Oxytocin Induces Dephosphorylation of Eukaryotic Elongation Factor 2 in Human Myometrial Cells
http://www.100md.com
内分泌学杂志 2005年第5期
Departments of Medicine (D.D., M.G., M.-E.C., C.R., H.H.Z.), Pharmacology and Therapeutics (D.D., M.-E.C., H.H.Z.), and Obstetrics and Gynecology (H.H.Z.), McGill University, Montreal, Quebec, Canada H3A 1A1
Address all correspondence and requests for reprints to: Hans H. Zingg, M.D., Ph.D., Laboratory of Molecular Endocrinology, Royal Victoria Hospital, Room H7.63, 687 Pine Avenue West, Montreal, Quebec, Canada H3A 1A1. E-mail: hans.zingg@mcgill.ca.
Abstract
The oxytocin (OT) receptor (OTR) mediates a wide spectrum of biological actions and is expressed in a large number of different tissues, including uterine, breast, and lung tumors. To define more completely the intracellular signaling mechanisms linked to OTR activation, we have used a phosphoproteomics approach and have characterized changes in the phosphorylation states of intracellular proteins in response to OTR activation in OTR-expressing cell lines. Using a specific antiphosphothreonine antibody, we observed several distinct changes in the threonine phosphorylation patterns. The most prominent change involved dephosphorylation of a 95-kDa moiety. Purification by ion exchange chromatography combined with one- and two-dimensional polyacrylamide gel electrophoresis followed by N-terminal micro-sequence analysis revealed that the 95-kDa moiety corresponded to eukaryotic elongation factor 2. This protein is a key regulator of cellular protein synthesis and mediates, upon dephosphorylation, the translocation step of peptide chain elongation. Dose-response curves in myometrial cells expressing the endogenous OTR indicated a significant effect of OT on eukaryotic elongation factor 2 dephosphorylation at 1 nM, a concentration close to the dissociation constant (Kd) of OT. Time course analysis indicates that the effect is rapid with a significant effect occurring at 5 min. To determine directly the effect of OT on protein synthesis, the incorporation of [35S]Met into total protein was assessed. In myometrial cells, OTR activation led to significant 29% increase in total protein synthesis over a 2-h period. These findings establish a novel link between OTR activation and cellular protein synthesis and thus define a mechanism by which OT assumes a so far unrecognized, physiologically relevant trophic function.
Introduction
THE NONAPEPTIDE HORMONE oxytocin (OT) assumes a wide spectrum of physiological functions that are mediated through the OT receptor (OTR), a member of the heptahelical G protein-coupled receptor family (1, 2). The classical actions of OT include uterine smooth muscle contraction at parturition and mammary gland myoepithelial cell contraction during lactation (1, 2). However, the spectrum of OT actions has been greatly enlarged through the discovery of novel sites of OTR expression. There is evidence that OT is involved in the regulation of affiliative behavior, pituitary function, sodium excretion, T-cell function, cardiomyocyte differentiation, and the growth control of different cancer cells (3, 4, 5). The OTR is primarily associated with Gq/11, and OTR activation results in a phospholipase C-mediated increase in intracellular calcium and inositol trisphosphate production (6). However, coupling to Gi and Gh has also been reported (7, 8). In addition, OTR activation results in phosphorylation of ERK1/2 involving Gq, G?/, and epidermal growth factor receptor kinase activation (9). The specific signaling pathways by which the OTR exerts its manifold demonstrated and postulated physiological and pathophysiological actions remain to be clarified. In an attempt to define further the spectrum of intracellular events that are set into motion upon OTR activation, we have used a phosphoproteomics approach. We characterized changes in the phosphorylation states of intracellular proteins in response to OTR activation, using one- and two-dimensional gel electrophoresis in conjunction with phosphospecific antibodies. Here, we report the characterization of a 95-kDa protein (pT95) that was dephosphorylated on threonine after OTR activation and was identified as eukaryotic elongation factor 2 (eEF2). eEF2 is a key regulator of cellular protein synthesis and, upon dephosphorylation, mediates the translocation step of peptide chain elongation at the level of the ribosome (10). Our present findings establish a link between OTR activation and cellular protein synthesis and may represent an important mechanism by which OT promotes cell growth and differentiation.
Materials and Methods
Tissue culture and immunoblotting
Chinese hamster ovary (CHO) cells stably expressing the rat OTR (CHO-OTR cells) were obtained from Steve J. Lolait (University of Bristol, Bristol, UK) (11). They were maintained in -MEM (Invitrogen, Carlsbad, CA; Sigma-Aldrich, St. Louis, MO), supplemented with 5% fetal bovine serum (Bio-Media, Montreal, Quebec) and penicillin and streptomycin. Myometrial M11 cells, derived from primary human myometrial cells by repeated passage, were obtained from John A. Copland (Mayo Clinic College of Medicine, Jacksonville, FL) and were maintained in DMEM high glucose (Invitrogen), supplemented with 10% fetal bovine serum and antibiotics. Cells were plated onto 100-mm culture dishes and cultured at 37 C in 5% CO2. At 90% confluency, cells were maintained in the absence of serum for 24 h (for CHO-OTR) or 48 h (for M11 cells) before exposure to test agents. At the end of the test period, cells were washed twice with ice-cold PBS and flash frozen in liquid nitrogen. The frozen plates were kept at –60 C until use. Cells were lysed in 1 ml of lysate buffer [25 mM HEPES/NaOH (pH 7.4), 150 mM NaCl, 5 mM EDTA, 10% glycerol, and 1% Triton X-100) and a mix of protease inhibitors (1 μg/ml each aprotinin, pepstatin A, and leupeptin and 0.5 mM phenylmethylsulfonylfluoride) and phosphatase inhibitors (50 mM NaF, 30 mM sodium pyrophosphate, and 1 mM sodium orthovanadate). Lysates were clarified by centrifugation at 15,000 x g for 10 min at 4 C in a microcentrifuge. Total protein concentration was determined by a colorimetric assay using BSA as standard (BCA Protein Assay Reagent Kit, Pierce, Rockford, IL.). Proteins were denatured by boiling in Laemmli buffer [50 mM Tris-HCl (pH 6.8), 2% sodium dodecyl sulfate (SDS), 10% glycerol, and 0.1 M ?-mercaptoethanol] for 5 min and subjected to SDS-PAGE and Western blotting. Immunodetection involved antiphospho-eEF2 and antiphosphothreonine antibodies (Cell Signaling Technology, Beverly, MA) in conjunction with a second horseradish peroxidase C-conjugated antibody and a chemiluminescence detection system (SuperSignal, Pierce).
