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• W2086707089 abstract "Ser-53 has previously been considered the major phosphorylation site in eukaryotic initiation factor (eIF)-4E, and this appeared to be supported by studies using a S53A mutant. Recently, however, several lines of evidence have indicated that Ser-53 might not be the true phosphorylation site. This prompted us to re-examine the phosphorylation site in eIF-4E using factor purified from 32P-labeled, serum-treated Chinese hamster ovary cells. Isoelectric focusing and phosphoamino acid analysis indicated the existence of a single phosphorylated serine. Edman degradation of the major radiolabeled tryptic product from 32P-labeled eIF-4E showed that the phosphorylated site was positioned three residues from the N terminus of this peptide. There are three serines in the sequence of eIF-4E that are three residues away from a tryptic cleavage site (i.e. lysine or arginine). 32P-Labeled eIF-4E was digested with trypsin, Lys-C, or trypsin followed by Glu-C and subjected to two-dimensional mapping; the data obtained eliminated two of these potential sites, leaving Ser-209. Comigration of the synthetic peptide SGS(P)209TTK with the radiolabeled tryptic product on (i) reverse-phase chromatography and (ii) two-dimensional mapping at different pH values confirmed that Ser-209 is the major phosphorylation site in eIF-4E in serum-stimulated Chinese hamster ovary cells. Ser-53 has previously been considered the major phosphorylation site in eukaryotic initiation factor (eIF)-4E, and this appeared to be supported by studies using a S53A mutant. Recently, however, several lines of evidence have indicated that Ser-53 might not be the true phosphorylation site. This prompted us to re-examine the phosphorylation site in eIF-4E using factor purified from 32P-labeled, serum-treated Chinese hamster ovary cells. Isoelectric focusing and phosphoamino acid analysis indicated the existence of a single phosphorylated serine. Edman degradation of the major radiolabeled tryptic product from 32P-labeled eIF-4E showed that the phosphorylated site was positioned three residues from the N terminus of this peptide. There are three serines in the sequence of eIF-4E that are three residues away from a tryptic cleavage site (i.e. lysine or arginine). 32P-Labeled eIF-4E was digested with trypsin, Lys-C, or trypsin followed by Glu-C and subjected to two-dimensional mapping; the data obtained eliminated two of these potential sites, leaving Ser-209. Comigration of the synthetic peptide SGS(P)209TTK with the radiolabeled tryptic product on (i) reverse-phase chromatography and (ii) two-dimensional mapping at different pH values confirmed that Ser-209 is the major phosphorylation site in eIF-4E in serum-stimulated Chinese hamster ovary cells. INTRODUCTIONPhosphorylation of eukaryotic initiation factor (eIF) 1The abbreviations used are: eIFeukaryotic initiation factorCHOChinese hamster ovarym7GTP7-methylguanosine triphosphateHPLChigh pressure liquid chromatography. -4E (also known as eIF-4α), which binds to the 5′-cap structure on eukaryotic mRNAs, has been shown to correlate positively with changes in the rate of translation under a wide range of conditions (for reviews, see (1Proud C.G. Curr. Top. Cell. Regul. 