©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Phosphorylation of Eukaryotic Protein Synthesis Initiation Factor 4E at Ser-209 (*)

Bhavesh Joshi (1), Ai-Li Cai (1), Brett D. Keiper (1), Waldemar B. Minich (1)(§), Raul Mendez (1), Carol M. Beach (3), Janusz Stepinski (2), Ryszard Stolarski (2), Edward Darzynkiewicz (2), Robert E. Rhoads (1)(¶)

From the (1)Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, Shreveport, Louisiana 71130-3932, the Structure Analysis Facility, University of Kentucky Medical Center, Lexington, Kentucky 40536, and the (2)Departments of Biophysics and Chemistry, University of Warsaw, 02-089 Warsaw, Poland

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Initiation factor 4E (eIF-4E) binds to the mGTP-containing cap of eukaryotic mRNA and facilitates the entry of mRNA into the initiation cycle of protein synthesis. eIF-4E is a phosphoprotein, and the phosphorylated form binds to mRNA caps 3-4-fold more tightly than the nonphosphorylated form. A previous study indicated that the major phosphorylation site was Ser-53 (Rychlik, W., Russ, M. A., and Rhoads, R. E.(1987) J. Biol. Chem. 262, 10434-10437). In the present study, we synthesized the phosphopeptide expected to result from tryptic digestion of eIF-4E, O-phosphoseryllysine. Surprisingly, the tryptic and synthetic phosphopeptides did not co-migrate electrophoretically. Accordingly, we redetermined the phosphorylation site by isolating a chymotryptic phosphopeptide on reverse phase high performance liquid chromatography. The peptide was sequenced by Edman degradation and corresponded to QSHADTATKSGSTTKNRF. The site of phosphorylation was determined to be Ser-209 by four methods: the increase in the ratio of dehydroalanine to serine derivatives during Edman degradation, the release of P, the further digestion of the chymotryptic phosphopeptide with trypsin, Glu-C, and Asp-N, and site-directed mutagenesis of eIF-4E cDNA. The S209A variant was not phosphorylated in a rabbit reticulocyte lysate system, whereas the wild-type, S53A, and S207A variants were. This site falls within the consensus sequence for phosphorylation by protein kinase C.


INTRODUCTION

The initiation of protein synthesis in eukaryotic cells is divided into discrete stages, each catalyzed by a different initia-tion factor (eIF-)()or group of initiation factor polypeptides(1, 2, 3) . Binding of mRNA to the 43 S initiation complex to form the 48 S complex is catalyzed by the eIF-4 factors. These factors consist of eIF-4A, a 46-kDa ATP-dependent RNA helicase, eIF-4B, a 70-kDa RNA-binding protein that stimulates the activity of eIF-4A, eIF-4E, a 25-kDa cap-binding protein, and eIF-4, a 154-kDa protein that forms complexes with the other eIF-4 polypeptides as well as with eIF-3. By promoting formation of these complexes, eIF-4 may serve to bring the 43 S initiation complex, the cap-binding function of eIF-4E, and the RNA helicase activity of eIF-4A and eIF-4B into immediate proximity so as to begin the dual processes of unwinding and scanning of the mRNA from the cap in the direction of the first AUG (4).

Many of the initiation factor polypeptides exist as phosphoproteins, and several lines of evidence support the view that the overall rate of translation is regulated by phosphorylation and dephosphorylation of initiation and elongation factors(5, 6) . In the case of eIF-4E, there is a correlation between the rate of protein synthesis and phosphorylation of the protein. Dephosphorylation of eIF-4E occurs under conditions where translation of normal cellular mRNAs is inhibited, such as heat shock, mitosis, and adenovirus infection. Conversely, phosphorylation of eIF-4E increases under conditions that stimulate protein synthesis such as exposure of cells to insulin, serum, epidermal growth factor, platelet-derived growth factor, nerve growth factor, phorbol esters, lipopolysaccharide, tumor necrosis factor-, and interleukin-1 or overexpression of oncoproteins such as p21 or pp60 (for review, see Ref. 6). The phosphorylated and nonphosphorylated forms of eIF-4E are resolved by chromatography on RNA-Sepharose, and the phosphorylated form binds to cap analogs and mRNA caps 3-4-fold more tightly than the nonphosphorylated form(7) . Since eIF-4E appears to be the least abundant of the initiation factors and acts at the rate-limiting step of initiation (for review, see Ref. 8), the phosphorylation of eIF-4E may directly regulate the rate of protein synthesis initiation.

Although there are at least five forms of mammalian eIF-4E separable by IEF, two forms predominate, having pI values of 5.9 and 6.3(9, 10, 11) . Several lines of evidence suggest that these represent nonphosphorylated and monophosphorylated forms of the protein. When eIF-4E is labeled in vivo with [P]P, the pI of the major radioactive form is 5.9, whereas that of the major nonradioactive form is 6.3, a difference that is consistent with the presence of a single phosphate residue(9, 10) . Phosphatase treatment converts the pI 5.9 form to pI 6.3 but has no effect on the pI 6.3 form, suggesting that there are no phosphate residues that are metabolically inactive and hence not labeled in vivo(10, 7) . Tryptic digestion produces a single major phosphopeptide(12, 13, 14) . Finally, the only phosphoamino acid detected in the in vivo labeled protein is P-Ser(9, 15) , although treatment of cells with okadaic acid produces eIF-4E containing P-Thr as well(16) .

