medline] [Order article via Infotrieve]
  24. Cockcroft, S., Thomas, G. M., Fensome, A., Geny, B., Cunningham, E., Gout, I., Hiles, I., Totty, N. F., Truong, O., and Hsuan, J. J. (1994) Science 263, 523-526 [Medline] [Order article via Infotrieve]
  25. Low, D. A., Scott, R. W., Baker, J. B., and Cunningham, D. D. (1982) Nature 298, 476-478 [Medline] [Order article via Infotrieve]
  26. Rangan, L. A., Wright, T. M., and Raben, D. M. (1991) Cell Regul. 2, 311-316 [Medline] [Order article via Infotrieve]
  27. Raben, D. M., Yasuda, K. M., and Cunningham, D. D. (1987) J. Cell. Physiol. 130, 466-473 [Medline] [Order article via Infotrieve]
  28. Wright, T. M., Shin, H. S., and Raben, D. M. (1990) Biochem. J. 267, 501-507 [Medline] [Order article via Infotrieve]
  29. Johansen, T., Bjorkoy, G., Overvatn, A., Diaz-Meco, M. T., Traavik, T., and Moscat, J. (1994) Mol. Cell Biol. 14, 646-654 [Abstract]
  30. Macara, I. G. (1989) Mol. Cell Biol. 9, 325-328 [Medline] [Order article via Infotrieve]
  31. Weinstein, I. B. (1990) Adv. Second Messenger Phosphoprotein Res. 24, 307-316 [Medline] [Order article via Infotrieve]
  32. Waterfield, M. D. (1989) Br. Med. Bull. 45, 570-581 [Abstract]
  33. Pessin, M. S., Altin, J. G., Jarpe, M., Tansley, F., Bradshaw, R. A., and Raben, D. M. (1991) Cell Regul. 2, 383-390 [Medline] [Order article via Infotrieve]
  34. Pessin, M. S., Baldassare, J. J., and Raben, D. M. (1990) J. Biol. Chem. 265, 7959-7966 [Abstract/Free Full Text]
  35. Pessin, M. S., and Raben, D. M. (1989) J. Biol. Chem. 264, 8729-8738 [Abstract/Free Full Text]
  36. Carnero, A., Dolfi, F., and Lacal, J. C. (1994) J. Cell. Biochem. 54, 478-486 [Medline] [Order article via Infotrieve]
  37. Carnero, A., Cuadrado, A., del Peso, L., and Lacal, J. C. (1994) Oncogene 9, 1387-1395 [Medline] [Order article via Infotrieve]
  38. Preiss, J., Loomis, C. R., Bishop, W. R., Stein, R., Niedel, J. E., and Bell, R. M. (1986) J. Biol. Chem. 261, 8597-8600 [Abstract/Free Full Text]
  39. Raben, D. M., Pessin, M. S., Rangan, L. A., and Wright, T. M. (1990) J. Cell. Biochem. 44, 117-125 [Medline] [Order article via Infotrieve]
  40. Faure, M., Voyno-Yasenetskaya, T. A., and Bourne, H. R. (1994) J. Biol. Chem. 269, 7851-7854 [Abstract/Free Full Text]
  41. Crespo, P., Xu, N., Simonds, W. F., and Gutkind, J. S. (1994) Nature 369, 418-420 [CrossRef][Medline] [Order article via Infotrieve]
  42. Gupta, S. K., Gallego, C., and Johnson, G. L. (1992) Mol. Biol. Cell 3, 123-128 [Medline] [Order article via Infotrieve]
  43. McCormick, F. (1995) Mol. Reprod. Dev. 42, 500-506 [Medline] [Order article via Infotrieve]
  44. Weinberg, R. A. (1996) Cell 85, 457-459 [Medline] [Order article via Infotrieve]
  45. Collu, R., Bouvier, C., Lagace, G., Unson, C. G., Milligan, G., Goldsmith, P., and Spiegel, A. M. (1988) Endocrinology 122, 1176-1178 [Abstract]
  46. Austruy, E., Candon, S., Henry, I., Gyapay, G., Tournade, M. F., Mannens, M., Callen, D., Junien, C., and Jeanpierre, C. (1995) Genes Chromosomes & Cancer 14, 285-294 [Medline] [Order article via Infotrieve]