Two-dimensional gel electrophoresis
Cytosols of CHO-OTR were prepared by disrupting cells in ice-cold cytosol buffer-1 [20 mM HEPES (pH 7.4), 0.25 M sucrose, 1 mM EDTA, and protease and phosphatase inhibitors, as described above], followed by passing the homogenate 5 times through a 26-gauge syringe. The samples were ultracentrifuged at 100,000 x g for 45 min at 4 C, and cytosolic proteins were concentrated using a spin column concentrator (Amicon Microcon, 3000 molecular weight cutoff, Millipore, Bedford, MA). Fifty micrograms protein was mixed with urea buffer [120 mM urea, 0.001% 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate, 2 mM tributylphosphine (Bio-Rad, Hercules, CA), 0.2% bromophenol blue, Biolytes (3–10, Bio-Rad), and protease and phosphatase inhibitors] and rehydrated passively overnight onto an isoelectric focusing strip (ReadyStrip IPG 3–10, Bio-Rad) according to the manufacturer’s recommendations. Isoelectric focusing (first dimension) was achieved using a Bio-Rad Protean IEF cell under the following conditions: 250 V for 15 min, voltage ramp from 250-4000 V for 2 h, and 4000 V for 5 h. Strips were then placed for 10 min each in equilibration buffer-1 [1% SDS, 0.125 M Tris-HCl (pH 6.8), 20% glycerol, and 130 mM dithiothreitol] and in equilibration buffer-2 [2% SDS, 0.375 M Tris-HCl (pH 6.8), 20% glycerol, and 2.5% iodoacetamide]. Each strip was laid on top of a 7.5% SDS-PAGE, and proteins were resolved at 30 V for 3 h. Proteins were blotted onto a polyvinylidene difluoride (PVDF) membrane (Immobilon-P, Millipore), and phosphothreonine proteins were detected using an antiphosphothreonine antibody (Cell Signaling Technology).
Protein purification and amino acid sequence determination
Cytosolic extracts were prepared as described above from 40 15-cm culture dishes of confluent CHO-OTR cells, using cytosol buffer-2 [20 mM HEPES (pH 7.4), 5 mM EDTA, 50 mM NaCl, and protease and phosphatase inhibitors]. The extract was dialyzed three times against 400 ml of low-salt buffer [20 mM Tris (pH 8.0), 100 mM NaCl, 1 mM EDTA, 10 mM NaF, and 1 mM phenylmethylsulfonylfluoride]. The dialyzed cytosol was clarified at 7500 x g at 4 C and loaded at 0.5 ml/min onto a 10-ml Q Sepharose high-performance column (Amersham Biosciences Inc., Piscataway, NJ) equilibrated in low-salt buffer at 4 C. The column was first washed with 200 ml low-salt buffer, and proteins were eluted using a salt gradient 100–500 mM NaCl at 1 ml/min. Fractions of 2.5 ml were collected. The phosphothreonine protein content of each fraction was assessed by immunoblot analysis. Fractions containing pT95 were pooled and concentrated to a volume of 350 μl. Ten micrograms protein from the pool was used to confirm the nature of the purified protein by two-dimensional gel/phosphothreonine immunoblot analysis as described above. One milligram of the pooled proteins was boiled in Laemmli buffer and loaded onto a 7.5% SDS-PAGE, run in 25 mM Tris, 200 mM glycine, 0.1% SDS, and 1 mM sodium thioglycolate (preparative blot). One tenth of the sample was applied to a separated track for phosphothreonine immunoblot analysis (analytical blot). Proteins were transferred onto an amino acid sequencing quality PVDF membrane (Sequiblot PVDF, Bio-Rad). The analytical blot portion of the membrane was cut for antiphosphothreonine immunoblot analysis. The resulting autoradiographic film was overlaid on top of the preparative portion of the membrane, and the zone containing pT95 was excised. N-terminal amino acid sequence determination was achieved directly on the membrane by the Sheldon Biotechnology Centre (McGill University, Montreal, Quebec) using Edman degradation chemistry on a gas-phase/pulsed-liquid automated sequencer (LC 492 Procise Protein Sequencing System, Applied Biosystems, Foster City, CA) and the Model 610A Protein Sequencing Software Package (Applied Biosystems) for sequence assignment.
[35S]methionine incorporation into nascent proteins
M11 cells were seeded in six-well plates. Once confluency was reached, cells were grown in the absence of serum in DMEM low glucose (Specialty Media, Phillipsburg, NJ) for 12 h, followed by a 2-h incubation in L-methionine-free DMEM labeling media (Specialty Media). Next, 1 μCi [35S] methionine (PerkinElmer, Boston, MA) was added, and incubation was continued for an additional 2 h in the presence or absence of 100 nM insulin or 100 nM OT. Cells were then washed twice with PBS and flash frozen in liquid nitrogen until use. Cells were lysed in 250 μl lysate buffer, and protein concentration in the lysates was determined by a bicinchoninic acid protein quantitation assay (Pierce). To denature proteins, 50 μl total protein lysate was mixed with 50 μl 1 M NaOH/2% H2O2 and incubated for 10 min at 37 C. Proteins were then precipitated on ice for 30 min after addition of 1 ml 25% trichloroacetic acid/2% casamino acids. The precipitates were recovered by filtration through G4 fiberglass filters (Fisher Scientific Co., Pittsburgh, PA). Filters were washed three times with 1 ml ice-cold 5% trichloroacetic acid and once by 3 ml acetone, dried at room temperature, and radioactivity was measured by scintillation counting.