1992; 32: 243-369Crossref PubMed Scopus (164) Google Scholar, 2Rhoads R.E. J. Biol. Chem. 1993; 268: 3017-3020Abstract Full Text PDF PubMed Google Scholar, 3Redpath N.T. Proud C.G. Biochim. Biophys. Acta. 1994; 1220: 147-162Crossref PubMed Scopus (87) Google Scholar)). eIF-4E is the least abundant of the components of the eIF-4F complex(4Duncan R. Milburn S.C. Hershey J.W. J. Biol. Chem. 1987; 262: 380-388Abstract Full Text PDF PubMed Google Scholar), which also contains the RNA helicase eIF-4A and eIF-4γ, and translation of messages with a high degree of secondary structure is enhanced by overexpression of eIF-4E(5Koromilas A.E. Lazaris-Karatzas A. Sonenberg N. EMBO J. 1992; 11: 4153-4158Crossref PubMed Scopus (336) Google Scholar). Phosphorylation of eIF-4E could therefore provide an important mechanism for regulating cellular translation and particularly that of certain mRNAs. Although it remains unclear how phosphorylation of eIF-4E affects its activity, it has been reported to lead to increased association of eIF-4E with high molecular mass complexes, perhaps including the other subunits of eIF-4F(6Morley S.J. Rau M. Kay J.E. Pain V.M. Eur. J. Biochem. 1993; 218: 39-48Crossref PubMed Scopus (60) Google Scholar, 7Bu X. Haas D.W. Hagedorn C.H. J. Biol. Chem. 1993; 268: 4975-4978Abstract Full Text PDF PubMed Google Scholar). Recent work also suggests that phosphorylation of eIF-4E may increase its affinity for the cap(8Minich W.B. Ballasta M.L. Goss D.J. Rhoads R.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7668-7672Crossref PubMed Scopus (260) Google Scholar), although phosphorylation is certainly not a prerequisite for it to bind.Several years ago, Rychlik et al.(9Rychlik W. Russ M.A. Rhoads R.E. J. Biol. Chem. 1987; 262: 10434-10437Abstract Full Text PDF PubMed Google Scholar) presented evidence that the major site of phosphorylation of eIF-4E in rabbit reticulocytes was Ser-53. This apparent identification of Ser-53 as the site of phosphorylation was backed up by several studies employing a site-directed mutant of eIF-4E in which Ser-53 was altered to alanine. Such a mutant showed a number of differences from the wild-type protein when overexpressed in intact cells or when studied in vitro in that it did not induce cell transformation or promote abnormal morphology(10De Benedetti A. Rhoads R.E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8212-8216Crossref PubMed Scopus (254) Google Scholar, 11Lazaris-Karatzas A. Montine K.S. Sonenberg N. Nature. 1990; 345: 544-547Crossref PubMed Scopus (800) Google Scholar), and it was not incorporated into 48 S initiation complexes(12Joshi-Barve S. Rychlik W. Rhoads R.E. J. Biol. Chem. 1990; 265: 2979-2983Abstract Full Text PDF PubMed Google Scholar).Evidence has, however, been presented that other phosphorylation sites exist in eIF-4E based on the fact that the S53A mutant still underwent phosphorylation(11Lazaris-Karatzas A. Montine K.S. Sonenberg N. Nature. 1990; 345: 544-547Crossref PubMed Scopus (800) Google Scholar, 12Joshi-Barve S. Rychlik W. Rhoads R.E. J. Biol. Chem. 1990; 265: 2979-2983Abstract Full Text PDF PubMed Google Scholar, 13Kaufman R.J. Murtha-Riel P. Pittman D.D. Davies M.V. J. Biol. Chem. 1993; 268: 11902-11909Abstract Full Text PDF PubMed Google Scholar, 14Bu X. Hagedorn C.H. FEBS Lett. 1991; 283: 219-222Crossref PubMed Scopus (26) Google Scholar). In addition, Kaufman et al.(13Kaufman R.J. Murtha-Riel P. Pittman D.D. Davies M.V. J. Biol. Chem. 1993; 268: 11902-11909Abstract Full Text PDF PubMed Google Scholar) found no discernible differences in the ability of wild-type or S53A mutant eIF-4E to support translation of selected mRNAs or to become incorporated into the eIF-4F complex. Furthermore, we recently observed that the major insulin-stimulated eIF-4E kinase in Chinese hamster ovary (CHO) cells was able to phosphorylate recombinant eIF-4E (S53A) to a similar extent compared with the wild-type recombinant protein. 