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Department of Reproductive Medicine (K.A.N., S.J.S., M.K.K., A.L.D., R.R., M.A.L.), The Center for the Study of Reproductive Biology and Disease (M.A.L.), and Biomedical Sciences Graduate Program (K.A.N, A.L.D.), University of California, San Diego, La Jolla, California 92093-0674
Address all correspondence and requests for reprints to: Mark A. Lawson, Department of Reproductive Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0674. E-mail: mlawson{at}ucsd.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Cell models of fully committed and differentiated gonadotropes 
T3–1, LßT2, and LßT4 cells (6, 7), derived by targeted tumorigenesis in mouse pituitary, have been developed. These cell lines allow the characterization of signaling pathways activated in response to ligand binding and GnRH receptor activation. Studies using these gonadotrope cell models and primary rat pituitary cultures to investigate the transcriptional response of gonadotropin genes to GnRH have shown that transcriptional changes in gene expression require 6–24 h to reach maximal response levels (3, 8, 9). In addition, studies in pituitary fragments showed no transcriptional responses within a 24-h period of tonic GnRH treatment (10). Similarly, microarray analysis of LßT2 cells detected no significant changes (<1.3 fold change) in gonadotropin gene expression in response to either 1 or 6 h of tonic GnRH treatment (11, 12, 13). These observations corroborate in vivo analysis of steady-state LHß mRNA levels in which a less than 50% increase (1.4-fold change) occurs in response to GnRH stimulation within 6 h (14). In contrast, this same study found maximal (100-fold) increases in serum gonadotropin levels within 6 h of GnRH treatment. Furthermore, it has been shown that increases in LHß protein synthesis in response to GnRH occur within 4 h in LßT2 cells (15). The discrepancy between measurements of transcriptional activity and protein production may be attributed, in part, to translational regulation of protein synthesis.
Translational regulation through extracellular signaling mechanisms commonly occurs through activation of receptor tyrosine kinases such as the insulin and epidermal growth factor receptors (16, 17). Regulation of translation by these receptors proceeds through phosphatidylinositol 3-kinase (PI3 kinase)/AKT and/or ERK signaling pathways. These pathways target the function of the N7-methylguanosine mRNA cap-binding protein eIF4E (eukaryotic initiation factor 4E) as well as eIF4G (eukaryotic initiation factor 4G), a scaffold protein required for the assembly of the translation initiation complex eIF4F. The association of these initiation factors with the mRNA cap is the rate-limiting step in translation initiation and is essential for initiation of capped mRNA translation (18). Phosphorylation of initiation factors controls the rate of mRNA binding to ribosomes. eIF4E is negatively regulated by a family of binding proteins known as the 4E-binding proteins (4E-BP) or protein, heat, and acid stable (PHAS) (19, 20). Phosphorylation of 4E-BP by activated receptor tyrosine kinase signaling cascades disables eIF4E binding activity, allowing eIF4E to associate with the N7-methylguanosine cap and initiate translation (21).
Regulation of translation via G protein-coupled receptors (GPCRs) is not commonly observed. To date, the µ-opioid receptor and the receptors for endothelin, phenylephrine, angiotensin II, and lysophosphatidic acid have been shown to regulate translation in other cell systems (16, 22, 23, 24). We recently demonstrated that the GnRH receptor regulates translation in the gonadotrope-derived cell line, T3–1, which expresses the GnRH receptor but not gonadotropin subunit genes (25). Therefore, to determine whether translational control is relevant to the production of gonadotropins, we examined the impact of GnRH-induced translational activation on LHß synthesis in LßT2 cells, a cell line that endogenously expresses both the GnRH receptor and gonadotropin genes. Our studies address the reported discrepancy between acute increased gonadotropin production and mRNA synthesis.
Using the LßT2 cell model, we show that acute synthesis of LHß and LH secretion in response to GnRH are not exclusively dependent on transcription. We further show that activation of the translational initiation proteins 4E-BP, eIF4E, and eIF4G occurs in a dose- and time-dependent manner in LßT2 cells in response to acute GnRH administration. Moreover, we demonstrate GnRH activation of translation is inhibited by the MAPK kinase (MEK) inhibitor, PD 98059. Based on these findings, we conclude that translational regulation is an important component of the acute response to GnRH receptor activation.