Results
OT-induced changes in threonine phosphorylation
Changes in protein phosphorylation on threonine residues in response to OT were assessed by immunoblotting using OTR-expressing cells (CHO-OTR cells) and a phosphothreonine-specific antibody. As shown in Fig. 1, the most striking change was the dephosphorylation of a band at molecular mass 95 kDa. The putative phosphoprotein corresponding to this band was termed pT95. Maximum dephosphorylation of pT95 was observed at 20 min after OT addition. Concomitantly, the de novo phosphorylation of a band at molecular mass 65 kDa (termed pT65) was observed with a maximum of phosphorylation coincident with the maximal dephosphorylation of pT95. Additional bands were observed at 110 and 120 kDa that were rapidly phosphorylated within the first 2 min after OT addition (Fig. 1).
FIG. 1. OT-induced changes in the overall threonine-phosphorylation pattern in CHO cells stably transfected with the human OTR (CHO-OTR cells). Cells were serum-starved for 24 h and treated for the times indicated with 100 nM OT, 1 μM OTA (a specific OT antagonist) (19 ), or both. Proteins in cell lysates were separated by 7.5% SDS-PAGE and analyzed by immunoblotting using a specific antiphosphothreonine antibody (Cell Signaling Technology). The positions of the major dephosphorylated substrate pT95 and the major phosphorylated substrate pT65 are indicated.
Identification of the pT95 band as eEF2
The overall strategy of the purification of pT95 is summarized in Fig. 2. Ion exchange chromatography was used as a first step. We determined by two-dimensional gel electrophoresis that the isoelectric point of pT95 was 5.9 (Fig. 3). To ensure that the target protein pT95 was present in ionized form, ion exchange chromatography was performed at pH 8. Proteins were eluted using a salt gradient from 100–500 mM NaCl, and fractions were collected and analyzed by antiphosphothreonine immunoblotting (Fig. 4). pT95 eluted in fractions six to 12, whereas pT65 eluted in fractions 24–32 (Fig. 4B). The fractions containing pT95 were pooled, and pT95 was further purified by preparative SDS-PAGE. After transfer to a PVDF membrane, the membrane portion containing the pT95 protein was excised and subjected to N-terminal microsequencing. The analysis yielded the N-terminal amino acid sequence VNFTVDQIRA. A database search of the NBCI protein database using the BLAST algorithm indicated that this amino acid sequence was unique and corresponded to amino acids two to 11 of human and rat elongation factor 2 (eEF2). Human eEF2 represents an 858-amino acid protein with a molecular mass of 95,337 kDa (GenBank protein no. NP_001952.1).
FIG. 2. Schematic diagram of the strategy followed for the purification and characterization of pT95. Preparative steps are indicated on the left, analytical steps on the right.
FIG. 3. Determination of isoelectric point of pT95 by two-dimensional polyacrylamide gel electrophoresis. CHO-OTR cells were treated (right panel) or not (left panel) with OT (100 nM). Proteins were first separated according to their isoelectric point (first dimension, horizontal) and then by their electrophoretic mobility in 7.5% SDS-PAGE (second dimension, vertical). Proteins were transferred onto a PVDF membrane and immunoblotted with a specific antiphosphothreonine antibody as in Fig. 1. Asterisk indicates position of pT95.
FIG. 4. Initial purification of pT95 by ion exchange chromatography. CHO-OTR cells were treated for 10 min with OT (100 mM) to allow for the concomitant detection of pT65. Cell lysates were loaded onto a Q Sepharose ion exchange column (Amersham Biosciences Inc.) and eluted with a salt gradient (100–500 mM). A, Total protein elution was monitored by absorbance at 280 nm and 2.5-ml fractions were collected. B, Phosphothreonine immunoreactivity was assessed in each second fraction by immunoblotting as in Fig. 1. Fractions 6–12 containing pT95 (pool 1) and fractions 24–32 containing pT65 (pool 2) were pooled. C, Immunoblot of total lysates of CHO-OTR cells, as well as of pools 1 and 2 shown in B. Antiphospho-eEF2 antibody (lanes 4–6) and antiphosphothreonine antibody (lanes 1–3) were used sequentially for the same membrane.
Confirmation of the identity of pT95 with eEF2
To confirm that pT95 corresponded to eEF2, we performed immunoblot analysis using a commercially available antiphospho eEF2 antibody. As shown in Fig. 4C, the phospho eEF2 antibody recognized a 95-kDa band that comigrated exactly with the pT95 band recognized by the antiphosphothreonine antibody. The 95-kDa band was detected by the phospho-eEF2 antibody in total cell lysates and was strongly enriched in pool 1 (the pT95-containing fractions) but was absent in pool 2 (the pT65-containing fractions). These findings lend further support to the idea that pT95 corresponds to the phosphorylated form of eEF2. Furthermore, the findings indicate that pT65 is unrelated to eEF2 and is unlikely to correspond to an eEF2 degradation product because phospho-eEF2 immunoreactivity was absent from the pT65-enriched pool 2.
OT-induced eEF2 dephosphorylation in myometrial cells
If pT95 corresponded indeed to phospho-eEF2, then OT should induce a decrease in phospho-eEF2 immunoreactivity that corresponded to the decrease observed for pT95. Moreover, we wished to determine whether this phenomenon was restricted to CHO-OTR cells or whether it could also be observed in untransformed myometrial cells. As shown in Fig. 5A, OT induced a rapid decrease in phospho-eEF2 immunoreactivity in myometrial M11 cells. The time course of dephosphorylation corresponded to the one observed for pT95 in CHO-OTR cells. This finding provided further confirmation that the pT95 band corresponded to eEF2 and indicated that OT-induced eEF2 dephosphorylation occurs in nontransformed myometrial cells.