2A. Flynn and C. G. Proud, unpublished data. Taken together, these findings prompted us to reinvestigate the site of phosphorylation in eIF-4E. 3Early in this study, we learned that Prof. R. E. Rhoads (Shreveport, LA) had also obtained data indicating that Ser-53 was not the major phosphorylation site in eIF-4E. Our data show that the major site of phosphorylation in eIF-4E in serum-stimulated CHO cells is Ser-209, close to the C terminus of the protein.EXPERIMENTAL PROCEDURESMaterialsm7GTP-Sepharose was from Pharmacia Biotech Inc. Carrier-free orthophosphate was purchased from Amersham Corp. Chinese hamster ovary (CHO.K1) cells were kindly provided by Dr. L. Ellis (Houston, TX). Materials for tissue culture were obtained from Life Technologies, Inc. Modified trypsin and sequencing-grade Lys-C and Glu-C were from Promega. Microcystin-LR was obtained from Calbiochem. Unless otherwise stated, all other reagents were from Sigma.Cell Culture, Treatment, and LabelingCHO.K1 cells were grown and maintained in culture as described by Dickens et al.(15Dickens M. Chin J.E. Roth R.A. Ellis L. Denton R.M. Tavaré J.M. Biochem. J. 1992; 287: 201-209Crossref PubMed Scopus (36) Google Scholar). Cells were seeded at an initial density of 5 × 105/60-mm dish and grown to near confluence (3-4 days) in Ham's F-12 medium containing 10% (v/v) serum before being “stepped down” to 0.1% serum for 16 h. For 32P labeling, the cells were washed (2 × 5 ml) with phosphate-free Dulbecco's modified Eagle's medium and then incubated for 3 h at 37°C in 2 ml of the same medium containing 1-1.5 mCi/ml 32Pi. Fetal calf serum was then added to a final concentration of 10% (v/v), and the cells were incubated for a further 10 min. Cell extracts were prepared as described previously (15Dickens M. Chin J.E. Roth R.A. Ellis L. Denton R.M. Tavaré J.M. Biochem. J. 1992; 287: 201-209Crossref PubMed Scopus (36) Google Scholar) and clarified by centrifugation (10 min at 10,000 × g) at 4°C.Isolation and Analysis of eIF-4EeIF-4E was purified by tumbling clarified cell extracts (700 μl) with m7GTP-Sepharose CL-4B (~30-μl packed volume) for 30 min at 4°C. The beads were pelleted by a short centrifugation and washed once with 1 ml of extraction buffer (see (15Dickens M. Chin J.E. Roth R.A. Ellis L. Denton R.M. Tavaré J.M. Biochem. J. 1992; 287: 201-209Crossref PubMed Scopus (36) Google Scholar)) containing 1% Triton X-100 and then twice with 1 ml of the same buffer containing only 0.1% Triton X-100. The pelleted beads were boiled with concentrated SDS sample buffer, and the entire sample was subjected to SDS-polyacrylamide gel electrophoresis on a 15% (w/v) acrylamide gel. The gel was transferred to Immobilon-P polyvinylidene difluoride (Millipore Corp.) using a Semi-dry transfer cell (Bio-Rad), and the membrane was washed extensively with deionized water before drying and subjecting to autoradiography overnight. 32P-Labeled eIF-4E was excised from the membrane, and the amount of radioactivity in each membrane piece was estimated by Cerenkov counting.Isoelectric Focusing and ImmunoblottingFor analysis of the phosphorylation state of eIF-4E by isoelectric focusing, cells were grown as described above, but with the omission of 32Pi. Extracts were prepared from either serum-stimulated or control (no addition) cells. eIF-4E was purified as described above, except that it was eluted from the m7GTP-Sepharose with 20 μl of m7GTP (100 μM, 30 min, 4°C). Following centrifugation, the supernatants were mixed with concentrated (7×) isoelectric focusing sample buffer (16Redpath N.T. Anal. Biochem. 1992; 202: 340-343Crossref PubMed Scopus (23) Google Scholar) and urea to a final concentration of 9 M. Isoelectric focusing was performed using a modification of the reversed polarity method described by Maurides et al.(17Maurides P.A. Akkaraju G.R. Jagus R. Anal. Biochem. 1989; 183: 144-151Crossref PubMed Scopus (42) Google Scholar). The gel was prepared using ampholyte carriers in the pH range 3.5-10 (Pharmacia Biotech Inc.). Western blotting was carried out as described (18Oldfield S. Jones B.L. Tanton D. Proud C.G. Eur. J. Biochem. 1994; 221: 399-410Crossref PubMed Scopus (46) Google Scholar) using an anti-eIF-4E antiserum prepared as described previously(11Lazaris-Karatzas A. Montine K.S. Sonenberg N. Nature. 