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RESULTS |
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). LßT2 cells were incubated in the presence of vehicle, actinomycin D, or cycloheximide for 1 h. Media were then supplemented with vehicle or 10 nM GnRH. After 4 h, cells were processed for immunocytochemistry using antibody directed against the LHß subunit (Fig. 1A). Alternatively, media were removed for analysis by LH immunoradiometric assay (Fig. 1C). Intensity of staining was compared between untreated and GnRH-treated cells. Quantification of digital images showed significantly increased staining intensity for LHß subunit, after stimulation by GnRH in both vehicle and actinomycin D-treated cells (Fig. 1B). In contrast, cycloheximide significantly decreased GnRH induction of LHß staining. Similarly, GnRH-stimulated LH secretion was not significantly decreased in the presence of actinomycin D but was significantly decreased in the presence of cycloheximide (Fig. 1C). Based on these observations, we conclude that increases in LHß subunit protein and LH secretion after 4 h of GnRH treatment are more dependent on new protein synthesis than new mRNA synthesis.
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by evaluating the effects of GnRH stimulation on reporter genes designed to evaluate the differential role of transcriptional vs. translational stimulation. To this end, we examined the activation of a reporter gene under the transcriptional control of the rat 1.8-kb LHß promoter in comparison with a bicistronic reporter gene responsive to increased cap-dependent translational activity. The bicistronic reporter gene directs the synthesis of a single mRNA encoding two independently translated reading frames. The first reading frame, encoding the firefly luciferase reporter enzyme, is translated by a cap-dependent translation initiation mechanism. The second reading frame, encoding the ß-galactosidase reporter enzyme, is translated independently of the first reading frame through internal ribosomal entry directed by the encephalomyocarditis virus (EMCV) 5'-noncoding region. Activity of the LHß promoter in a vector containing the firefly luciferase gene was measured directly relative to the control cytomegalovirus (CMV) promoter in an identical vector background encoding the Escherichia coli ß-galactosidase. After 6 h stimulation with 10 nM GnRH, no significant increase in rat 1.8-kb LHß promoter activity was detected (Fig. 2); this is in agreement with observations by others examining stimulation of endogenous mRNA for similar time periods (11, 12, 13, 14). However, 4 h activation of cap-dependent translation measured with the bicistronic reporter gene under the transcriptional control of the CMV promoter resulted in significant activation of cap-dependent translation as measured by the ratio of luciferase to ß-galactosidase activity produced by the bicistronic mRNA (Fig. 2). These results confirm that increased synthesis of LHß after GnRH stimulation may be attributed to activation of translation, rather than transcription directed by the LHß promoter.
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T3–1, causes phosphorylation of the translational regulatory factor 4E-BP1. The 4E-BP family of proteins are phosphorylated at five distinct serine/threonine residues causing a marked alteration in their electrophoretic mobility, with the 
-hyperphosphorylated inactive isoform showing the lowest mobility (26). It is known that phosphorylation of 4E-BP prevents interaction with eIF4E, thereby allowing eIF4E to associate with the scaffolding protein eIF4G (27, 28, 29). Phosphorylation of 4E-BP, eIF4E, and eIF4G has been shown to result in an increase of cap-dependent protein synthesis (19, 20, 30, 31). To determine whether GnRH activates these translational initiation factors, the phosphorylation status of each factor after 30 min of GnRH treatment (0.3–100 nM) in LßT2 cells was examined by Western blotting (Fig. 3). Quantitative luminescent image analysis shows that 3 nM GnRH was sufficient to maximally phosphorylate 4E-BP1; although 10 nM GnRH treatment resulted in the highest fold induction of 4E-BP1 phosphorylation, there were no significant differences found between 3 nM and 10 nM GnRH treatments (Fig. 3A). As for eIF4E, 10 nM GnRH caused maximal activation, and this was sustained even in the presence of 100 nM GnRH (Fig. 3B). Similar to 4E-BP1, eIF4G was maximally stimulated in the presence of 3 nM GnRH but at 100 nM, significant stimulation was not observed (Fig. 3C).