A previous study presented evidence that the major phosphorylation site in eIF-4E was Ser-53(12) . This was based on labeling eIF-4E in vivo with [P]P, isolating tryptic phosphopeptides either directly or after alkylation of the protein with citraconic anhydride, and determining their amino acid compositions. Comparison with the cDNA-derived amino acid sequence of eIF-4E suggested that the phosphorylated residue lay in the peptide WALWFFKNDKSKTWQANLR, which contains only a single Ser residue, Ser-53. Several studies have subsequently demonstrated the importance of Ser-53 to the activity of eIF-4E, and these have been interpreted to mean that phosphorylation enhances its activity. The wild-type protein (eIF-4E) is incorporated into the 48 S initiation complex along with mRNA(17) , whereas the eIF-4E(18) and eIF-4E (8) variants are not. Overexpression or microinjection of eIF-4E, but not eIF-4E, in cultured mammalian cells causes accelerated protein synthesis and cell growth, leading to oncogenesis in some cases (19, 20) and to aberrant cellular morphology such as multiple nuclei in others(21, 8) . Overexpression of eIF-4E but not eIF-4E causes increased expression of cyclin D1 (22) and chloramphenicol acetyl transferase encoded by an mRNA with high 5` secondary structure(23) . The ability to relieve inhibition of protein synthesis in early sea urchin embryo extracts is a property of eIF-4E but not eIF-4E(24) . Overexpression of eIF-4E in Xenopus embryos induces the differentiation of mesodermal tissues in 90% of the cases, as opposed to 53% for eIF-4E(25) . In contrast to the above, however, overexpressed eIF-4E and eIF-4E are equally phosphorylated and competent to form eIF-4 complexes in COS cells(26) .

The kinase(s) responsible for in vivo phosphorylation of eIF-4E has not been identified, although rat brain protein kinase C (27) and bovine kidney protamine kinase (28) can phosphorylate eIF-4E in vitro at the physiological site, based on generation of a tryptic phosphopeptide that co-migrates with the in vivo phosphopeptide. Another study indicated that protein kinase C phosphorylates both Ser and Thr in eIF-4E in vitro(29) , whereas casein kinase I phosphorylates predominantly Thr residues(30) .

In order to aid in isolation of the physiological kinase, we undertook the chemical synthesis of P-Ser-Lys, the peptide expected from complete tryptic digestion of the region surrounding Ser-53 in eIF-4E. Such a peptide would be useful as a standard in studies to determine whether a given kinase phosphorylates eIF-4E at the physiological site. Surprisingly, the synthetic peptide did not co-migrate with the natural tryptic phosphopeptide. Consequently, we have redetermined the phosphorylation site of eIF-4E.


EXPERIMENTAL PROCEDURES

Materials

Z-Ser, Lys(Z)OBn hydrochloride, L-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin and 1-chloro-3-tosylamido-7-amino-2-heptone-treated -chymotrypsin were purchased from Sigma. Protease Glu-C and endoproteinase Asp-N were purchased from Boehringer Mannheim.

NMR and Mass Spectroscopy

C NMR spectra were obtained with a Jeol FX90Q spectrometer. H NMR spectra were recorded at 400 MHz on a Jeol GX-400 and at 500 MHz on a Bruker AM 500 spectrometer in HO. Chemical shifts were determined relative to sodium 3-trimethylsilyl-[2,2,3,3-H]propionate as internal standard. Liquid matrix secondary ions mass spectrometry was performed on a AMD-604 Intectra GmbH spectrometer.

Synthesis and Characterization of P-Ser-Lys

The scheme of synthesis was as follows.

On-line formulae not verified for accuracy

On-line formulae not verified for accuracy

On-line formulae not verified for accuracy

On-line formulae not verified for accuracy

On-line formulae not verified for accuracy

Z-Ser-Lys(Z)OBn (1) was prepared from Z-Ser and Lys(Z)OBn hydrochloride(31) . C NMR spectra and other physiochemical data were in accordance with the predicted structure. This compound (1, 101 mg, 0.17 mmol) was treated with trimethyl phosphate (1 ml), and then phosphorous oxychloride (32 µl, 0.34 mmol) was added. The mixture was stirred at 0 °C overnight, poured into water (20 ml), and neutralized with 1 N NHOH. The precipitate obtained was purified by semipreparative HPLC on a Supelco LC-18-DB reverse phase column monitored at 260 nm over 15 min with a linear 40-100% gradient of buffer A (0.03 M NHOAc, pH 5.0) and buffer B (33% buffer A, 67% acetonitrile (v/v)). The material eluting at 6.6 min was collected and lyophilized (yield, 35 mg; 29% as calculated for the diammonium salt). The C NMR spectrum of product 2 and other physiochemical data confirmed the presence of a phosphate group on the serine moiety. Next, 2 (27 mg) was dissolved in ethanol (8 ml), palladium on charcoal (100 mg) was added, and hydrogen was passed through the mixture at room temperature for 8 h. The catalyst and product were collected on a sintered glass funnel, the ethanol filtrate being discarded, and the product was eluted by washing twice with water. The water solution was evaporated to dryness to obtain 3 (10 mg, 78.8%). Analytical HPLC was performed on a Supelco LC-18-T reversed phase column (250 5 mm) using 0.01 M KHPO, pH 3.0, at 2.0 ml/min with monitoring at 210 nm. This produced a single peak with retention time of 1.4 min. Mass spectrometry revealed peaks at 314 (M+H), 627 (2M+H), and 940, (3M+H); mass calculated for CHNOP + 1 was 314.

