THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 265, No. 5, Issue of February 15, pp. 2979~2983,lSSO 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S. A.

Alteration of the Major Phosphorylation Site of Eukaryotic Protein Synthesis Initiation Factor 4E Prevents Its Association with the 48 S Initiation Complex*

(Received for publication, June 28, 1989)

Swati Joshi-Barve, Wojciech Rychlik, and Robert E. RhoadsS From the Department of Biochemistry, University of Kentucky, Lexington, Kentucky 40536

Site-directed mutagenesis was used to replace the serine residue at the primary phosphorylation site of human eukaryotic initiation factor (eIF) 4E with an alanine residue. The mutated cDNA was transcribed in vitro, and the transcript was used to direct protein synthesis in a reticulocyte lysate system. The variant protein (eIF-4E*‘“) was retained on a 7-methylguano- sine 5’-triphosphate (m’GTP)-Sepharose affinity col- umn and was specifically eluted by m’GTP. Examina- tion of eIF-4E*‘” by isoelectric focusing revealed two species which had the same p1 values as the phosphoryl- ated and nonphosphorylated forms of unaltered eIF- 4E (here designated eIF-4ES”‘). However, conversion of unphosphorylated eIF-4E*‘” to the putative phos- phorylated eIF-4E*‘” in the reticulocyte lysate system was slower than the corresponding conversion of eIF- 4ESer. The possibility that the more acidic form of eIF- 4E*“’ was due to NH&erminal acetylation was ruled out by an experiment in which the acetyl-CoA pool of the reticulocyte lysate system was depleted with oxal- oacetate and citrate synthase. The more acidic form of eIF-4E*‘” was, however, eliminated by treatment with calf intestine alkaline phosphatase, suggesting that it results from a second-site phosphorylation. When translation reaction mixtures were resolved on sucrose density gradients, the ?%labeled eIF-4ES”’ was found on the 48 S initiation complex in the presence of gua- nylyl imidodiphosphate, as reported earlier (Hiremath, L. S., Hiremath, S. T., Rychlik, W., Joshi, S., Domier, L. L., and Rhoads, R. E. (1989) J. Biol. Chem. 264, 1132-1138). eIF-4E*‘*, by contrast, was not found on the 48 S complex, suggesting that phosphorylation of eIF-4E is necessary for it to carry out its role of trans- ferring mRNA to the 48 S complex. Supporting this interpretation was the finding that eIF-4E’“’ isolated from 48 S initiation complexes consisted predomi- nantly of the phosphorylated form.

Eukaryotic initiation factor 4E(eIF-4E)’ plays a central role

* This work was supported by Research Grant GM20818 from the National Institute of General Medical Science. The costs of publica- tion of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertise- ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be addressed. ’ The abbreviations used are: EIF, eukaryotic initiation factor; eIF-

4E, the 25-kDa mRNA cap-binding protein, also referred to as CBP, CBP I, cap-binding protein, cap site factor, and the 24-kDa cap- binding protein; eIF-4ES”, the natural form of eIF-4E containing Ser at position 53; eIF-4EA’“, the variant form of eIF-4ES” containing Ala at position 53: GMP-PNP, guanylyl imidodiphosphata; IEF, isoelec- tric focusing; m’GTP, 7-methylguanosine 5’-triphosphate; SDS- PAGE, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate; HEPES, 4-(2-hydroxyethyl)-l-piperazineethansul- fonic acid; TEMED, N,N,N’,N-tetramethylenediamine.