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-isoform (Fig. 4A). Analysis of phosphorylation of 4E-BP1 in comparison to control by quantitative luminescent image analysis shows that maximal stimulation occurs within 15 min of GnRH treatment and is maintained up to 60 min, indicating that 10 nM GnRH treatment results in sustained phosphorylation of 4E-BP1 in LßT2 cells. After 4E-BP1 phosphorylation, maximal activation of eIF4E occurs at 15 min and is also maintained for up to 60 min (Fig. 4B). Unlike 4E-BP1 and eIF4E, eIF4G is not maximally activated until 30 min of GnRH treatment. Additionally, eIF4G activation is not sustained by prolonged GnRH stimulation; rather, phospho-eIF4G is significantly decreased after 60 min of treatment (Fig. 4C). This implicates a time-sensitive response to GnRH stimulation that is specific to each initiation factor.
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T3–1 cell line attenuates GnRH-stimulated translation, suggesting the involvement of mTOR in translational control (25). However, GnRH has also been shown to activate the ERK pathway in LßT2 cells, and inhibition of ERK activation by the MEK inhibitor PD 98059 prevents acute GnRH-induced increase in LHß synthesis (15, 32). To determine whether the ERK pathway is involved in the GnRH regulation of translation, we measured GnRH activation of the cap-dependent translational reporter activity in the presence of the MEK inhibitor PD 98059 (Fig. 5). The presence of 30 µM PD 98059 significantly represses the GnRH-induced activation of the translation reporter gene, as measured by the ratio of luciferase to ß-galactosidase activity. Interestingly, translational reporter activation was not affected by rapamycin inhibition of mTOR, the upstream regulator of 4E-BP1 phosphorylation (data not shown). These findings implicate the ERK rather than mTOR as the key pathway in the GnRH activation of translation in LßT2 cells.
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). Western blot analysis shows that GnRH activation of Mnk1 was significantly inhibited by the ERK inhibitor alone (Fig. 6A), whereas activation of eIF4E was significantly inhibited by the ERK, PI3 kinase, and mTOR inhibitors (Fig. 6B). Nevertheless, none of the inhibitors had an effect on GnRH activation of eIF4G (data not shown), suggesting that Mnk and eIF4E are the direct targets of the ERK pathway. This observation, along with the observation that the ERK and not mTOR inhibition leads to translational repression, suggests that Mnk may be the key factor in GnRH regulation of translation in LßT2 cells. This is consistent with the finding that a kinase-deficient Mnk1 results in the impairment of cap-dependent translation in the human embryonic kidney 293 cell line (31).
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T3–1 cells and other studies showing GPCR modulation of translation through 4E-BP1 and PI3 kinase (16, 22, 25). It is noted that significant levels of the -isoform of 4E-BP1 exist in unstimulated LßT2 cells (Fig. 3A). This may be a consequence of a high level of endogenous PI3 kinase activity providing sufficient basal levels of mTOR activity, leading to the maintenance of inactive 4E-BP1 in unstimulated cells. To test this hypothesis, we examined the endogenous levels of AKT, a target of PI3 kinase activity that lies upstream of mTOR. We examined AKT phosphorylation in serum-starved unstimulated LßT2 cells and found that, indeed, AKT is highly activated as determined by phosphorylation at Ser 473 (Fig. 7). Levels of AKT activity in untreated control extracts are as high as in GnRH-stimulated extracts and are inhibited in the presence of the PI3 kinase inhibitor, LY 294002. This finding suggests that PI3 kinase activity is endogenously active in our cell model and provides a possible explanation for the involvement of ERK rather than 4E-BP1/PI3 kinase in translational activation of LßT2 cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). It is of interest that there are no significant differences between groups treated with GnRH in the presence of actinomycin D or cycloheximide in Fig. 1, B and C. If transcription were not involved in the response of GnRH activation, we would expect to see significant differences between these two groups. The lack of difference suggests a transcriptional component in the GnRH-stimulated increase of hormone production. This finding is consistent with other studies showing a small (
1.4 fold) transcriptional activation at 6 h of GnRH stimulation (11, 12, 13, 14).