Preparation of P-Labeled eIF-4E

Rabbit reticulocytes were incubated with [P]P, and the eIF-4E purified by mGTP-Sepharose chromatography as described previously (12) with the exception that the cells were resuspended in 2 volumes of the labeling buffer and incubated in the presence of 0.4 mCi/ml of radiolabel (method 1) or phosphate-free Dulbecco's modified Eagle's medium containing 10% fetal calf serum and at 0.2 mCi/ml of radiolabel (method 2). Similar results were obtained with both methods. The P- eIF-4E was then mixed with 100 µg of unlabeled rabbit reticulocyte eIF-4E and further purified by reverse-phase HPLC as described previously(12) , except that a C column (4.6 150 mm, 5 µm particle size, Vydac, Hesperia, CA) was used.

Proteolytic and Acid Hydrolysis

P-labeled eIF-4E was dried by vacuum centrifugation and resuspended at 0.5 mg/ml in 50 mM NHHCO. Proteolysis was carried out for 20 h at 37 °C with either trypsin (25 µg/ml) or chymotrypsin (50 µg/ml). Peptides were digested with trypsin (80 µg/ml), Glu-C (16 µg/ml), or Asp-N (8 µg/ml) for 12 h under the same conditions except in the case of Glu-C, where 50 mM potassium phosphate buffer, pH 8.0, was used. Phosphoamino acid analysis was performed essentially as described previously(9) .

Thin-layer Electrophoresis

TLE was performed as described previously(9) . Following electrophoresis, peptides or amino acids were visualized by staining with 0.5% ninhydrin in 1-butanol. Radiolabeled phosphopeptides were visualized by autoradiography and, when necessary, were recovered from the plates by scraping and extracting the cellulose with 8% acetic acid, 2% formic acid.

Isolation of a Chymotryptic Phosphopeptide from eIF-4E

P-labeled eIF-4E was digested with chymotrypsin, and the resulting peptides were diluted to 1 ml with 0.1% trifluoroacetic acid and resolved by reverse phase HPLC using a Vydac C column (4.6 150 mm, 5 µm particle size). Buffer C was 0.1% aqueous trifluoroacetic acid, and buffer D was 0.1% trifluoroacetic acid in 95% acetonitrile. Buffer D increased linearly from 0 to 15% over 30 min and then to 85% over 19 min. P-Labeled peptides were identified by Cerenkov counting. For rechromatography of isolated peaks, fractions were pooled, concentrated to approximately 200 µl by vacuum centrifugation, and diluted to 500 µl with buffer C prior to injection.

Edman Degradation

All peptide sequencing was performed on an Applied Biosystems 477A peptide sequencer (Foster City, CA). A polybrene-coated glass fiber disc was the sequencing support for samples sequenced by the standard noncovalent method. The ratios of PTH-DHA-DTT to PTH-Ser were calculated at Ser cycles from measurements of peak heights for the two species. When covalent sequencing was performed, the peptides were linked through their carboxyl groups to an arylamine-derivitized polyvinylidine difluoride disc according to the protocol provided by the manufacturer (Sequelon AA reagent kit, Millipore, Bedford, MA). The disc was washed after coupling with first water and then methanol until radioactivity in the wash solutions remained at background levels. Then the disc was minced and loaded into a standard sequencer reaction cartridge above an Immobilon-P disc (Millipore). Ninety percent methanol was substituted for butyl chloride at the S3 and X2 positions. Approximately one-third of the PTH sample was analyzed by HPLC. After sample injection, the conversion flask was washed with 90% methanol from the X2 bottle, and this wash was used to push the remaining two-thirds of the sample to a fraction collector for quantitation of radioactivity by Cerenkov detection.