FIG. 5. OT-induced eEF2 dephosphorylation in untransformed myometrial M11 cells. A, Time course of OT-induced dephosphorylation. Cells were treated as in Fig. 1 with OT for different times, and eEF2 phosphorylation was assessed by immunoblotting using a phospho-eEF2 antibody (Cell Signaling Technology). Autoradiograms resulting from three independent experiments were analyzed by densitometric analysis using ImageQuant 5.1 (Amersham Biosciences Inc.). The control values were set to 100%. Each point represents the mean ± SEM. *, P < 0.05 vs. time 0. A representative autoradiogram is shown in the top panel. B, Dose-response curve of OT-induced eEF2 dephosphorylation. eEF2 phosphorylation was assessed as in A, and the means ± SEM from three independent experiments were plotted against the OT concentration used. C, Control; *, P < 0.05 vs. control. A representative autoradiogram is shown in the top panel.
Dose-response relationship of OT-induced eEF2 dephosphorylation
We next determined the dose-response relationship of OT-induced eEF2 dephosphorylation. As shown in Fig. 5B, the maximum effective concentration of OT was 10–8 M, and the efficiency of OT induced dephosphorylation decreased with concentrations above 10–7 M. The fact that the same dephosphorylation was observed in the nontransformed myometrial cells as in OTR-transfected CHO cells indicated that the observed OT effect is physiologically relevant because it can be mediated by the endogenous OTR in a physiologically relevant cell type.
OT-induced stimulation of total protein synthesis
Because it is widely accepted that eEF2 dephosphorylation is accompanied by an increase in the rate of peptide chain elongation, and, as a result, of protein synthesis, we wished next to determine to what extent OTR activation was leading to a measurable increase in overall protein synthesis. To this end, we determined the effect of 100 nM OT on the amount of [35S]methionine incorporation into proteins in myometrial cells. As shown in Fig. 6, OT induced a significant 29% increase in the rate of total protein synthesis over a 2-h period. This stimulatory effect was similar to the one induced by insulin (32%). This finding indicates that the observed OT-induced dephosphorylation of eEF2 is functionally meaningful and supports a novel role for OT as a trophic agent.
FIG. 6. Effect of OT on protein synthesis in myometrial M11 cells in vitro. Cells were incubated in presence of 1 μCi [35S]methionine for 2 h in the presence of absence of 100 nM OT. Data were expressed as counts per minute per microgram of protein. Each bar represents the mean ± SEM of three experiments. *, P < 0.01 vs. control.
Discussion
In the present study, a phosphoproteomics approach was taken to identify additional intracellular targets of OTR-linked signaling pathways. The successful identification of eEF2 as a novel target of OTR signaling attests to the efficiency of this approach. As a result, we have identified not only a novel target of OTR signaling, but we have also been able to provide the basis for further characterization of a novel trophic role of OT at the level of the myometrium and possibly other OT-responsive tissues or cell types. There has been much discussion of the possible mitogenic roles of OT in different cancer tissues (4, 5). Although OT stimulates proliferation of certain cancer cells, the role of OT as a trophic factor has not been directly addressed in any study.
The present finding of OT’s effect on protein synthesis may shed new light on a so far unrecognized mechanism supporting a role of OT in cell differentiation. In support of this idea is the observation that OT induces differentiation of developing mammary gland myoepithelial cells in culture (12) and that OT-deficient OT–/– mice are deficient in postpartum development and differentiation of mammary gland alveoli (13). Moreover, OT stimulates growth of endothelial cells (14) and of the ovarian follicle (15) and induces differentiation of embryonic stem cells to cardiomyocytes (16). It remains to be explored to what extent these effects also involve an eEF2-mediated trophic action.
eEF2 is a monomeric 95-kDa GTP binding protein that is critically involved in the process of protein elongation at the level of the ribosome where eEF2 specifically mediates the translocation step (10). eEF2 is inactivated by specific phosphorylation on threonine 56, a residue located in the GTP binding site. Phosphorylation at this site prevents binding of eEF2 to the ribosome. The phosphorylation status of eEF2 is regulated, on the one hand, by the activity of a specific eEF2 kinase and, on the other hand, by less well-characterized phosphatases, likely including the phosphatase PP2A or a closely related enzyme (10). Although the mechanism of action remains to be determined, the relatively rapid effect of OT on eEF2 dephosphorylation suggests OT-induced activation of a phosphatase. It is well established that trophic growth factors such as insulin and IGF-I stimulate protein synthesis via eEF2 dephosphorylation and that this pathway involves the mammalian target of rapamycin (10). On the other hand, recent reports have indicated that eEF2 dephosphorylation can also be induced by ERK1/2 activation (16, 17). This latter pathway has been implicated in mediating the effect of angiotensin II, endothelin, and 1-adrenergic receptor agonists (17, 18). Interestingly, our preliminary experiments indicate that neither blockage of the ERK 1/2 pathway nor blockage of the mammalian target of rapamycin pathway are able to fully abrogate the action of OT on eEF2 dephosphorylation (Devost, D., M.-E. Carrier, and H. H. Zingg, unpublished data). Therefore, the precise pathways via which OTR activation leads to eEF2 dephosphorylation and increased protein synthesis remain to be determined. The dose-response curve indicates that the maximal OT effect is obtained at 10 nM and that the effect is suppressed at higher concentrations. It is possible that, at higher concentrations, the OT effect on phosphatases is overridden by a calcium-/calmodulin-dependent stimulation of eEF2 kinase (10). It is thus conceivable that OT subserves a trophic role in the uterus during pregnancy but that, with the onset of labor, maximal OTR activation may lead to an inhibition of protein synthesis in the interest of energy preservation for the high-energy demands of myometrial contractions.
Additional proteins that were threonine-phosphorylated in response to OT remain to be characterized. This includes proteins in the range of 110–120 kDa that were rapidly phosphorylated within the first 2 min after OT addition as well a 65-kDa moiety (pT65). The phosphorylation of pT65 was maximal 20 min after OT addition and coincided exactly with the dephosphorylation of eEF2. Because pT65 is threonine-phosphorylated but not recognized by the antiphospho eEF2 antibody used here, it is, however, unlikely to represent an eEF2 breakdown product.
In summary, our studies on OT-induced changes in intracellular phosphorylation patterns have led to the identification of a novel OTR-linked signaling pathway that may underlie a so-far understudied role of OT as a trophic factor. More work is needed to fully define the pathways underlying OT-induced eEF2 dephosphorylation and the role of OT’s trophic actions in OT target tissues.