To further examine the significance of the translational component observed in Fig. 1, B and C, we performed transient tranfections using reporter genes that distinguish between specific activation of the LHß promoter and activation of translation. We observed no significant increase in the rLHß promoter activity driving the reporter gene in LßT2 cells at 4 h of GnRH stimulation (Fig. 2). However, increases in promoter activity are observed in 8 h of GnRH treatment (Lawson, M. A., unpublished observations). In spite of this, increases in translational activity are observed within 4 h of GnRH treatment when changes in cap-dependent translational activity of a bicistronic reporter gene under transcriptional control of the nonspecific CMV immediate-early gene promoter are measured (Fig. 2). These observations suggest that the transcriptional activation may not be as significant as translational activation within the 4-h time period. The differences between our data and previous observations of increased promoter activity reported (37) may be attributed to differences in GnRH treatment. The use of a GnRH analog rather than GnRH peptide and the use of truncated promoters to examine GnRH regulation of promoter activity in other studies may contribute to the observed differences.
To elucidate the mechanism of GnRH regulation of translation, we examined the phosphorylation status of translation initiation factors, 4E-BP1, eIF4E, and eIF4G, in response to GnRH stimulation. Hyperphosphorylation of 4E-BP1 prevents binding to eIF4E, thereby activating translation through increased availability of eIF4E binding to eIF4G to form the cap-binding complex eIF4F. The formation of this cap-binding complex is necessary for ribosome assembly, recruitment, and initiation. Although it is not clearly understood how phosphorylation of eIF4E and eIF4G leads to increased translation, it is known that phosphorylation of eIF4E and eIF4G does not increase their association (28). Rather, it has been postulated that phosphorylation of eIF4E leads to the promotion of ribosome loading (28, 29). Analyses of these initiation factors show that GnRH stimulation results in increased phosphorylation of all three factors. We found this action to be dose dependent with 3–10 nM GnRH as optimal concentrations for maximal initiation factor activation. In addition, we also observed an order of maximal initiation factor activation in response to GnRH treatment: 4E-BP1 and eIF4E are the first to be maximally phosphorylated at 15 min, followed by eIF4G at 30 min (Fig. 4). These findings establish a dose- and time-sensitive factor response that will allow us to establish a pattern of differential regulation by GnRH.
Specificity was also established for the signal cascades that affect each factor’s phosphorylation status. Functional tests of the pathways involved in translational regulation found that GnRH-stimulated cap-dependent translation was inhibited by blockade of the ERK pathway with PD 98059 (Fig. 5) but was unaffected when mTOR activity was blocked with rapamycin (data not shown). Only recently has evidence showing regulation of translation by GPCRs been presented (16, 22, 24, 25). However, these studies show GPCR translational activation through PI3 kinase, mTOR, and 4E-BP1. In contrast, our present study shows that, unlike other GPCRs, GnRH activation of translation in LßT2 cells is more dependent upon activation of eIF4E, rather than inactivation of 4E-BP1. The importance of eIF4E in translational activation has also been demonstrated in human embryonic kidney 293 cells, i.e. eIF4E mutants that cannot be phosphorylated have been found to drastically inhibit cap-dependent translation (31). The dependence of eIF4E rather than 4E-BP1 regulation in LßT2 cells may be attributed to the high level of endogenous PI3 kinase activity in these cells, as evidenced by the high level of AKT phosphorylation found in the unstimulated state (Fig. 7). Endogenously activated AKT may in turn provide sufficient mTOR activation and maintain a level of inactive 4E-BP1 that allows sufficient, unbound eIF4E to be available for interaction with eIF4G in unstimulated cells. Although not sequestered by 4E-BP1, eIF4E still requires activation by Mnk1 and thus renders eIF4E sensitive to inhibition of ERK activity.
GnRH-mediated control of translation initiation has implications for the control of gonadotropes and, ultimately, reproductive function. An important difference in measuring overall translational responses as opposed to measuring transcriptional responses of gonadotropin genes to GnRH is that the first mechanism is a very general response, whereas the second is a very specific response. The question remains whether a generalized stimulation of the translational control apparatus can elicit the specificity in the observed GnRH response. Although many eukaryotic genes are translationally regulated, it is not yet clearly understood how specific genes are controlled through this mechanism. However, regulation of specific genes through alteration of translation rather than transcription has been demonstrated in other model systems (38, 39). It is possible that the structure of the mRNA is a principal contributor to this specificity. Examination of mRNA structure or sequence similarities between genes sensitive to cap-dependent translational control may reveal a common mechanism of regulation.