in the recognition of the 7-methylguanosine cap structure of messenger RNA and the formation of initiation complexes in protein synthesis. eIF-4E can be isolated both free and in complexes with other initiation-related polypeptides includ- ing eIF-4A, eIF-4B, and ~220, reflecting the fact that its role requires its interaction with both the mRNA cap and the other proteins (Rhoads, 1988; Sonenberg, 1988). The eIF-4E polypeptide exists in both phosphorylated and nonphosphor- ylated forms (Rychlik et al., 1986; Buckley and Ehrenfeld, 1986; Duncan et al., 1987), and the primary site of phosphoryl- ation has been identified as Ser-53 (Rychlik et al., 1987b). The dephosphorylation of the protein coincides with a reduc- tion in protein synthesis after heat shock (Duncan et al., 1987) and during mitosis (Bonneau and Sonenberg, 1987), whereas its phosphorylation coincides with an increase of protein synthesis upon mitogen stimulation and serum activation of T lymphocytes or 3T3 cells, respectively (Kaspar et al., 1990), and fertilization of sea urchin eggs (Waltz and Lopo, 1987). Phosphorylation of eIF-4E is also stimulated in reticulocytes by phorbol esters (Morley and Traugh, 1989) and in 3T3 cells by insulin (Morley and Traugh, 1988). These findings suggest that phosphorylation of eIF-4E may promote its function and thus constitute a form of regulation of protein synthesis initiation.

In examining how phosphorylation might affect eIF-4E function, one must first ask what the function of eIF-4E is. The most demonstrable activity of eIF-4E is binding to the mRNA cap or its analogues. However, previous studies have indicated that phosphorylation of eIF-4E does not alter rec- ognition of the cap structure (Duncan et al., 1987; Bonneau and Sonenberg, 1987; Hiremath et al., 1989). Recently we demonstrated another activity for eIF-4E, namely binding to the small ribosomal subunit coincident with mRNA binding (Hiremath et al., 1989). A model was proposed whereby eIF- 4E recognizes mRNA via the cap and then conducts it to the 43 S initiation complex to form the 48 S initiation complex.

In the present study, we have examined the effect of eIF- 4E phosphorylation on this process by constructing a modified form of eIF-4E in which Ser-53 was changed to Ala-53, thus preventing phosphorylation of the major site. Our results indicate that the altered protein fails to bind to the 48 S initiation complex.

EXPERIMENTAL PROCEDURES

Materials-RNasin, T? RNA polymerase, GMP-PNP, m’GTP- Sepharose, and ampholytes were purchased from Pharmacia LKB Biotechnology Inc. The Sequenase kit was purchased from United States Biochemical Corp. Citrate synthase was purchased from Sigma. Globin mRNA was purchased from Bethesda Research Lab- oratories. Phosphoseryl-lysine was a gift from Drs. Zbigniew Korty- lewicz and Richard Galardy, University of Kentucky. Calf intestine alkaline phosphatase was obtained from Boehringer Mannheim.

2979

by guest on April 16, 2018

http://ww

w.jbc.org/

Dow

nloaded from

2980 Role of eIF-4E Phosphorylation

Site-directed Mutagenesis-The method of Kunkel et al. (1987) was used. An ung- dut- strain of Escherichia coli (BD2399) was transformed with pTCEEC (Hiremath et al., 1989), and single- stranded uridine-substituted DNA was obtained by infection with the helper phage M13K07. The oligonucleotide used to alter the serine at position 53 to an alanine residue, 5’-AAT GAT AAA GCC AAA ACT TGG-3’, was synthesized with a model 380B DNA synthesizer (Applied Biosystems, Inc., Foster, CA). Clones were screened by sequencing DNA in the region of the mutation. One, designated pTCALA, was selected and its DNA sequenced through the entire coding region of eIF-4E to confirm that other undesired mutations had not been introduced.

H,N+ - - -COO- +

AcN ~~ COO- + H,N'- IL-COO- + AC,, !-COO- +

In Vitro Transcription and Translation-The in vitro transcrip- tions were performed as described by Hiremath et al. (1989). The transcripts were used without further treatment to program a retic- ulocyte cell-free protein synthesis system. In some cases, amino- terminal N-acetylation of the newly synthesized protein was blocked by carrying out cell-free translation in the presence of 1 mM oxaloac- etate and 30 units/ml citrate synthase, as described by Palmiter (1977).