Preparation of cDNA Encoding wild-type, S53A, S207A, and S209A eIF-4E

The plasmids pTCEEC (17) and pTCALA(18) , which contain most of the human eIF-4E cDNA, were modified to form pT4EA+ and pT4ES53A, respectively, by excision of the NsiI to HindIII region of the 3`-untranslated region (nucleotides 1085-1353) and replacement with the 3`-untranslated and poly(A) sequence (nucleotides 456-651) derived from the Xenopus ribosomal protein S22 (32) using an EcoRI, HindIII, BamHI adaptor (Oligos Etc., Wilsonville, OR). pT4EA+ served as the template for site-directed mutagenesis by polymerase chain reaction (33) to introduce a single-base change (T625G) converting Ser-209 to Ala or a double-base change (A619G/G620C) converting Ser-207 to Ala. Briefly, 0.1 µg of linear plasmid was amplified in two separate reactions (10 cycles of 45 s at 94 °C, 45 s at 50 °C, and 90 s at 72 °C; 50 pmol of each oligonucleotide) with the upstream primer, CBP440 (5`-ATGATGTATGTGGCGCTG-3`), and mut624 (5`-AGTGGTGGCGCCGCTCTTAG-3`) or a downstream primer, S22-3` (5`-CAATCCACATTACTTTCTTT-3`) and mut624s (5`-CTAAGGCCGGCTCCACCACT-3`) using PfuI thermostable DNA polymerase (Stratagene, La Jolla, CA). mut624s and mut624 are complementary with the exception of three mismatches. The gel-purified products of the initial reactions were mixed, heated 5 min at 94 °C, and then annealed to one another by slow cooling to 45 °C. Annealed fragments were subjected to a second round of amplification (20 cycles of 45 s at 94 °C, 45 s at 45 °C, and 90 s at 72 °C) with PfuI using only the external primers, CBP440 and S22-3`. The product was digested with AccI and NsiI and used to replace the corresponding 539 base pairs of wild-type eIF-4E sequence in pT4EA+. The resulting plasmids, pT4ES207A and pT4ES209A, were sequenced in the amplified region from the AccI site of the insertion through the termination codon using the antisense oligonucleotide, CBP 695 (5`-TCTCGATTGCTTGACGCAGT-3`) and a Sequenase T7 DNA polymerase dITP kit (U. S. Biochemical Corp.) as described by the manufacturer. Each of the induced mutations creates a new restriction endonuclease site (NaeI in pT4ES207A and NarI in pT4ES209A), which was used for independent verification of the correct mutation. pT4ES53A was also sequenced to verify the site of mutation with the antisense oligonucleotide, CBP 258 (5`-GTGAGTAGTCACAGCCAGGC-3`).

Cell-free Transcription

Plasmids were linearized with EcoRI and transcribed with T7 RNA polymerase in vitro as described previously(4) . The resulting mRNA transcripts, which contain a poly(A) tail, were approximately 5-fold more efficient in directing eIF-4E synthesis in vitro than transcripts derived from pTCEEC, which do not contain poly(A).

Cell-free Translation and IEF

Cell-free translation was performed as described previously(4) . Following synthesis, 3-µl samples from the reactions were diluted with 70 µl of buffer containing 9.2 M urea, 0.5% Nonidet P-40, 80 mM DTT, 10% glycerol, 1.75% Pharmalytes, pH 5-8, and 0.75% Pharmalytes, pH 4-6.5 (Pharmacia Biotech, Inc.). IEF (10 watts, 16 h, 20 °C) was performed in polyacrylamide gels (5% acrylamide, 0.15% N,N`-bisacrylamide) prepared in the same buffer, and using 10 mM NaOH and 20 mM phosphoric acid at the cathode and anode, respectively.


RESULTS

Based on the assignment of Ser-53 as the major phosphorylation site of eIF-4E, we predicted a tryptic phosphopeptide of P-Ser-Lys. To provide an electrophoretic standard for investigating the in vivo phosphorylation of eIF-4E, we synthesized this dipeptide. Initial attempts to synthesize 3 by coupling of either Z-P-Ser or N-t-butyloxycarbonyl-P-Ser to Lys(Z)OBn using N, N`-dicyclohexylcarbodiimide and 1-hydroxybenzotriazole were unsuccessful. Yields were extremely low due to side reactions in which the phosphate instead of the carboxyl reacted and also to difficulties with isolation of the coupling product because of its mixed hydrophilic-hydrophobic properties. The successful synthesis of 3 was achieved by phosphorylation of Z-Ser-Lys(Z)OBn (1) with POCl followed by deprotection using a palladium catalyst, the structure being established by H NMR spectroscopy (Fig. 1). We also synthesized Ser-Lys to permit comparison of H NMR spectra. Ser-Lys has a simple NMR pattern, which enables one to make a straightforward assignment of the resonances. In particular, the Ser methine proton appears as a triplet coupled to the Ser methylene protons, the two protons having almost identical chemical shifts (Fig. 1a). Phosphorylation shifts the signals so that the Ser proton overlaps with one of the Ser protons, whereas the other Ser proton (now with a different chemical shift) overlaps with the Lys proton (Fig. 1b). Other regions of the P-Ser-Lys spectrum did not differ significantly from that of Ser-Lys (not shown). Several selective decouplings and an additional spectrum at pH 2 were used to simplify both multiplets so that the couplings with the phosphorus were observable. For example, Fig. 1c shows a portion of the spectrum with decoupling of Ser H- and H-. The signal of the second proton appeared in this case as a doublet at 4.12 ppm with a 5.5-Hz coupling constant due to coupling with phosphorus through three chemical bonds.


Figure 1: H NMR spectra of Ser-Lys and P-Ser-Lys. a, spectrum of Ser-Lys, showing the region for H- of Ser and Lys and H- of Ser. b, spectrum of P-Ser-Lys for the corresponding protons. Note that a different region of the spectrum is shown. c, spectrum of P-Ser-Lys with decoupling of Ser H- and H-.