Our observations suggest that translational control plays an important role in the acute GnRH response of the LßT2 gonadotrope cell line. The rapidity of the translational response explains the ability to detect increased LHß subunit protein in cells within 4 h of GnRH treatment (15). We have confirmed that this activation may indeed be a result of translational regulation that occurs concomitantly with, but independently of, transcriptional activation. Our interpretation is that translational stimulation is a component of the acute GnRH response. In the absence of significant acute changes in LHß gene transcription, cap-dependent translational activation provides a potent, direct, and nongenomic regulatory response to GnRH that results in a rapid increase in hormone synthesis. This regulatory mechanism is one that functions on a time scale that more closely matches the hourly changes that occur during the preovulatory phase of the estrous cycle and may therefore provide a regulatory pathway that is responsive to short-term changes in GnRH pulsatility.
In summary, we have demonstrated that the acute response to GnRH includes a component of translation regulation. We show that this involves GnRH regulation of translation initiation factors. We further show the ERK cascade as the key pathway in the GnRH activation of translation in the LßT2 cell line. Acute activation of translation by GnRH provides a mechanism for rapid response to hypothalamic signals controlling gonadotrope function, thereby providing a mechanism for fine temporal control of gonadotrope function. This may contribute to an increase in LHß protein synthesis that is not solely dependent on transcriptional regulation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Immunocytochemistry
For immunocytochemistry, LßT2 cells were plated at a density of 7 x 104 cells/cm2 on Lab-Tek chamber slides (Nalge Nunc, Naperville, IL) in DMEM containing 10% fetal bovine serum. After 48 h of incubation, medium was changed to serum-free DMEM. After overnight incubation, the media were replaced with fresh serum-free DMEM containing vehicle, 400 µM actinomycin D, or 100 µg/ml cycloheximide. Effectiveness of actinomycin D inhibition of transcription was verified by blockade of increased early-growth response factor-1 mRNA expression in response to GnRH stimulation using Northern blot analysis (40, 41). Cycloheximde inhibition of translation was verified by blockade of protein synthesis as measured by metabolic labeling with 35S-methionine or 35S-cystine. After 1 h of inhibitor treatments, media were adjusted to 10 nm GnRH or an equivalent volume of vehicle. After 4 h, cell viability was verified using Trypan Blue, and no effects by inhibitor treatments were observed. Alternatively, after 4 h of treatment, cells were washed 5 min in PBS once and fixed with 4% formaldehyde in PBS for 20 min. The cells were subsequently washed twice and blocked with 2.0% normal goat serum and 0.3% Triton X-100 in PBS blocking buffer for 20 min. Fixed cells were then washed and incubated for 1 h with 1:2000 dilution of rabbit antirat LHß primary antibody (supplied by the National Hormone and Pituitary Program), washed three times, and incubated with biotinylated antirabbit IgG 1.5 µg/ml (Vector Laboratories, Burlingame, CA) for 30 min. After antibody treatments, fluorescein isothiocyanate Avidin D was then applied at a final concentration of 1 µg/ml (Vector Laboratories). Finally, cells were cover slipped with Vectashield containing 4',6-diamidino-2-phenylindole (Vector Laboratories) for visualization of nuclei and examined by fluorescence microscopy on a Nikon E800 microscope (Nikon, Melville, NY). Images of cells were acquired using a Microfire digital camera (Olympus Corp., Lake Success, NY) using the manufacturer’s software. Image analysis, correction for background fluorescence, and measurement of cell staining intensity was carried out using Sigma Scan Pro v5.0 Software (SPSS, Inc., Chicago, IL). Approximately 10 images per treatment were analyzed for each experiment. Total intensity of staining was normalized to control to yield relative intensity.