Affinity Chromatography-The cap-binding activity of the in uitro- synthesized variant protein (eIF-4EA’“) was determined by retention on an m’GTP-Sepharose affinity column (Webb et al., 1984). 35S- Labeled eIF-4ES” (unaltered) and eIF-4EA’” proteins were synthesized in uitro, and the total reaction mixture in each case was loaded directly onto a 0.5-ml m’GTP-Sepharose column, pre-equilibrated with B20 buffer (20 mM HEPES, pH 7.6, 20 mM KOAc, 0.1 mM EDTA) containing 1 mM ATP and 0.2 mM GTP. The unbound fraction was passed over the column a second time. The column was washed with 10 column volumes of B20 containing nucleotides and then with 5 volumes of B20 without nucleotides. The eIF-4E bound to the column was specifically eluted with 5 volumes of B20 containing 70 /IM m7GTP. Equivalent aliquots of each fraction were analyzed by electrophoresis on 10% SDS-PAGE (Laemmli, 1970).

FIG. 1. Forms of eIF-4ES” and eIF-4EA’“. 3”S-labeled proteins were synthesized in cell-free translation reactions under different conditions and resolved by vertical tube IEF. An aliquot of 20 ~1 of each reaction mixture was loaded on each gel. Lane 1, a reaction programmed with the pTCEEC transcript; lane 2, a reaction pro- grammed with the pTCALA transcript; lane 3,20 ,ul each of Reactions 1 and 2; lane 4, a reaction programmed with pTCEEC transcript and carried out under conditions that block N-acetylation (see text); lane 5, 20 ~1 each of Reactions 1 and 4; lane 6, a reaction programmed with pTCALA transcript carried out under conditions that block N- acetylation; lane 7, 20 ~1 each of Reactions 2 and 6; lane 8, 35S-labeled eIF-4ES” was added to a translation reaction mixture programmed with globin mRNA in the presence of GMP-PNP, and the initiation complexes were separated by sucrose density gradient sedimentation as indicated in Fig. 4. Fractions which corresponded to the 48 S initiation complex were pooled, acetone-precipitated with reticulocyte lysate as a carrier, and loaded on the IEF gel.

alkaline phosphatase (1.1 unit/r1 reaction volume) in 50 mM Tris- HCl buffer, pH 8.5. Protein was precipitated with acetone and ana- lyzed by horizontal slab IEF as described above.

RESULTS

Sucrose Density Gradient Analysis-“5S-Labeled eIF-4ES” and eIF- 4EA” were synthesized, purified by affinity chromatography, and added to a 75-/11 translation reaction mixture containing globin mRNA. In some cases, the reaction mixtures also contained 3 mM GMP-PNP and 1.5 mM MgC12 as described previously (Hiremath et al., 1989). In other experiments, the concentrations of GMP-PNP and MgCl, were 1 and 2.5 mM, respectively. The distribution of 35S- labeled eIF-4E was the same in both cases. Reaction mixtures were incubated at 37 “C for 30 min, diluted with a solution containing 10 mM Tris-HCl, pH 7.5, 3 mM MgClz, 85 mM KCl, and 10 pM GMP- PNP and analyzed on linear 4.5 ml lo-34% sucrose gradients as described by Hiremath et al. (1989).

Previous studies indicated that the major phosphorylation site of eIF-4E was Ser-53 (Rychlik et al., 1987b). In order to study the role of this phosphrylation, the site was changed from serine to alanine by site-directed mutagenesis. Direct sequencing of DNA showed that nucleotides A’57 and G’58 of plasmid pTCEEC (Hiremath et al., 1989) had been changed to G’57 and C15’, respectively, in plasmid pTCALA’.