1990; 345: 544-547Crossref PubMed Scopus (800) Google Scholar). Blots were developed using enhanced chemiluminescence (Amersham Corp.) and scanned by laser densitometry on a Joyce-Loebl Chromoscan 3 apparatus.Protein Chemical ProceduresTryptic digestion of 32P-labeled eIF-4E on polyvinylidene difluoride chips was performed by incubation with 20 μg of modified trypsin in 100 mMN-ethylmorpholine (pH 8) at 30°C for 16 h. For digestion with Lys-C, the membrane was incubated with 1 μg of the protease in 50 mM NaHCO3 (pH 9) for 1 h at 30°C; then another 1 μg of endoproteinase Lys-C was added; and incubation was continued for 15 h. The recovery of 32P counts from each membrane chip was monitored by Cerenkov counting. Radioactive tryptic fragments that did not elute immediately from the treated membrane were removed using a mixture of 0.1% trifluoroacetic acid/acetonitrile (20% for the first extraction and then 60%). Typically, ~60% of the radioactivity in the membrane chip was recovered in this way. The elution washes were pooled and dried by vacuum centrifugation and then washed (5 × 50 μl) and resuspended in water.Glu-C digestion of the major radiolabeled tryptic product of eIF-4E was performed by scraping radioactive material from a thin-layer cellulose plate following two-dimensional mapping (see below) and washing extensively with water. The pooled supernatants were dried under vacuum, resuspended in water, and incubated with 1 μg of endoproteinase Glu-C in 50 mM ammonium bicarbonate (pH 7.8) for 16 h before processing as described above. Both the Glu-C and Lys-C preparations used digested completely ~1 μg of rabbit reticulocyte eIF-2 (data not shown) under the same conditions.HPLCSeparation of 32P-labeled tryptic products of eIF-4E by HPLC was performed using a C18 column (Brownlee, Spheri-5, 2.1-mm diameter, 5-μm particles). Samples were prepared as described above and centrifuged through a 10-kDa cutoff Ultrafree-MC unit (Millipore Corp.). The column was pre-equilibrated in 0.1% trifluoroacetic acid, 2% acetonitrile and developed using a gradient of 2-30% acetonitrile in 0.1% trifluoroacetic acid over 30 min, 30-60% in 5 min, and to 95% in 1 min. 100-μl fractions were collected, and the radioactive material was located by Cerenkov counting. The same method was used to compare the behavior of the unlabeled synthetic peptide SGS(P)TTK on the C18 column, except that the protein peaks were detected by monitoring absorbance at 215 nm.Phosphoamino Acid AnalysisPhosphoamino acid analysis was performed on intact 32P-labeled eIF-4E that had been resolved by SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membrane. The sample was hydrolyzed in 6 N HCl at 105°C for 75 min. Electrophoresis was performed in one dimension (pH 3.5) for 1.5 h at 400 V, and the dried thin-layer cellulose plate was subjected to autoradiography. 1 μg each of nonradioactive phosphoserine and phosphothreonine were loaded as standards and carriers and detected by ninhydrin staining.Radiosequencing by Manual Edman DegradationThe peak fraction of counts following separation of tryptic products from 32P-labeled eIF-4E by reverse-phase chromatography (see above) was dried under vacuum and washed and resuspended in water. A sample was removed for analysis by manual Edman degradation, performed exactly as described earlier(19Boyle W.J. van der Geer P. Hunter T. Methods Enzymol. 1991; 201: 110-149Crossref PubMed Scopus (1273) Google Scholar). An aliquot of starting material and samples from each round of degradation were loaded onto a thin-layer cellulose plate and electrophoresed in one dimension (pH 3.5) for 25 min at 1 kV. 32Pi was loaded as a standard, and the dried plate was subjected to autoradiography.Two-dimensional Peptide MappingSamples were analyzed by two-dimensional peptide mapping, as described previously(19Boyle W.J. van der Geer P. Hunter T. Methods Enzymol. 