The synthetic dipeptide was compared by TLE to the product of tryptic digestion of eIF-4E labeled in vivo with [P]P (Fig. 2). Tryptic digestion resulted in all of the radioactivity in the eIF-4E migrating as a single species (lane1). However, the peptide migrated more slowly than P-Ser-Lys (lane3). This was not due to the presence of salt or other contaminants that might have retarded mobility of one of the peptides; when the two peptides were mixed before TLE, the result was the same (not shown). This suggested that Ser-53 was not the major phosphorylation site in eIF-4E.


Figure 2: TLE of phosphopeptides and phosphoamino acids. The anode in each panel is indicated by +, and the cathode is indicated by -. For peptides, the running time was 50 min, and for phosphoamino acids it was 90 min. A, autoradiograms of tryptic peptides derived from either P-eIF-4E (lane1) or from the chymotryptic phosphopeptide (lane2). B, ninhydrin staining of synthetic P-Ser-Lys. PanelsA and B are from the same TLE run. C, autoradiogram of phosphoamino acids obtained by acid hydrolysis of P-labeled eIF-4E (lane4) or the chymotryptic phosphopeptide (lane5). The dashedcircles represent the positions of ninhydrin-stained P-Ser (upper) and P-Thr + P-Tyr (lower), added as internal standards to the samples before hydrolysis. D, the P-labeled chymotryptic peptide was further digested by Asp-N, and the resultant radioactive peptide was purified by TLE. A portion was run without further digestion (lane6) or with tryptic digestion (lane7). An autoradiogram is shown. E, same as D except that the order of Asp-N and trypsin treatments was reversed. Proof that the enzymes were active in panelsD and E came from inclusion of a synthetic peptide, CQSHADTATKSGSTTKN, in the reaction mixtures. Cleavage by trypsin and Asp-N was verified by ninhydrin staining of the TLE plates prior to autoradiography. The peptide was synthesized by Fmoc (9-fluorenylmethyloxycarbonyl) chemistry at the University of Kentucky Macromolecular Structure Analysis Facility.



The tryptic phosphopeptide of eIF-4E is too hydrophilic to be retained on C or C columns(12) . Attempts to purify and analyze the peptide by other means (hydrophilic interaction chromatography, ion-exchange chromatography) or to generate a longer, more hydrophobic peptide with other proteases (Glu-C, Asp-N, subtilisin, Arg-C) were unsuccessful. However, chymotryptic digestion generated radioactive peptides that could be retained on C (Fig. 3). Three major peaks of radioactivity were observed (Fig. 3A, fractions1-3). When each of these was subjected to a second round of chromatography on C, the radioactivity co-eluted with a peak of optical density (Fig. 3, B-D). The peak fractions (bracketed) were then subjected to Edman degradation. Fraction 1 produced the sequence QSHADTATKSGSTTKN, which exactly matches residues 198-213 of rabbit eIF-4E, as determined from the cDNA(35) . Based on the specificity of chymotrypsin, the peptide is likely to be QSHADTATKSGSTTKNRF, but low yields in late Edman cycles prevented exact identification of the C terminus. The sequence of Fraction 2 was the same as that of Fraction 1; the difference in elution time from C is not understood but may represent C-terminal heterogeneity. Fraction 3 produced the sequence XPQVQSXX, which does not match eIF-4E but which is similar to residues 124-131 of the chain of rabbit hemoglobin (TPQVQSAW; apparently this protein was a contaminant in the eIF-4E preparation).


Figure 3: Resolution of chymotryptic phosphopeptides. eIF-4E was labeled by incubation of rabbit reticulocytes with [P]P and purified by affinity chromatography and HPLC on C. A, the protein was digested with chymotrypsin, applied to a C reverse phase column, and both absorbance and radioactivity were monitored. B, fractions eluting at 20.7-22.2 min (panelA, bracket1) were reapplied to the same C column. Fractions eluting at 21.2-21.7 min (bracketed) were subjected to Edman degradation. C, same as B except fraction eluting at 22.2-23.7 min in panelA were subjected to rechromatography. D, same as B except that the fractions eluting from 23.7-24.7 min in panelA were used.



Subsequent characterization was performed on Fraction 1 from this and other preparations of eIF-4E. Since the chromatogram of Fig. 3A exhibited several peaks of radioactivity, it was important to show that Fraction 1 contained the major phosphorylation site. Hence, Fraction 1 was digested with trypsin and subjected to TLE (Fig. 2, lane2). The resultant peptide migrated identically to the tryptic peptide derived from the entire eIF-4E molecule (lane1).