Isoelectric Focusing (IEF) of “‘S-labeled eIF-4ESer and eIF-4EA’“- The products of in vitro translation reactions or fractions from an affinity column or sucrose gradients were precipitated with 10 vol- umes of 80% acetone at -20 “C. The samples were analyzed by one of two methods. In the first, vertical tube IEF, the samples were solubilized in 2% Nonidet P-40, 8.0 M urea, and 2% ampholytes (pH 3.5-10) and separated on vertical tube IEF gels prepared with 8% polyacrylamide (30:0.8 of acrylamide:bisacrylamide), 9.0 M urea, 2% Nonidet P-40, 0.1% TEMED, 0.02% ammonium persulfate and con- taining 2% ampholytes, pH 3.5-10. The cathode solution was 10 mM ethanolamine (degassed), and the anode solution was 10 mM phos- phoric acid. After prefocusing (30 min at 100 V, 30 min at 200 V, 1 h at 400 V), the samples were applied to the cathode end of the gels and focused for 40 h at 400 V. The gels were subjected to fluorography using Fluoro-Hance (Research Products International Corp., Mt. Prospect, IL) according to the protocol recommended by the manu- facturer and exposed to Kodak XAR film. In the second method, horizontal slab IEF, the acetone-precipitated samples were solubilized in a buffer containing 8 M urea, 100 mM dithiothreitol, and 1% sodium decyl sulfate and separated on a 0.3-mm horizontal slab isoelectric focusing gel (Multiphor II system, Pharmacia) prepared with 5% polyacrylamide (24:l of acrylamide:bisacrylamide), 8.0 M urea, 0.5% ampholytes, pH 3-10, 0.5% ampholytes, pH 5-8, 2.4% ampholytes, pH 4-6.5, 0.04% TEMED, and 0.04% ammonium persulfate. The cathode solution was 1 M NaOH, and the anode solution was 1 M phosphoric acid. After prefocusing (30 min at 5 watts/cm’ of cross- sectional area), the samples were applied at the cathode end and focused for 3 h at 17 watts/cm*. The gels were dried and exposed to Kodak XAR film. The autoradiograms obtained in both IEF methods were quantitated using an LKB Ultroscan densitometer.

Both plasmids pTCEEC and pTCALA are transcription templates for T7 RNA polymerase (Hiremath et al., 1989). In vitro transcription yielded the same amount of transcript from each plasmid (20 rg of RNA/pg of DNA). When translated in a reticulocyte lysate system, the two transcripts produced the same amount of trichloroacetic acid-precipitable radioactiv- ity. In both cases, the predominant translation products were polypeptides of 25 kDa which comigrated on SDS-PAGE with eIF-4E. These were designated eIF-4ES” for the original plas- mid pTCEEC and eIF-4E*” for the mutated plasmid pTCALA.

eIF-4E synthesized in a reticulocyte lysate system under- goes a time-dependent phosphorylation (Hiremath et al., 1989). To characterize the phosphorylation state of eIF-4E*‘“, the cell-free translation product was analyzed by IEF and compared with eIF-4ES”. Surprisingly, two major spots were observed for eIF-4E*‘“, with isoelectric points in the p1 range of 5.8-6.4 (Fig. 1, lane 2, the double spots are an artifact due to the gel splitting during drying). A similar pattern was observed for eIF-4ES”’ (lane 1). In order to determine whether the charged species comprising eIF-4E*‘” had precisely the same p1 values as those of eIF-4ES”‘, the two radiolabeled proteins were mixed together and subjected to IEF (Fig. 1, lane 3). The appearance of the same two major spots indicated that the pIs were indistinguishable by this technique.

Several possible explanations for this unexpected finding can be put forth: 1) eIF-4E*‘” becomes phosphorylated, but at a second site; 2) a different post-translational modification, like N-acetylation, is responsible for the two forms of eIF-

Phosphutuse Treatment-Cell-free translation reactions contain- * Nucleotide residue numbers refer to the cDNA sequence of eIF- ing ?‘S-labeled eIF-4ES” and eIF-4E*‘” were treated with calf intestine 4E (Rychlik et al., 1987a).

by guest on April 16, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Role of eIF-4E Phosphorylation 2981

4EA’“; and 3) the earlier assignment of the major phosphoryl- ation site (Rychlik et al., 1987b) is in error.