1991; 201: 110-149Crossref PubMed Scopus (1273) Google Scholar), at pH 3.5 and 1.9. The thin-layer cellulose plates were dried and either subjected to autoradiography (for 32P-labeled samples) or sprayed with ninhydrin (to detect the unlabeled synthetic phosphopeptide). A mixture of 2,4-dinitrophenyllysine/cyanol (~1 μg of each) was loaded as a marker.RESULTS AND DISCUSSIONAs we have previously reported, 4A. Flynn and C. G. Proud, submitted for publication. isoelectric focusing analysis reveals the existence of only two distinct species of eIF-4E in CHO cells as detected by immunoblotting, and treatment of isolated eIF-4E with alkaline phosphatase causes conversion of all the factor to the more basic (i.e. unphosphorylated) form. Fig. 1A shows an immunoblot of eIF-4E from control and serum-treated CHO.K1 cells: in the former case, only 20% of the eIF-4E is phosphorylated, while in serum-treated cells, almost all the factor (~80%) is in the more acidic (phosphorylated) form. These findings are consistent with the data from a range of other cell types indicating that eIF-4E contains one major phosphorylation site(20Sonenberg N. Biochimie (Paris). 1994; 76: 839-846Crossref PubMed Scopus (113) Google Scholar).To determine which type of amino acid was phosphorylated in eIF-4E in CHO.K1 cells, serum-starved cells were incubated with radioactive inorganic phosphate and briefly treated with serum, and eIF-4E was then isolated and subjected to phosphoamino acid analysis. Only phosphoserine was observed (Fig. 1B), which is consistent with several other reports using a range of different cell types (see (20Sonenberg N. Biochimie (Paris). 1994; 76: 839-846Crossref PubMed Scopus (113) Google Scholar), for a review), although phosphothreonine has been reported in eIF-4E under some conditions (see, for example, (7Bu X. Haas D.W. Hagedorn C.H. J. Biol. Chem. 1993; 268: 4975-4978Abstract Full Text PDF PubMed Google Scholar)).It thus appeared likely that eIF-4E was phosphorylated at a single serine residue in serum-treated CHO.K1 cells. To investigate further the location of the phosphoserine residue in eIF-4E, radiolabeled factor was isolated from serum-treated cells and subjected to tryptic digestion followed by separation of the resulting peptides by reverse-phase chromatography (Fig. 2). Two separate experiments were performed, and in both cases, one major peak of radioactive material was observed. This peak eluted at ~8% acetonitrile, indicating that the material was very hydrophilic. Some additional radioactive material eluted later, at 20-30% acetonitrile, and may have represented products of incomplete trypsinolysis of radiolabeled eIF-4E. In both experiments, the first peak contained ~75% of the total radioactive material, and this was contained within one fraction, whereas the later material emerged as a broad smear.Figure 2:HPLC analysis of the tryptic phosphopeptides from 32P-labeled eIF-4E. Reverse-phase chromatography on a C18 column was performed for the products of trypsinolysis of 32P-labeled eIF-4E from serum-treated CHO.K1 cells (A) or for a nonradiolabeled synthetic phosphopeptide, SGS(P)TTK (B), as described under “Experimental Procedures.” The flow rate was 0.1 ml/min; 100-μl fractions were collected.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Two-dimensional phosphopeptide mapping was performed to characterize radiolabeled material contained in the main peak from reverse-phase chromatography. Fig. 3A (panel i) shows that one positively charged phosphopeptide was obtained when electrophoresis was performed at pH 1.9; this phosphopeptide migrated slightly faster than cyanol (which has a net charge of +1 at this pH). The same result was obtained at pH 3.5, except that the peptide and cyanol did not migrate as quickly toward the cathode (data not shown). Consistent with the findings of other groups(14Bu X. Hagedorn C.H. FEBS Lett. 1991; 283: 219-222Crossref PubMed Scopus (26) Google Scholar, 19Boyle W.J. van der Geer P. Hunter T. Methods Enzymol. 