Since the chymotryptic peptide isolated in Fig. 3contained numerous hydroxyamino acid residues, it was necessary to determine which of them was phosphorylated. Four independent methods were used. In the first, the release of radioactivity during Edman degradation was followed. P-Ser and P-Thr are unstable to Edman degradation and break down during cleavage to release free phosphate. The acidic phosphate cannot be extracted from the ionic polybrene matrix used for a sequencing support by butyl chloride, the usual extraction solvent, and more hydrophilic washes would extract the peptide. Covalent attachment prior to sequencing eliminates the necessity for using a polybrene-coated glass fiber disc, so the free phosphate may be easily extracted from the peptide without fear of peptide loss. Fig. 4shows that there was an increase in released radioactivity at cycle 12, which corresponds to Ser-209. However, released radioactivity was also above background in cycles 1, 2, 5, 13, and 14. It is likely that this represented extraction of noncovalently bound peptide in the case of the earlier cycles, or incomplete Edman chemistry in cycle 12, resulting in carryover into cycles 13 and 14. Cycles 13 and 14 contained Thr, which could potentially be phosphorylated in eIF-4E. We therefore analyzed the phosphoamino acid content of the chymotryptic peptide (Fig. 2, lane5) as well as that of intact eIF-4E (lane4). Only P-Ser was detected, confirming earlier results (9, 15) and indicating that the radioactivity in cycles 13 and 14 does not represent P-Thr but rather carryover from cycle 12. This analysis, however, could not exclude the possibility that Ser-199 (cycle 2) also contained radioactivity.()


Figure 4: Radioactivity released during Edman degradation. A preparation of the chymotryptic phosphopeptide similar to that shown in Fig. 3B (bracketedfractions) was subjected to Edman degradation, except that in this case, the peptide was covalently attached to the solid support as described under ``Experimental Procedures.'' Radioactivity released at each cycle was determined by Cerenkov counting (bars). Superimposed on each bar is the amino acid sequence of human, rabbit and mouse eIF-4E, beginning at residue 198 (7). At the top (DHA/SER) is given the ratio of PTH-DHA-DTT to PTH-Ser for cycles 2, 10 and 12 of a noncovalent Edman degradation of fraction 1 (Fig. 3B).



The second method relied on the formation of dehydroalanine. Automated Edman degradation of Ser and Thr normally produces some breakdown products, notably the DTT adduct of PTH-dehydroalanine (PTH-DHA-DTT) for Ser and PTH-dehydroaminobutyric acid for Thr. The ratio of the amount of breakdown product to the amount of PTH-Ser or PTH-Thr has been found to be elevated in cycles containing P-Ser or P-Thr(36, 37) , and this effect has been employed as a nonradioactive means of identifying phosphorylation sites(38, 39) . Fig. 4presents the ratios of PTH-DHA-DTT to PTH-Ser for each of the cycles in which Ser was detected. The ratio was considerably lower in cycles 2 and 10 than in cycle 12, again suggesting that only Ser-209 is phosphorylated.

The third method involved analysis of the labeled chymotryptic peptide by further proteolytic digestion and separation of peptides by TLE (Fig. 2). The chymotryptic phosphopeptide (QSHADTATKSGSTTKNRF) was further digested with Asp-N. This resulted in a shift in mobility of the radiolabel, indicating that it contains an Asp-N site (data not shown). From the specificity of Asp-N, this should produce two peptides, QSHA and DTATKSGSTTKNRF, in which Ser-199 and Ser-209 are separated. The results indicated that only one of these peptides was labeled. To determine which peptide this represented, the sample was recovered from the TLE plate and further incubated without and with trypsin (Fig. 2, lanes 6 and 7). The fact that the phosphopeptide changed mobility indicates that DTATKSGSTTKNRF, which contains tryptic sites, was radioactive. The converse experiment was also performed. The chymotryptic phosphopeptide was digested first with trypsin, which should produce QSHADTATK, SGSTTK, NR, and F. Only one of these peptides was labeled (lane8). Further digestion with Asp-N did not change its mobility (lane9), indicating that the radioactivity was in SGSTTK. Similar results were obtained when Glu-C was used in place of Asp-N (not shown). In all cases, cleavage activity of the enzymes was verified with a synthetic peptide of the same sequence as the chymotryptic peptide, which was used as an internal standard with products detected by ninhydrin (see the legend to Fig. 2). These results demonstrate that radioactivity was not associated with Ser-199.

The fourth method was to alter the codons for positions 207 and 209 in human eIF-4E from Ser to Ala using site-directed mutagenesis. These mutations represent single or double base changes in the cDNA coding region that convert codon 207 from AGC (Ser) to GCC (Ala) and codon 209 from TCC (Ser) to GCC (Ala). The mutations, and that previously generated at codon 53 (AGC to GCC; Ref. 18) were confirmed by dideoxynucleotide sequencing (Fig. 5, A and B). RNA transcribed from these plasmid constructs was translated in rabbit reticulocyte lysate in the presence of [S]Met. Exogenous S-labeled human eIF-4E has been shown to be phosphorylated in this system(17, 18) . The S-labeled eIF-4E (wild-type) contained nearly equal amounts of the basic (pI 6.3) and acidic (pI 5.9) forms, as indicated by IEF, indicating efficient phosphorylation (Fig. 5C, lanes2 and 3). The more acidic form was found to co-migrate with in vivoP-labeled rabbit eIF-4E (data not shown). Likewise, in vitro synthesized eIF-4E (lanes4 and 5) and eIF-4E (lanes6 and 7) were resolved into acidic and basic forms, indicating that each could be phosphorylated. eIF-4E could not be modified during incubation, however, resulting in only the basic form (lanes8 and 9). This further supports the assignment of Ser-209 as the major site of phosphorylation in eIF-4E. Interestingly, even though the amount of protein synthesized was similiar, the extent of phosphorylation of eIF-4E was lower than that of either eIF-4E or eIF-E. Since this residue is just two amino acids from the phosphorylation site, it is possible that the change to Ala at position 207 affected the accessibility of the kinase(s) or phosphatase(s) involved.