With regard to the second possibility, earlier attempts in our laboratory to sequence the NH2 terminus of eIF-4E proved unsuccessful, suggesting that the protein was N-acetylated. If only partial N-acetylation of eIF-4EA’” occurs in the reticulo- cyte lysate system, the lower spot in Fig. 1, lane 2, could conceivably be the acetylated species. In order to explore this possibility, translation reactions were carried out in the pres- ence of citrate synthase and oxaloacetate, conditions which reduce the degree of N-acetylation by lowering the acetyl- CoA concentration (Palmiter, 1977). Translation of the pTCEEC transcript under these conditions (Fig. 1, lane 4) resulted in the appearance of two new spots, accounting for about 65% of the total. The remaining 35% consisted of two spots which comigrated with the phosphorylated and non- phosphorylated forms seen in the control reaction (lane 1). This comigration was confirmed by mixing the translation reactions carried out with (lane 4) and without (lane 1) citrate synthase and oxaloacetate and subjecting the mixture to IEF (lane 5). Similar experiments were carried out with the tran- script of pTCALA. In the presence of oxaloacetate and citrate synthase, two new spots appeared (lane 6). Mixing of these reaction products with those of lane 2 demonstrated that the new spots were shifted to more basic p1 compared with the original two (lane 7), suggesting that they were derived from the original two. When the results with the pTCEEC and pTCALA are taken together, they demonstrate that the two spots seen in lane 2 do not represent N-acetylated and nona- cetylated forms of the same protein. The most probable as- signment of structures is given on the left in Fig. 1.

To deal with the third possibility, that Ser-53 was not the major phosphorylation site, we obtained a chromatographic standard consisting of the tryptic phosphopeptide which would be expected if Ser-53 were the site of phosphorylation, namely phosphoseryl-lysine. eIF-4E was labeled in uiuo in HeLa cells using [32P]orthophosphoric acid in the culture medium as described previously (Rychlik et al., 198713). The 32P-labeled protein was purified by affinity chromatography followed by high pressure liquid chromatography (Rychlik et al., 1986), digested with trypsin, and the tryptic peptides resolved by thin layer electrophoresis (Rychlik et al., 1987b). The comigration of most of the radioactivity with the standard dipeptide confirmed that Ser-53 was the major phosphoryla- tion site (data not shown).

Elimination of possibilities 2 and 3 suggested that eIF- 4E A’a was indeed undergoing phosphorylation at a second site. Further evidence for this conclusion was obtained by treating eIF-4EA’” (and eIF-4ES”) with alkaline phosphatase. Examination of the products by horizontal slab IEF revealed the disappearance of the more acidic form of the protein in both cases (data not shown).

A minor phosphorylation might be expected to occur at a slower rate. To examine the kinetics of phosphorylation, translation reactions were incubated for 30 min, and further incorporation of radioactivity was prevented by addition of nonradioactive methionine. Aliquots were taken at various times of incubation and analyzed on IEF gels (Fig. 2). With no additional incubation beyond the initial 30 min, both eIF- 4ES” and eIF-4EA’” consisted of approximately 40% of the acidic species, which was earlier shown to be the phosphoryl- ated form of the protein (Rychlik et al., 1986). With longer incubation times, increasing amounts of the phosphorylated form appeared in the case of eIF-4ES”, reaching about 75% by 150 min. By contrast, eIF-4EA’” remained mostly unphos- phorylated until 90 min of incubation, after which phos-

FIG. 2. Kinetics of phosphorylation of eIF-4ES” and eIF- 4EALA. Cell-free protein synthesis reactions (250 ~1 each) pro- grammed with either pTCEEC or pTCALA transcripts were incu- bated for 30 min. Nonradioactive methionine was added to a final concentration of 20 mM, and aliquots were taken directly or after additional incubations of 30, 90, 120, and 150 min. Reactions were stopped by acetone precipitation, and samples were subjected to IEF as described under “Experimental Procedures.” The phosphorylated form of each protein, expressed as percent of total eIF-4E, is plotted against time of incubation.