1991; 201: 110-149Crossref PubMed Scopus (1273) Google Scholar, 21Morley S.J. Traugh J.A. J. Biol. Chem. 1989; 264: 2401-2404Abstract Full Text PDF PubMed Google Scholar), the radiolabeled tryptic product from eIF-4E did not move from the origin on chromatography, again testifying to its extremely hydrophilic nature.Figure 3:Analysis of the location of the phosphate label in eIF-4E. A, two-dimensional peptide mapping and autoradiography were performed for the products of digestion of 32P-labeled eIF-4E with trypsin (panel i), Lys-C (panel ii), or trypsin followed by Glu-C (panel iii). Panel iv shows a schematic representation of the map obtained for the nonradiolabeled synthetic phosphopeptide SGS(P)TTK and serves as a key for the position of 2,4-dinitrophenyllysine (DNP-lysine) and cyanol in panels i-iii. o, origin. Electrophoresis was performed at pH 1.9. Only the parts of the map containing marker or peptide are shown; no other radiolabeled material was observed. B, manual Edman degradation was performed using a sample of the HPLC-purified tryptic product of 32P-labeled eIF-4E. Samples of starting material (lane 0) or following each round of degradation (lanes 1-3) were analyzed by one-dimensional electrophoresis at pH 3.5, followed by autoradiography. Pi, [32P]orthophosphate marker.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To determine the position of the phosphoserine residue in the tryptic product, manual Edman analysis was performed, with samples being retained after every cycle for further analysis by one-dimensional mapping. The initial peptide and the products obtained after one or two cycles had a net positive charge (Fig. 3B). However, after the third cycle, ~75% of the radioactivity no longer migrated with positively charged material, but instead moved in the opposite direction and migrated with inorganic phosphate. Furthermore, the fourth round of degradation yielded [32P]orthophosphate only, with no counts remaining in the position of the original phosphopeptide (data not shown). These data indicate that the third residue in the peptide bears the radiolabel, and given the conclusions drawn from the phosphoamino acid analysis (Fig. 1B), the tryptic peptide therefore contains a phosphoserine at position 3.There are no sequence data available for eIF-4E from the Chinese hamster. However, there is a very high degree of identity between rabbit, mouse, and human eIF-4E(22Rychlik W. Domier L. Gardner P.R. Hellmann G.M. Rhoads R.E. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 945-949Crossref PubMed Scopus (85) Google Scholar, 23Rychlik W. Rhoads R.E. Nucleic Acids Res. 1992; 20: 6415Crossref PubMed Scopus (14) Google Scholar, 24McCubbin W.D. Edery I. Altmann M. Sonenberg N. Kay C.M. J. Biol. Chem. 1988; 263: 17663-17671Abstract Full Text PDF PubMed Google Scholar); the only significant differences are in the extreme N terminus of the factor (residues 12, 16, and 17), while the remainder of the eIF-4E sequence is 99.5% identical. We therefore examined the sequences of these proteins to identify possible candidates for the tryptic peptide obtained in our studies. In each case, there are three serines that lie three residues C-terminal to a tryptic cleavage site (Lys or Arg). Attempts to purify sufficient amounts of the tryptic phosphopeptide from eIF-4E in CHO.K1 cells were unsuccessful. Therefore, to distinguish between these three possible phosphorylation sites, further proteolytic digests were performed. In the case of Ser-64 in the tryptic peptide LIS64K, digestion of the intact 32P-labeled