Figure 5: Production and analysis of variant eIF-4E forms by site-directed mutagenesis, cell free translation, and IEF. A, the plasmids pT4EA+ (WT) and pT4ES53A were sequenced using the antisense oligonucleotide primer, CBP258 (see ``Experimental Procedures''). The region of the gel surrounding the mutation site is shown. The bases complementary to the respective dideoxynucleotide triphosphate included in each lane are shown at the bottom of each panel. B, plasmids pT4ES207A, pT4EA+, and pT4ES209A were sequenced using the antisense oligonucleotide primer CBP695. The GCC codon of pT4ES207A lies within a GC-rich region, which causes some termination of the polymerase in all four lanes. This sequence was independently verified by digestion with NaeI. C, mRNA-dependent reticulocyte lysate translation systems containing [S]Met (1 mCi/ml, 1100 Ci/mmol, ICN Biomedicals, Costa Mesa, CA) were programmed with either no mRNA (lane1), or in vitro transcribed mRNA from the plasmids described above encoding wt eIF-4E (lanes2 and 3), eIF-4E (lanes4 and 5), eIF-4E (lanes6 and 7), or eIF-4E. Following incubation (30 °C, 1.5 h) in the presence or absence of 5 mM 2-aminopurine as indicated, samples were removed for the separation of S-labeled eIF-4E isoforms by IEF. The positions of nonphosphorylated and phosphorylated eIF-4E are indicated on the left.



Each reaction was conducted in the presence or absence of 2-aminopurine, a kinase inhibitor. Although the amount of protein synthesized was greater in the presence of this compound, the ratio of phosphorylated versus nonphosphorylated eIF-4E was decreased about 30%. A similar effect was reported by Huang and Schneider (40) when human embroynic kidney cells were treated with 2-aminopurine, but that treatment resulted in an inhibition of translation. Since the drug has been shown to inhibit the action of two eIF-2 kinases, heme-controlled repressor and protein kinase R(41) , which, when active, lead to an inhibition of translation, there may be a mixed effect of this drug on translation rates: stimulation due to prevention of eIF-2 phosphorylation and inhibition due to prevention of eIF-4E phosphorylation.

The effect of changing Ser-209 to Ala on the association of eIF-4E with the 48 S initiation complex was tested. Two different protocols were used, one in which S-labeled eIF-4E was synthesized by cell-free translation and the reaction mixture was then added to a second translation reaction containing globin mRNA(4) , and the other in which S-labeled eIF-4E was purified on mGTP-Sepharose before being added to the second translation reaction(18) . With either method, wild-type eIF-4E and eIF-4E accumulated to the same degree on the 48 S initiation complex (data not shown). This result is not entirely unexpected; our previous study showed that phosphorylation of eIF-4E increased the affinity of the protein for caps(7) , but the relationship between the cap-binding and ribosome-binding activities of eIF-4E has not yet been elucidated.


DISCUSSION

The evidence presented in this study supports the existence of a single phosphorylation site in eIF-4E at Ser-209. The release of radioactivity during Edman sequencing (Fig. 4), the increase in the PTH-DHA-DTT/PTH-Ser ratio (Fig. 4), the susceptibility to site-specific proteases (Fig. 2), and the results of site-directed mutagenesis (Fig. 5) all support the assignment of Ser-209 as the major phosphorylation site of eIF-4E. The previous evidence favoring Ser-53 consisted of (i) amino acid composition data(12) , (ii) the fact that eIF-4E was not phosphorylated (24) or was phosphorylated more slowly (18) than eIF-4E in a rabbit reticulocyte translation system, and (iii) the fact that protein kinase C phosphorylated eIF-4Ein vitro but was inactive with eIF-4E(42) . In our original study(12) , the citraconylated phosphopeptide was purified by C reverse phase HPLC. To correct for background amino acids, a second run was performed on the same column but with no injected peptide. Corresponding fractions were collected, and amino acid analysis was performed on both peptide and blank fractions. The difference was used to deduce the peptide's composition and choose the best candidate among the eIF-4E peptides. Apparently there was sufficient experimental error in this method that we incorrectly chose peptide 5 (see of Ref. 12) instead of peptide 4, which has a similar composition. With regard to the other studies, however, it is not clear why phosphorylation of eIF-4E would be impaired in a complete translation system or with purified protein kinase C, unless perhaps the conformation of eIF-4E is altered by the S53A modification in a way that affects the accessibility of Ser-209.

Reassignment of the major phosphorylation site does not negate the evidence that Ser-53 is important for the activity of eIF-4E. This has been demonstrated by the activity of eIF-4E compared with the inactivity of eIF-4E in a variety of biological systems: the stimulation of NIH 3T3 and Rat2 cells to grow in soft agar and form tumors in nude mice(19) , the acceleration of growth of HeLa (21) or HBL100 (8) cells, leading to multiple nuclei and cell death, the stimulation of DNA synthesis in 3T3 cells(20) , the binding of eIF-4E to the 43 S initiation complex(18) , the specific stimulation of translation of mRNAs containing high secondary structure (22, 23), and the relief of inhibition of protein synthesis in early sea urchin embryo extracts(24) . These studies were conducted with cDNAs encoding both human (18) and mouse (19) eIF-4E, independently subjected to site-directed mutagenesis to convert Ser-53 to Ala-53. Also, these effects were seen with both integrating (19, 8) and episomal (21) vectors. Thus, although Ser-53 is not the major phosphorylation site, this position appears to be important for the proper functioning of the protein.