200 -

92.5-

69 - -

46 -

elF-4EAla elF-4ESe’

FIG. 3. m’GTP-Sepharose affinity chromatography of eIF- 4ES”’ and eIF-4EA’“. 35S-Labeled eIF-4ES” and eIF-4EA’” were syn- thesized in uitro and passed over an m’GTP-Sepharose affinity col- umn as described under “Experimental Procedures.” Equivalent ali- quots of each column fraction were acetone-precipitated, solubilized, and resolved on 10% SDS-PAGE. The gel was subjected to fluorog- raphy, dried, and exposed on x-ray film. Total represents the material in a translation reaction before affinity chromatography. The Un- bound fraction contains the material not, retained on the column. Wash 1 is the wash with nucleotides, and Wash 2 is the wash without nucleotides. The material retained on the column and specifically eluted with m7GTP is marked Eluute.

phorylation increased. Thus eIF-4EA’” was phosphorylated considerably more slowly than eIF-4ES”, supporting the ar- gument that the former protein undergoes phosphorylation at a secondary site.

Given the correlation between phosphorylation of eIF-4E and the elevated rate of protein synthesis (see the Introduc- tion), one might predict that eIF-4EA’” would bind with lower affinity to caps. Accordingly, translation reactions pro- grammed with the transcript of pTCEEC or pTCALA were passed over m7GTP-Sepharose (Fig. 3). Most of the eIF-4EA’” was retained on the column and was specifically eluted with m7GTP (lane labeled Elude), similar to eIF-4ES”. Based on

by guest on April 16, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Role of eIF-4E Phosphorylation

L OTOP

\ \ ,+\ elF-4ESer

10 20 BOTTOM

FRACTION NUMBER

FIG. 4. Localization of eIF-4ES”’ and eIF-4E*‘” on initiation complexes. %-Labeled eIF-4ES” and eIF-4E*‘” were purified by affinity chromatography and added to translation reactions pro- grammed with globin mRNA and containing, in some cases, GMP- PNP. The samples were then analyzed by sucrose density gradient sedimentation and the radioactivity in the fractions determined. The positions of the ribosomal subunits and monosomes are indicated by &rows. Top panel, translation reaction with ‘%-labeled eIF-4ES” in the absence (Cl) and presence (A) of GMP-PNP. Bottom panel, translation reaction with ‘?S-labeled eIF-4E*‘” in the absence (0) and presence (A) of GMP-PNP.

this criterion, cap binding was not altered by the Ala-53 substitution.

Finally, the effect of altering the major phosphorylation site of eIF-4E on mRNA binding and transfer to ribosomes was tested. Previously we demonstrated that both eIF-4E and mRNA bind to the 43 S initiation complex to form the 48 S initiation complex (Hiremath et al., 1989). In that study, identification of 35S-labeled eIF-4E on 48 S initiation complex required ultracentrifugal separation followed by SDS-PAGE and fluorography. The electrophoretic separation was neces- sary because unincorporated [35S]methionine in the transla- tion reaction mixtures was nonspecifically carried into the 48 S region, obscuring detection of eIF-4E. In the present study, we streamlined this technology by first purifying 35S-labeled eIF-4ES”’ and eIF-4E*‘” on m7GTP-Sepharose. Since all radio- activity was now confined to eIF-4E (see Fig. 3), it was necessary only to measure the radioactivity of the sucrose gradient fractions directly to determine the distribution of the protein.

The distribution of eIF-4ES”’ and eIF-4E*‘” in translation reactions programmed with globin mRNA is shown in Fig. 4. GMP-PNP was used to increase the concentration of the 48 S complexes. In the absence of GMP-PNP, both eIF-4ES”’ and eIF-4E*‘” remained at the top of the gradient, with some trailing into the heavier fractions in the case of eIF-4ES”. In the presence of GMP-PNP, eIF-4ES”’ was observed in the 48 S region. A corresponding peak did not appear, however, with eIF-4E*‘“. This result was obtained in four separate experi-

ments with different batches of transcripts and eIF-4E*‘“. The most straightforward interpretation of these results is that eIF-4E must be phosphorylated in order to bind to the 43 S initiation complex, carrying with it the mRNA.