Acknowledgments
We thank Drs. John A. Copland and Steven L. Young for the gift of M11 myometrial cells and Dr. Steve Lolait for the gift of CHO-OTR cells.
References
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Address all correspondence and requests for reprints to: Hans H. Zingg, M.D., Ph.D., Laboratory of Molecular Endocrinology, Royal Victoria Hospital, Room H7.63, 687 Pine Avenue West, Montreal, Quebec, Canada H3A 1A1. E-mail: hans.zingg@mcgill.ca.
Abstract
The oxytocin (OT) receptor (OTR) mediates a wide spectrum of biological actions and is expressed in a large number of different tissues, including uterine, breast, and lung tumors. To define more completely the intracellular signaling mechanisms linked to OTR activation, we have used a phosphoproteomics approach and have characterized changes in the phosphorylation states of intracellular proteins in response to OTR activation in OTR-expressing cell lines. Using a specific antiphosphothreonine antibody, we observed several distinct changes in the threonine phosphorylation patterns. The most prominent change involved dephosphorylation of a 95-kDa moiety. Purification by ion exchange chromatography combined with one- and two-dimensional polyacrylamide gel electrophoresis followed by N-terminal micro-sequence analysis revealed that the 95-kDa moiety corresponded to eukaryotic elongation factor 2. This protein is a key regulator of cellular protein synthesis and mediates, upon dephosphorylation, the translocation step of peptide chain elongation. Dose-response curves in myometrial cells expressing the endogenous OTR indicated a significant effect of OT on eukaryotic elongation factor 2 dephosphorylation at 1 nM, a concentration close to the dissociation constant (Kd) of OT. Time course analysis indicates that the effect is rapid with a significant effect occurring at 5 min. To determine directly the effect of OT on protein synthesis, the incorporation of [35S]Met into total protein was assessed. In myometrial cells, OTR activation led to significant 29% increase in total protein synthesis over a 2-h period. These findings establish a novel link between OTR activation and cellular protein synthesis and thus define a mechanism by which OT assumes a so far unrecognized, physiologically relevant trophic function.
Introduction
THE NONAPEPTIDE HORMONE oxytocin (OT) assumes a wide spectrum of physiological functions that are mediated through the OT receptor (OTR), a member of the heptahelical G protein-coupled receptor family (1, 2). The classical actions of OT include uterine smooth muscle contraction at parturition and mammary gland myoepithelial cell contraction during lactation (1, 2). However, the spectrum of OT actions has been greatly enlarged through the discovery of novel sites of OTR expression. There is evidence that OT is involved in the regulation of affiliative behavior, pituitary function, sodium excretion, T-cell function, cardiomyocyte differentiation, and the growth control of different cancer cells (3, 4, 5). The OTR is primarily associated with Gq/11, and OTR activation results in a phospholipase C-mediated increase in intracellular calcium and inositol trisphosphate production (6). However, coupling to Gi and Gh has also been reported (7, 8). In addition, OTR activation results in phosphorylation of ERK1/2 involving Gq, G?/, and epidermal growth factor receptor kinase activation (9). The specific signaling pathways by which the OTR exerts its manifold demonstrated and postulated physiological and pathophysiological actions remain to be clarified. In an attempt to define further the spectrum of intracellular events that are set into motion upon OTR activation, we have used a phosphoproteomics approach. We characterized changes in the phosphorylation states of intracellular proteins in response to OTR activation, using one- and two-dimensional gel electrophoresis in conjunction with phosphospecific antibodies. Here, we report the characterization of a 95-kDa protein (pT95) that was dephosphorylated on threonine after OTR activation and was identified as eukaryotic elongation factor 2 (eEF2). eEF2 is a key regulator of cellular protein synthesis and, upon dephosphorylation, mediates the translocation step of peptide chain elongation at the level of the ribosome (10). Our present findings establish a link between OTR activation and cellular protein synthesis and may represent an important mechanism by which OT promotes cell growth and differentiation.
Materials and Methods
Tissue culture and immunoblotting
Chinese hamster ovary (CHO) cells stably expressing the rat OTR (CHO-OTR cells) were obtained from Steve J. Lolait (University of Bristol, Bristol, UK) (11). They were maintained in -MEM (Invitrogen, Carlsbad, CA; Sigma-Aldrich, St. Louis, MO), supplemented with 5% fetal bovine serum (Bio-Media, Montreal, Quebec) and penicillin and streptomycin. Myometrial M11 cells, derived from primary human myometrial cells by repeated passage, were obtained from John A. Copland (Mayo Clinic College of Medicine, Jacksonville, FL) and were maintained in DMEM high glucose (Invitrogen), supplemented with 10% fetal bovine serum and antibiotics. Cells were plated onto 100-mm culture dishes and cultured at 37 C in 5% CO2. At 90% confluency, cells were maintained in the absence of serum for 24 h (for CHO-OTR) or 48 h (for M11 cells) before exposure to test agents. At the end of the test period, cells were washed twice with ice-cold PBS and flash frozen in liquid nitrogen. The frozen plates were kept at –60 C until use. Cells were lysed in 1 ml of lysate buffer [25 mM HEPES/NaOH (pH 7.4), 150 mM NaCl, 5 mM EDTA, 10% glycerol, and 1% Triton X-100) and a mix of protease inhibitors (1 μg/ml each aprotinin, pepstatin A, and leupeptin and 0.5 mM phenylmethylsulfonylfluoride) and phosphatase inhibitors (50 mM NaF, 30 mM sodium pyrophosphate, and 1 mM sodium orthovanadate). Lysates were clarified by centrifugation at 15,000 x g for 10 min at 4 C in a microcentrifuge. Total protein concentration was determined by a colorimetric assay using BSA as standard (BCA Protein Assay Reagent Kit, Pierce, Rockford, IL.). Proteins were denatured by boiling in Laemmli buffer [50 mM Tris-HCl (pH 6.8), 2% sodium dodecyl sulfate (SDS), 10% glycerol, and 0.1 M ?-mercaptoethanol] for 5 min and subjected to SDS-PAGE and Western blotting. Immunodetection involved antiphospho-eEF2 and antiphosphothreonine antibodies (Cell Signaling Technology, Beverly, MA) in conjunction with a second horseradish peroxidase C-conjugated antibody and a chemiluminescence detection system (SuperSignal, Pierce).