Similarly, the evidence that phosphorylated eIF-4E binds to cap analogs and mRNA caps 3-4-fold more strongly than nonphosphorylated eIF-4E (7) is not contradicted by the change in the phosphorylation site. In that study, in vivo phosphorylated eIF-4E was chromatographically resolved from the nonphosphorylated form, and the binding affinity of each was measured fluorimetrically. Knowledge that phosphorylation increases the affinity for caps combined with the localization of the phosphorylation site at the C terminus suggests that phosphorylation may induce a conformational change in the protein that makes the cap-binding site more accessible. C-terminal phosphorylation on Ser/Thr is correlated with modulation of activity of a number of proteins, such as glycogen synthase, cAMP-dependent protein kinase, and the -adrenergic receptor(43) .

Although the physiological eIF-4E kinase(s) has not been isolated, the context surrounding Ser-209 indicates that it would be an optimal phosphorylation acceptor for protein kinase C based on the consensus sequence (R/K,X)(S/T)(X,R/K) (44). Furthermore, the pseudosubstrate sites of all known isozymes of protein kinase C, with the exception of protein kinase C, contain Gly just before the phosphorylation site, which would correspond to Gly-208 in eIF-4E. Indeed, protein kinase C has been found to phosphorylate eIF-4E in vitro within the same tryptic phosphopeptide as is isolated from in vivo phosphorylated eIF-4E(45, 46) . In vivo, stimulation of reticulocytes (46) and 3T3 L1 (14) cells with phorbol esters, which activate protein kinase C, leads to increased phosphorylation of eIF-4E and correlates with an increase in translational rate. In contrast, long term treatment of B lymphocytes with phorbol ester failed to demonstrate a reduction in the rate eIF-4E phosphorylation(47) . This led the authors to question the involvement of protein kinase C. However, it is now known that long term phorbol ester treatment does not result in down-regulation of all protein kinase C isoforms (for review, see Ref. 48). Hence, protein kinase C is a likely candidate for the physiological kinase of eIF-4E.

The chymotryptic phosphopeptide also contains sequence contexts that are predicted to be phosphate acceptor sites for casein kinase I and II (49). One of the five casein kinase I consensus sites (D/E)XX(S/T) in eIF-4E lies within the chymotryptic phosphopeptide in the context DTAT, where Thr-205 would be the phosphate acceptor. If this region of the C terminus of eIF-4E were to be made accessible for phosphorylation upon phosphorylation of Ser-209, then the Thr phosphorylation by casein kinase I observed in vitro(30) may be at Thr-205. Ser-199, in the context SHAD, is predicted to be an acceptor for casein kinase II. Attempts to phosphorylate eIF-4E with purified casein kinase II, however, were unsuccessful(45) . No phosphorylation of any secondary sites was observed in the present study. However, the presence of numerous Ser and Thr residues in the C terminus of eIF-4E may explain the multiple minor phosphorylations of eIF-4E that have been previously observed(9, 16) .


FOOTNOTES

*
This work was supported by Research Grant GM20818 from the National Institute of General Medical Sciences, U. S. A. (to R. E. R.), Grant 3076 from the Council for Tobacco Research U.S.A., Inc. (to R. E. R.), and Project 4 0800 91 01 from the Polish Committee For Scientific Research (to E. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Abteilung Innere Medizin I, Medizinische Klinik und Polyklinik, Universität Ulm, D-7900 Ulm, Germany.

To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Louisiana State University Medical Center, 1501 Kings Highway, Shreveport, LA 71130-3932. Tel.: 318-675-5161: Fax: 318-675-5180.

The abbreviations are: eIF, eukaryotic initiation factor; IEF, isoelectric focusing; P-Ser, phosphoserine; P-Ser-Lys, O-phosphoseryllysine; P-Thr, phosphothreonine; TLE, thin-layer electrophoresis; Z, N-benzyloxycarbonyl; HPLC, high performance liquid chromatography; PTH, phenylthiohydantoin; DTT, dithiothreitol; PTH-DHA-DTT, the adduct of dithiothreitol and the phenylthiohydantoin derivative of dehydroalanine; PTH-Ser, the phenylthiohydantoin derivative of Ser; Bn, benzyl.

The reason why Ser and not P-Ser was detected at cycle 12 when fractions 1 and 3 of Fig. 3 were sequenced is that only 20-30% of rabbit eIF-4E is the phosphorylated form (7, 9). Apparently the phosphorylated and nonphosphorylated chymotryptic peptides co-elute from the C column.


ACKNOWLEDGEMENTS

We thank Dr. Robert Smith, Debra Waters, and Wei-Hua Wu, Department of Biochemistry and Molecular Biology, Lousiana State University Medical Center, for technical advice and assistance.


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