LH Immunoradiometric Assay
For the analysis of LH secretion, 1.2 x 106 cells were plated in six-well plates and treated as described for the immunohistochemistry experiments. After the 4-h GnRH treatment, 150 µl of media were collected and desalted using MicroSpin G-25 columns (Amersham Biosciences Corp, Piscataway, NJ). To prevent nonspecific protein binding to the beads, spin columns were rinsed twice with 0.1% BSA/PBS before sample application. Samples were collected according to the manufacture’s instructions. Samples were lyophilized and resuspended in 75 µl of sterile H2O. LH secretion levels were determined by immunoradiometric assay, performed at the Ligand Assay & Analysis Core Laboratory (University of Virginia, Charlottesville, VA).
Plasmids and Transfections
The bicistronic reporter plasmid was previously described (25). Briefly, the 664-bp SpeI fragment from pcDNAI (Invitrogen, Carlsbad, CA) containing the immediate-early CMV promoter was inserted into the NheI site of pGL3-basic (Promega Corp., Madison, WI) creating a new vector, pGL3-CMV. Downstream of the luciferase reporter gene coding sequence, the 5'-noncoding region of the EMCV was inserted, followed by the E. coli ß-galactosidase coding sequence derived from pSDK LacZ pA. The resultant plasmid directs the synthesis of a single transcript encoding the luciferase reporter followed by the EMCV 5'-untranslated region containing the internal ribosome entry site, and the ß-galactosidase coding sequence. Each reading frame is translated independently by either cap-dependent (luciferase) or cap-independent (ß-galactosidase) mechanisms (42).
The 1.8-kb rat LHß promoter-driven reporter plasmid previously described (43) was removed by HinDIII-XbaI restriction digest and inserted into the multiple cloning site of pBluescriptII KS+ (Stratagene, La Jolla, CA). This new plasmid, pBKS+-rLHß1.8, was then digested with KpnI and XbaI to liberate the 1.8-kb promoter fragment. This fragment was inserted into KpnI- and NheI-digested pGL3-Basic. The resultant plasmid, pGL3-rLHß-1.8, was then sequenced to confirm identity. The proximal sequence of this promoter matches that of the 797 bases reported in GenBank accession no. AF020505. To construct the internal control plasmid pGL3-CMV-ß-Gal, the luciferase coding sequences from NcoI to BamHI of pGL3-CMV were then substituted by replacement with the NcoI to BamHI region of the plasmid pSDK-Lac Z pA encoding E. coli ß-galactosidase.
For transfection, cells were plated at a density of 50,000 cells/cm2 and incubated 24 h before transfection. Cells were then changed into serum-free medium and transfected using Fugene 6 (Roche Applied Science, Indianapolis, IN) according to the manufacturer’s instructions using 0.1 µg total DNA per cm2 plate area and incubated 16–18 h before treatment with GnRH. For inhibitor experiments, cells were pretreated with PD 98059, LY 294002, or rapamycin (Calbiochem, San Diego, CA) at indicated concentrations for 30 min before 10 nM GnRH treatment (4 h). Cells were harvested by lysis in 100 mM PBS containing 0.1% Triton X-100, vortexed, and clarified by centrifugation. Cell lysates were assayed directly for luciferase and ß-galactosidase activity using the glow-type luciferase assay kit (Promega Corp.) and the Galacto-Light Plus kit (Tropix, Bedford, MA), respectively. Luminescence was measured in a 96-well plate using 20 µl of lysate in a LB96V luminometer (EG&G Berthold, Gaithersburg, MD). Plate transfection assays were performed in quadruplicate.
Western Blotting
For analysis of protein derived from static cultures, 1 x 107 cells were plated in 10-cm dishes and incubated for 24 h before treatment. LßT2 cells were placed in serum-free medium 24 h before harvest to eliminate hormone and growth factor influences on translation (44). To remove amino acid influences on initiation factor phosphorylation, cells were incubated in Earle’s balanced salt solution (Sigma Chemical Co., St. Louis, MO) to amino acid starve (with the exception of 4E-BP1 dose response and AKT experiments) for 1 h (44) before stimulation with GnRH at the indicated doses and times. For inhibitor experiments, cells were treated with 10 µM PD 98059, 10 nM rapamycin, or 1.0 µM LY 294002 (Calbiochem, San Diego, CA) for 30 min and treated with 10 nM GnRH for 15 min.