Two-dimensional gel electrophoresis
Cytosols of CHO-OTR were prepared by disrupting cells in ice-cold cytosol buffer-1 [20 mM HEPES (pH 7.4), 0.25 M sucrose, 1 mM EDTA, and protease and phosphatase inhibitors, as described above], followed by passing the homogenate 5 times through a 26-gauge syringe. The samples were ultracentrifuged at 100,000 x g for 45 min at 4 C, and cytosolic proteins were concentrated using a spin column concentrator (Amicon Microcon, 3000 molecular weight cutoff, Millipore, Bedford, MA). Fifty micrograms protein was mixed with urea buffer [120 mM urea, 0.001% 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate, 2 mM tributylphosphine (Bio-Rad, Hercules, CA), 0.2% bromophenol blue, Biolytes (3–10, Bio-Rad), and protease and phosphatase inhibitors] and rehydrated passively overnight onto an isoelectric focusing strip (ReadyStrip IPG 3–10, Bio-Rad) according to the manufacturer’s recommendations. Isoelectric focusing (first dimension) was achieved using a Bio-Rad Protean IEF cell under the following conditions: 250 V for 15 min, voltage ramp from 250-4000 V for 2 h, and 4000 V for 5 h. Strips were then placed for 10 min each in equilibration buffer-1 [1% SDS, 0.125 M Tris-HCl (pH 6.8), 20% glycerol, and 130 mM dithiothreitol] and in equilibration buffer-2 [2% SDS, 0.375 M Tris-HCl (pH 6.8), 20% glycerol, and 2.5% iodoacetamide]. Each strip was laid on top of a 7.5% SDS-PAGE, and proteins were resolved at 30 V for 3 h. Proteins were blotted onto a polyvinylidene difluoride (PVDF) membrane (Immobilon-P, Millipore), and phosphothreonine proteins were detected using an antiphosphothreonine antibody (Cell Signaling Technology).
Protein purification and amino acid sequence determination
Cytosolic extracts were prepared as described above from 40 15-cm culture dishes of confluent CHO-OTR cells, using cytosol buffer-2 [20 mM HEPES (pH 7.4), 5 mM EDTA, 50 mM NaCl, and protease and phosphatase inhibitors]. The extract was dialyzed three times against 400 ml of low-salt buffer [20 mM Tris (pH 8.0), 100 mM NaCl, 1 mM EDTA, 10 mM NaF, and 1 mM phenylmethylsulfonylfluoride]. The dialyzed cytosol was clarified at 7500 x g at 4 C and loaded at 0.5 ml/min onto a 10-ml Q Sepharose high-performance column (Amersham Biosciences Inc., Piscataway, NJ) equilibrated in low-salt buffer at 4 C. The column was first washed with 200 ml low-salt buffer, and proteins were eluted using a salt gradient 100–500 mM NaCl at 1 ml/min. Fractions of 2.5 ml were collected. The phosphothreonine protein content of each fraction was assessed by immunoblot analysis. Fractions containing pT95 were pooled and concentrated to a volume of 350 μl. Ten micrograms protein from the pool was used to confirm the nature of the purified protein by two-dimensional gel/phosphothreonine immunoblot analysis as described above. One milligram of the pooled proteins was boiled in Laemmli buffer and loaded onto a 7.5% SDS-PAGE, run in 25 mM Tris, 200 mM glycine, 0.1% SDS, and 1 mM sodium thioglycolate (preparative blot). One tenth of the sample was applied to a separated track for phosphothreonine immunoblot analysis (analytical blot). Proteins were transferred onto an amino acid sequencing quality PVDF membrane (Sequiblot PVDF, Bio-Rad). The analytical blot portion of the membrane was cut for antiphosphothreonine immunoblot analysis. The resulting autoradiographic film was overlaid on top of the preparative portion of the membrane, and the zone containing pT95 was excised. N-terminal amino acid sequence determination was achieved directly on the membrane by the Sheldon Biotechnology Centre (McGill University, Montreal, Quebec) using Edman degradation chemistry on a gas-phase/pulsed-liquid automated sequencer (LC 492 Procise Protein Sequencing System, Applied Biosystems, Foster City, CA) and the Model 610A Protein Sequencing Software Package (Applied Biosystems) for sequence assignment.
[35S]methionine incorporation into nascent proteins
M11 cells were seeded in six-well plates. Once confluency was reached, cells were grown in the absence of serum in DMEM low glucose (Specialty Media, Phillipsburg, NJ) for 12 h, followed by a 2-h incubation in L-methionine-free DMEM labeling media (Specialty Media). Next, 1 μCi [35S] methionine (PerkinElmer, Boston, MA) was added, and incubation was continued for an additional 2 h in the presence or absence of 100 nM insulin or 100 nM OT. Cells were then washed twice with PBS and flash frozen in liquid nitrogen until use. Cells were lysed in 250 μl lysate buffer, and protein concentration in the lysates was determined by a bicinchoninic acid protein quantitation assay (Pierce). To denature proteins, 50 μl total protein lysate was mixed with 50 μl 1 M NaOH/2% H2O2 and incubated for 10 min at 37 C. Proteins were then precipitated on ice for 30 min after addition of 1 ml 25% trichloroacetic acid/2% casamino acids. The precipitates were recovered by filtration through G4 fiberglass filters (Fisher Scientific Co., Pittsburgh, PA). Filters were washed three times with 1 ml ice-cold 5% trichloroacetic acid and once by 3 ml acetone, dried at room temperature, and radioactivity was measured by scintillation counting.