factor with Lys-C would yield a much larger and more hydrophobic phosphopeptide (TWQANLRLISK) than that obtained by tryptic digestion, which would migrate quite differently on two-dimensional peptide mapping. However, as shown in Fig. 3A (panel ii), the migration of the phosphopeptide produced with Lys-C was identical to that of the tryptic product at pH 1.9 (and also at pH 3.5; data not shown), ruling out Ser-64 as the phosphorylation site.In the case of Ser-24 in the tryptic peptide TES24NQEVANPEHYIK, treatment of the tryptic phosphopeptide with Glu-C would be expected to generate a smaller species with altered mobility on electrophoresis and chromatography. However, Fig. 3A (panel iii) shows that after Glu-C treatment, the migration of the phosphopeptide was unchanged on two-dimensional mapping at pH 1.9 (and also at pH 3.5; data not shown), indicating that Ser-24 is not the radiolabeled residue. By a process of elimination, then the phosphorylation site in eIF-4E in CHO cells appears to be Ser-209 in the tryptic peptide SGS209TTK, consistent with the recent findings of the Rhoads' laboratory.3As a final confirmation of the identity of the phosphorylation site in eIF-4E, a synthetic phosphopeptide (SGS(P)TTK) that corresponds to residues 207-212 of eIF-4E was prepared. This peptide showed identical behavior to that of the tryptic product from 32P-labeled eIF-4E on (i) reverse-phase chromatography (Fig. 2), (ii) electrophoresis at pH 1.9 (Fig. 3A (panel iv)) or (iii) at pH 3.5 (data not shown), and (iv) thin-layer chromatography (Fig. 3A (panel iv)). This strongly supports the identification of the labeled phosphopeptide and confirms that the phosphorylated residue is indeed Ser-209.In conclusion, all these data indicate that the major (and probably the only) phosphorylation site in CHO cells is Ser-209. Reappraisal of the role of phosphorylation of eIF-4E is therefore required using mutants based on Ser-209. This work also highlights the need for caution when using Ser to Ala mutants of proteins to investigate the role of phosphorylation, as this approach has given phenotypes not related to phosphorylation in two translation initiation factors, i.e. eIF-4E (discussed above) and eIF-2α(25Kaufman R.J. Davies M.V. Pathak V.K. Hershey J.W. Mol. Cell. Biol. 1989; 9: 946-958Crossref PubMed Scopus (332) Google Scholar, 26Choi S.Y. Scherer B.J. Schnier J. Davies M.V. Kaufman R.J. Hershey J.W. J. Biol. Chem. 1992; 267: 286-293Abstract Full Text PDF PubMed Google Scholar) INTRODUCTIONPhosphorylation of eukaryotic initiation factor (eIF) 1The abbreviations used are: eIFeukaryotic initiation factorCHOChinese hamster ovarym7GTP7-methylguanosine triphosphateHPLChigh pressure liquid chromatography. -4E (also known as eIF-4α), which binds to the 5′-cap structure on eukaryotic mRNAs, has been shown to correlate positively with changes in the rate of translation under a wide range of conditions (for reviews, see (1Proud C.G. Curr. Top. Cell. Regul. 1992; 32: 243-369Crossref PubMed Scopus (164) Google Scholar, 2Rhoads R.E. J. Biol. Chem. 1993; 268: 3017-3020Abstract Full Text PDF PubMed Google Scholar, 3Redpath N.T. Proud C.G. Biochim. Biophys. Acta. 1994; 1220: 147-162Crossref PubMed Scopus (87) Google Scholar)). eIF-4E is the least abundant of the components of the eIF-4F complex(4Duncan R. Milburn S.C. Hershey J.W. J. Biol. Chem. 1987; 262: 380-388Abstract Full Text PDF PubMed Google Scholar), which also contains the RNA helicase eIF-4A and eIF-4γ, and translation of messages with a high degree of secondary structure is enhanced by overexpression of eIF-4E(5Koromilas A.E. Lazaris-Karatzas A. Sonenberg N. EMBO J. 1992; 11: 4153-4158Crossref PubMed Scopus (336) Google Scholar). Phosphorylation of eIF-4E could therefore provide an important mechanism for regulating cellular translation and particularly that of certain mRNAs. Although it remains unclear how phosphorylation of eIF-4E affects its activity, it has been reported to lead to increased association of eIF-4E with high molecular mass complexes, perhaps including the other subunits of eIF-4F(6Morley S.J. Rau M. Kay J.E. Pain V.M. Eur. J. Biochem. 