For 4E-BP1, after cell treatment, the medium was removed and cells were washed once with ice-cold buffer A (50 mM Tris, pH 7.5; 150 mM KCl; 1 mM dithiothreitol; and 1 mM EDTA with phosphatase inhibitors, 50 mM ß-glycerophosphate, 1 mM EGTA, 50 mM sodium fluoride, 10 mM sodium pyrophosphate, 0.1 mM sodium orthovanadate, and 50 nM okadaic acid). Cells were then harvested into 1 ml of the same buffer and pelleted briefly. The supernatant was removed, and 150 µl fresh buffer A were added to the cell pellet. For eIF4E, Mnk, eIF4G, and AKT, cells were washed with ice-cold PBS after cell treatment, harvested with Laemmli sample buffer, and sonicated for 15–30 sec. Cell lysates were assayed for protein content using the BIO-RAD DC Protein Assay (Bio-Rad Laboratories, Hercules, CA). All samples were boiled for 5 min before 50–100 µg of protein were separated by SDS-PAGE on a 15% (4E-BP1), 10% (eIF4E, AKT, Mnk), or 7.5% (eIF4G) gel and transferred onto polyvinylidene difluoride membrane by semidry transfer. The membranes were blocked in 2x casein (Vector Laboratories) and incubated with primary antibodies. 4E-BP1, phospho-eIF4E, and AKT antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) whereas phospho-eIF4G, -AKT, -Mnk, and -eIF4E antibodies were from Cell Signaling Technology (Beverly, MA). Rabbit anti-4EBP1-R113 was used in a 1:400 dilution; rabbit phospho-eIF4G Ser1108 and phospho-AKT Ser 473 antibody were used at a 1:1000 dilution; and AKT antibody was used at 1:2000 dilution at room temperature for 60 min. Rabbit eIF4E, rabbit phospho-Mnk 1 Thr197/202, and phospho-eIF4E Ser209 antibodies were used in a 1:1000 dilution overnight at 4 C. Blots were developed by enhanced chemiluminescence using a 1:5000 dilution of biotinylated antirabbit secondary antibody and horseradish peroxidase-conjugated avidin-biotin complex (Vector Laboratories), and visualized using GeneSnap Bio Imaging System (Syngene, Frederick, MD). Chemiluminescent analysis was performed using GeneTools software (Syngene).
Statistical Analysis
All statistical analysis was performed using JMP v. 4.0 or v. 5.0 software (SAS Institute, Cary, NC). Data are expressed as means ± SEM of at least three samples per group. Results were analyzed for significant differences using ANOVA. Post hoc group comparison was made using Student’s t test, Dunnett’s comparison to control test, or Tukey’s honestly significant difference where appropriate. Analysis was conducted using untransformed data or data optimally transformed by the method of Box and Cox as indicated (45). A P 0.05 was the requirement for declaring significance.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: CMV, Cytomegalovirus; 4E-BP, 4E-binding protein; eIF4E, eukaryotic initiation factor 4E; eIF4G, eukaryotic initiation factor 4G; EMCV, encephalomyocarditis virus; GPCR, G protein-coupled receptor; MEK, MAPK kinase; Mnk1, MAPK-interacting kinase; mTOR, mammalian target of rapamycin; PI3 kinase, phosphatidylinositol 3-kinase.
Received for publication December 10, 2003. Accepted for publication January 23, 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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T. C. Vary and C. J. Lynch Meal feeding enhances formation of eIF4F in skeletal muscle: role of increased eIF4E availability and eIF4G phosphorylation Am J Physiol Endocrinol Metab, April 1, 2006; 290(4): E631 - E642. [Abstract] [Full Text] [PDF]
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F. Liu, M. S. Ruiz, D. A. Austin, and N. J. G. Webster Constitutively Active Gq Impairs Gonadotropin-Releasing Hormone-Induced Intracellular Signaling and Luteinizing Hormone Secretion in L{beta}T2 Cells Mol. Endocrinol., August 1, 2005; 19(8): 2074 - 2085. [Abstract] [Full Text] [PDF]
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