  47. Bardi, G., Johansson, B., Pandis, N., Mandahl, N., Bak-Jensen, E., Lindstrom, C., Tornqvist, A., Frederiksen, H., Andren-Sandberg, A., Mitelman, F., and Heim, S. (1993) Int. J. Cancer 55, 422-428 [Medline] [Order article via Infotrieve]
  48. Douglass, E. C., Rowe, S. T., Valentine, M., Parham, D., Meyer, W. H., and Thompson, E. I. (1990) Cytogenet. Cell Genet. 53, 87-90 [Medline] [Order article via Infotrieve]
  49. Huff, V., Reeve, A. E., Leppert, M., Strong, L. C., Douglass, E. C., Geiser, C. F., Li, F. P., Meadows, A., Callen, D. F., Lenoir, G., and Saunders, S. (1992) Cancer Res. 52, 6117-6120 [Abstract]
  50. Mandahl, N., Mertens, F., Willen, H., Rydholm, A., Brosjo, O., and Mitelman, F. (1994) J. Cancer Res. Clin. Oncol. 120, 707-711 [Medline] [Order article via Infotrieve]
  51. Newsham, I., Kindler-Rohrborn, A., Daub, D., and Cavenee, W. (1995) Genes Chromosomes & Cancer 12, 1-7 [Medline] [Order article via Infotrieve]
  52. Kato, K., Asano, T., Kamiya, N., Haimoto, H., Hosoda, S., Nagasaka, A., Ariyoshi, Y., and Ishiguro, Y. (1987) Cancer Res. 47, 5800-5805 [Abstract]
  53. Asano, T., Morishita, R., and Kato, K. (1988) Cancer Res. 48, 2756-2759 [Abstract]
  54. Uhara, H., Wang, Y. L., Matsumoto, S., Kawachi, S., and Saida, T. (1995) J. Cutan. Pathol. 22, 146-148 [Medline] [Order article via Infotrieve]
  55. Cai, H., Erhardt, P., Szeberenyi, J., Diaz-Meco, M. T., Johansen, T., Moscat, J., and Cooper, G. M. (1992) Mol. Cell Biol. 12, 5329-5335 [Abstract]
  56. Dominguez, I., Marshall, M. S., Gibbs, J. B., Garcia de Herreros, A., Cornet, M. E., Graziani, G., Diaz-Meco, M. T., Johansen, T., McCormick, F., and Moscat, J. (1991) EMBO J. 10, 3215-3220 [Abstract]
  57. Podo, F., Ferretti, A., Knijn, A., Zhang, P., Ramoni, C., Barletta, B., Pini, C., Baccarini, S., and Pulciani, S. (1996) Anticancer Res. 16, 1399-1412 [Medline] [Order article via Infotrieve]
  58. Cowen, D. S., Sowers, R. S., and Manning, D. R. (1996) J. Biol. Chem. 271, 22297-22300 [Abstract/Free Full Text]
  59. Cai, H., Erhardt, P., Troppmair, J., Diaz-Meco, M. T., Sithanandam, G., Rapp, U. R., Moscat, J., and Cooper, G. M. (1993) Mol. Cell Biol. 13, 7645-7651 [Abstract]
  60. Bjorkoy, G., Overvatn, A., Diaz-Meco, M. T., Moscat, J., and Johansen, T. (1995) J. Biol. Chem. 270, 21299-21306 [Abstract/Free Full Text]
  61. Ratnam, S., and Kent, C. (1995) Arch. Biochem. Biophys. 323, 313-322 [CrossRef][Medline] [Order article via Infotrieve]
  62. Tian, W. N., and Deth, R. C. (1993) Life Sci. 52, 1899-1907 [Medline] [Order article via Infotrieve]
  63. Watkins, D. C., Moxham, C. M., Morris, A. J., and Malbon, C. C. (1994) Biochem. J. 299, 593-596 [Medline] [Order article via Infotrieve]
  64. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  65. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4350-4354 [Abstract]
  66. Weber, J. D., Cheng, J., Raben, D. M., Gardner, A., and Baldassare, J. J. (1997) J. Biol Chem. 272, 17320-17326 [Abstract/Free Full Text]

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