Results
OT-induced changes in threonine phosphorylation
Changes in protein phosphorylation on threonine residues in response to OT were assessed by immunoblotting using OTR-expressing cells (CHO-OTR cells) and a phosphothreonine-specific antibody. As shown in Fig. 1, the most striking change was the dephosphorylation of a band at molecular mass 95 kDa. The putative phosphoprotein corresponding to this band was termed pT95. Maximum dephosphorylation of pT95 was observed at 20 min after OT addition. Concomitantly, the de novo phosphorylation of a band at molecular mass 65 kDa (termed pT65) was observed with a maximum of phosphorylation coincident with the maximal dephosphorylation of pT95. Additional bands were observed at 110 and 120 kDa that were rapidly phosphorylated within the first 2 min after OT addition (Fig. 1).
FIG. 1. OT-induced changes in the overall threonine-phosphorylation pattern in CHO cells stably transfected with the human OTR (CHO-OTR cells). Cells were serum-starved for 24 h and treated for the times indicated with 100 nM OT, 1 μM OTA (a specific OT antagonist) (19 ), or both. Proteins in cell lysates were separated by 7.5% SDS-PAGE and analyzed by immunoblotting using a specific antiphosphothreonine antibody (Cell Signaling Technology). The positions of the major dephosphorylated substrate pT95 and the major phosphorylated substrate pT65 are indicated.
Identification of the pT95 band as eEF2
The overall strategy of the purification of pT95 is summarized in Fig. 2. Ion exchange chromatography was used as a first step. We determined by two-dimensional gel electrophoresis that the isoelectric point of pT95 was 5.9 (Fig. 3). To ensure that the target protein pT95 was present in ionized form, ion exchange chromatography was performed at pH 8. Proteins were eluted using a salt gradient from 100–500 mM NaCl, and fractions were collected and analyzed by antiphosphothreonine immunoblotting (Fig. 4). pT95 eluted in fractions six to 12, whereas pT65 eluted in fractions 24–32 (Fig. 4B). The fractions containing pT95 were pooled, and pT95 was further purified by preparative SDS-PAGE. After transfer to a PVDF membrane, the membrane portion containing the pT95 protein was excised and subjected to N-terminal microsequencing. The analysis yielded the N-terminal amino acid sequence VNFTVDQIRA. A database search of the NBCI protein database using the BLAST algorithm indicated that this amino acid sequence was unique and corresponded to amino acids two to 11 of human and rat elongation factor 2 (eEF2). Human eEF2 represents an 858-amino acid protein with a molecular mass of 95,337 kDa (GenBank protein no. NP_001952.1).
FIG. 2. Schematic diagram of the strategy followed for the purification and characterization of pT95. Preparative steps are indicated on the left, analytical steps on the right.
FIG. 3. Determination of isoelectric point of pT95 by two-dimensional polyacrylamide gel electrophoresis. CHO-OTR cells were treated (right panel) or not (left panel) with OT (100 nM). Proteins were first separated according to their isoelectric point (first dimension, horizontal) and then by their electrophoretic mobility in 7.5% SDS-PAGE (second dimension, vertical). Proteins were transferred onto a PVDF membrane and immunoblotted with a specific antiphosphothreonine antibody as in Fig. 1. Asterisk indicates position of pT95.
FIG. 4. Initial purification of pT95 by ion exchange chromatography. CHO-OTR cells were treated for 10 min with OT (100 mM) to allow for the concomitant detection of pT65. Cell lysates were loaded onto a Q Sepharose ion exchange column (Amersham Biosciences Inc.) and eluted with a salt gradient (100–500 mM). A, Total protein elution was monitored by absorbance at 280 nm and 2.5-ml fractions were collected. B, Phosphothreonine immunoreactivity was assessed in each second fraction by immunoblotting as in Fig. 1. Fractions 6–12 containing pT95 (pool 1) and fractions 24–32 containing pT65 (pool 2) were pooled. C, Immunoblot of total lysates of CHO-OTR cells, as well as of pools 1 and 2 shown in B. Antiphospho-eEF2 antibody (lanes 4–6) and antiphosphothreonine antibody (lanes 1–3) were used sequentially for the same membrane.
Confirmation of the identity of pT95 with eEF2
To confirm that pT95 corresponded to eEF2, we performed immunoblot analysis using a commercially available antiphospho eEF2 antibody. As shown in Fig. 4C, the phospho eEF2 antibody recognized a 95-kDa band that comigrated exactly with the pT95 band recognized by the antiphosphothreonine antibody. The 95-kDa band was detected by the phospho-eEF2 antibody in total cell lysates and was strongly enriched in pool 1 (the pT95-containing fractions) but was absent in pool 2 (the pT65-containing fractions). These findings lend further support to the idea that pT95 corresponds to the phosphorylated form of eEF2. Furthermore, the findings indicate that pT65 is unrelated to eEF2 and is unlikely to correspond to an eEF2 degradation product because phospho-eEF2 immunoreactivity was absent from the pT65-enriched pool 2.
OT-induced eEF2 dephosphorylation in myometrial cells
If pT95 corresponded indeed to phospho-eEF2, then OT should induce a decrease in phospho-eEF2 immunoreactivity that corresponded to the decrease observed for pT95. Moreover, we wished to determine whether this phenomenon was restricted to CHO-OTR cells or whether it could also be observed in untransformed myometrial cells. As shown in Fig. 5A, OT induced a rapid decrease in phospho-eEF2 immunoreactivity in myometrial M11 cells. The time course of dephosphorylation corresponded to the one observed for pT95 in CHO-OTR cells. This finding provided further confirmation that the pT95 band corresponded to eEF2 and indicated that OT-induced eEF2 dephosphorylation occurs in nontransformed myometrial cells.
FIG. 5. OT-induced eEF2 dephosphorylation in untransformed myometrial M11 cells. A, Time course of OT-induced dephosphorylation. Cells were treated as in Fig. 1 with OT for different times, and eEF2 phosphorylation was assessed by immunoblotting using a phospho-eEF2 antibody (Cell Signaling Technology). Autoradiograms resulting from three independent experiments were analyzed by densitometric analysis using ImageQuant 5.1 (Amersham Biosciences Inc.). The control values were set to 100%. Each point represents the mean ± SEM. *, P < 0.05 vs. time 0. A representative autoradiogram is shown in the top panel. B, Dose-response curve of OT-induced eEF2 dephosphorylation. eEF2 phosphorylation was assessed as in A, and the means ± SEM from three independent experiments were plotted against the OT concentration used. C, Control; *, P < 0.05 vs. control. A representative autoradiogram is shown in the top panel.