1993; 218: 39-48Crossref PubMed Scopus (60) Google Scholar, 7Bu X. Haas D.W. Hagedorn C.H. J. Biol. Chem. 1993; 268: 4975-4978Abstract Full Text PDF PubMed Google Scholar). Recent work also suggests that phosphorylation of eIF-4E may increase its affinity for the cap(8Minich W.B. Ballasta M.L. Goss D.J. Rhoads R.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7668-7672Crossref PubMed Scopus (260) Google Scholar), although phosphorylation is certainly not a prerequisite for it to bind.Several years ago, Rychlik et al.(9Rychlik W. Russ M.A. Rhoads R.E. J. Biol. Chem. 1987; 262: 10434-10437Abstract Full Text PDF PubMed Google Scholar) presented evidence that the major site of phosphorylation of eIF-4E in rabbit reticulocytes was Ser-53. This apparent identification of Ser-53 as the site of phosphorylation was backed up by several studies employing a site-directed mutant of eIF-4E in which Ser-53 was altered to alanine. Such a mutant showed a number of differences from the wild-type protein when overexpressed in intact cells or when studied in vitro in that it did not induce cell transformation or promote abnormal morphology(10De Benedetti A. Rhoads R.E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8212-8216Crossref PubMed Scopus (254) Google Scholar, 11Lazaris-Karatzas A. Montine K.S. Sonenberg N. Nature. 1990; 345: 544-547Crossref PubMed Scopus (800) Google Scholar), and it was not incorporated into 48 S initiation complexes(12Joshi-Barve S. Rychlik W. Rhoads R.E. J. Biol. Chem. 1990; 265: 2979-2983Abstract Full Text PDF PubMed Google Scholar).Evidence has, however, been presented that other phosphorylation sites exist in eIF-4E based on the fact that the S53A mutant still underwent phosphorylation(11Lazaris-Karatzas A. Montine K.S. Sonenberg N. Nature. 1990; 345: 544-547Crossref PubMed Scopus (800) Google Scholar, 12Joshi-Barve S. Rychlik W. Rhoads R.E. J. Biol. Chem. 1990; 265: 2979-2983Abstract Full Text PDF PubMed Google Scholar, 13Kaufman R.J. Murtha-Riel P. Pittman D.D. Davies M.V. J. Biol. Chem. 1993; 268: 11902-11909Abstract Full Text PDF PubMed Google Scholar, 14Bu X. Hagedorn C.H. FEBS Lett. 1991; 283: 219-222Crossref PubMed Scopus (26) Google Scholar). In addition, Kaufman et al.(13Kaufman R.J. Murtha-Riel P. Pittman D.D. Davies M.V. J. Biol. Chem. 1993; 268: 11902-11909Abstract Full Text PDF PubMed Google Scholar) found no discernible differences in the ability of wild-type or S53A mutant eIF-4E to support translation of selected mRNAs or to become incorporated into the eIF-4F complex. Furthermore, we recently observed that the major insulin-stimulated eIF-4E kinase in Chinese hamster ovary (CHO) cells was able to phosphorylate recombinant eIF-4E (S53A) to a similar extent compared with the wild-type recombinant protein. 2A. Flynn and C. G. Proud, unpublished data. Taken together, these findings prompted us to reinvestigate the site of phosphorylation in eIF-4E. 3Early in this study, we learned that Prof. R. E. Rhoads (Shreveport, LA) had also obtained data indicating that Ser-53 was not the major phosphorylation site in eIF-4E. Our data show that the major site of phosphorylation in eIF-4E in serum-stimulated CHO cells is Ser-209, close to the C terminus of the protein." @default.
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• W2086707089 creator A5020542657 @default.
• W2086707089 creator A5030513468 @default.
• W2086707089 date "1995-09-01" @default.
• W2086707089 modified "2023-09-30" @default.
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