Dose-response relationship of OT-induced eEF2 dephosphorylation
We next determined the dose-response relationship of OT-induced eEF2 dephosphorylation. As shown in Fig. 5B, the maximum effective concentration of OT was 10–8 M, and the efficiency of OT induced dephosphorylation decreased with concentrations above 10–7 M. The fact that the same dephosphorylation was observed in the nontransformed myometrial cells as in OTR-transfected CHO cells indicated that the observed OT effect is physiologically relevant because it can be mediated by the endogenous OTR in a physiologically relevant cell type.
OT-induced stimulation of total protein synthesis
Because it is widely accepted that eEF2 dephosphorylation is accompanied by an increase in the rate of peptide chain elongation, and, as a result, of protein synthesis, we wished next to determine to what extent OTR activation was leading to a measurable increase in overall protein synthesis. To this end, we determined the effect of 100 nM OT on the amount of [35S]methionine incorporation into proteins in myometrial cells. As shown in Fig. 6, OT induced a significant 29% increase in the rate of total protein synthesis over a 2-h period. This stimulatory effect was similar to the one induced by insulin (32%). This finding indicates that the observed OT-induced dephosphorylation of eEF2 is functionally meaningful and supports a novel role for OT as a trophic agent.
FIG. 6. Effect of OT on protein synthesis in myometrial M11 cells in vitro. Cells were incubated in presence of 1 μCi [35S]methionine for 2 h in the presence of absence of 100 nM OT. Data were expressed as counts per minute per microgram of protein. Each bar represents the mean ± SEM of three experiments. *, P < 0.01 vs. control.
Discussion
In the present study, a phosphoproteomics approach was taken to identify additional intracellular targets of OTR-linked signaling pathways. The successful identification of eEF2 as a novel target of OTR signaling attests to the efficiency of this approach. As a result, we have identified not only a novel target of OTR signaling, but we have also been able to provide the basis for further characterization of a novel trophic role of OT at the level of the myometrium and possibly other OT-responsive tissues or cell types. There has been much discussion of the possible mitogenic roles of OT in different cancer tissues (4, 5). Although OT stimulates proliferation of certain cancer cells, the role of OT as a trophic factor has not been directly addressed in any study.
The present finding of OT’s effect on protein synthesis may shed new light on a so far unrecognized mechanism supporting a role of OT in cell differentiation. In support of this idea is the observation that OT induces differentiation of developing mammary gland myoepithelial cells in culture (12) and that OT-deficient OT–/– mice are deficient in postpartum development and differentiation of mammary gland alveoli (13). Moreover, OT stimulates growth of endothelial cells (14) and of the ovarian follicle (15) and induces differentiation of embryonic stem cells to cardiomyocytes (16). It remains to be explored to what extent these effects also involve an eEF2-mediated trophic action.
eEF2 is a monomeric 95-kDa GTP binding protein that is critically involved in the process of protein elongation at the level of the ribosome where eEF2 specifically mediates the translocation step (10). eEF2 is inactivated by specific phosphorylation on threonine 56, a residue located in the GTP binding site. Phosphorylation at this site prevents binding of eEF2 to the ribosome. The phosphorylation status of eEF2 is regulated, on the one hand, by the activity of a specific eEF2 kinase and, on the other hand, by less well-characterized phosphatases, likely including the phosphatase PP2A or a closely related enzyme (10). Although the mechanism of action remains to be determined, the relatively rapid effect of OT on eEF2 dephosphorylation suggests OT-induced activation of a phosphatase. It is well established that trophic growth factors such as insulin and IGF-I stimulate protein synthesis via eEF2 dephosphorylation and that this pathway involves the mammalian target of rapamycin (10). On the other hand, recent reports have indicated that eEF2 dephosphorylation can also be induced by ERK1/2 activation (16, 17). This latter pathway has been implicated in mediating the effect of angiotensin II, endothelin, and 1-adrenergic receptor agonists (17, 18). Interestingly, our preliminary experiments indicate that neither blockage of the ERK 1/2 pathway nor blockage of the mammalian target of rapamycin pathway are able to fully abrogate the action of OT on eEF2 dephosphorylation (Devost, D., M.-E. Carrier, and H. H. Zingg, unpublished data). Therefore, the precise pathways via which OTR activation leads to eEF2 dephosphorylation and increased protein synthesis remain to be determined. The dose-response curve indicates that the maximal OT effect is obtained at 10 nM and that the effect is suppressed at higher concentrations. It is possible that, at higher concentrations, the OT effect on phosphatases is overridden by a calcium-/calmodulin-dependent stimulation of eEF2 kinase (10). It is thus conceivable that OT subserves a trophic role in the uterus during pregnancy but that, with the onset of labor, maximal OTR activation may lead to an inhibition of protein synthesis in the interest of energy preservation for the high-energy demands of myometrial contractions.
Additional proteins that were threonine-phosphorylated in response to OT remain to be characterized. This includes proteins in the range of 110–120 kDa that were rapidly phosphorylated within the first 2 min after OT addition as well a 65-kDa moiety (pT65). The phosphorylation of pT65 was maximal 20 min after OT addition and coincided exactly with the dephosphorylation of eEF2. Because pT65 is threonine-phosphorylated but not recognized by the antiphospho eEF2 antibody used here, it is, however, unlikely to represent an eEF2 breakdown product.
In summary, our studies on OT-induced changes in intracellular phosphorylation patterns have led to the identification of a novel OTR-linked signaling pathway that may underlie a so-far understudied role of OT as a trophic factor. More work is needed to fully define the pathways underlying OT-induced eEF2 dephosphorylation and the role of OT’s trophic actions in OT target tissues.
Acknowledgments
We thank Drs. John A. Copland and Steven L. Young for the gift of M11 myometrial cells and Dr. Steve Lolait for the gift of CHO-OTR cells.
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