A prediction of this hypothesis is that the eIF-4E found in the 48 S initiation complex should be predominantly the phosphorylated species. This was tested by isolating the 48 S peak of radioactivity from sucrose gradients similar to those of Fig. 4 and examining the eIF-4E by vertical tube IEF. It can be seen in Fig. 1, lane 8, that the eIF-4E found in 48 S complexes was, in fact, predominantly the phosphorylated species.

DISCUSSION

The initial studies on structure of eIF-4E from human erythrocytes revealed at least five species separable by IEF (Rychlik et al., 1986). In rapidly growing cells (HeLa, reticu- locyte) two of these forms were found to be predominant (Rychlik et al., 1986; Buckley and Ehrenfeld, 1986; Duncan et al., 1987), and it was speculated that the other forms resulted from deamidation during the aging of erythrocytes. Nonethe- less, minor forms were present in rapidly growing cells, albeit in smaller amounts, and the fact that two of them, having pIs of 6.1 and 5.7, could be labeled with “P (Rychlik et al., 1986) argued against the deamidation hypothesis. Furthermore, eIF- 4E synthesized in uitro contained major as well as minor forms separable by IEF (Hiremath et al., 1989). In that study, the time course of appearance of the more acidic forms sug- gested two phosphorylation events: the ~15.9 species (major, putatively monophosphorylated) appeared with the same ki- netics as the p1 5.7 species (minor, putatively diphosphoryl- ated). We also observe, in the present study, minor forms which suggest multiple phosphorylations of eIF-4E (e.g. Fig. 1). Similarly, tryptic digestion of 32P-labeled rabbit reticulo- cyte eIF-4E produced both a major and minor radioactive peptide (Rychlik et al., 1987b). The major peptide was shown to be phosphoseryl-lysine, originating from residues 53 and 54 of eIF-4E, but the minor peptide was not characterized due to small quantities. All of these results suggest that eIF-4E is multiply phosphorylated.

In the present study we have confirmed, through the use of a synthetic dipeptide standard, that the major site of phos- phorylation in eIF-4E is Ser-53. Thus, the acidic species formed with time in reticulocyte lysates synthesizing eIF- 4EA1” (Fig. 1, lane 2) could not have arisen from phosphoryl- ation of the major site since eIF-4E*” lacks this site. None- theless, the phosphatase experiment indicated that the acidic species of eIF-4E*‘” was a phosphoprotein. Consequently, it must have resulted from phosphorylation of one of the minor sites mentioned above. It is interesting that preventing phos- phorylation at the major site appears to allow a greater extent of phosphorylation at the minor site, albeit at a slower rate.

The observation that eIF-4E lacking phosphate at position 53 fails to become incorporated into the 48 S initiation com- plex (Fig. 4) provides a plausible resolution of the apparent dilemma that eIF-4E phosphorylation was closely correlated with increased initiation rate in a variety of in uiuo systems, but no difference in affinity of phosphorylated and unphos- phorylated eIF-4E for caps could be demonstrated (see the Introduction). It should be noted that the reciprocal obser- vation, that 48 S initiation complexes contain essentially only the phosphorylated form (Fig. 1, lane B), provides independent verification of this phenomenon: the eIF-4E*‘” variant was not used in this experiment, so any hypothetical effects of increased phosphorylation at a minor site or altered tertiary

by guest on April 16, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Role of eIF-4E Phosphorylation 2983

structure caused by an alanyl residue at position 53 can be ruled out.

The precise mechanism by which mRNA is recruited to ribosomes is not known. One potential mechanism is that eIF-4E, as a free protein, binds the mRNA cap and then binds, carrying with it the mRNA, to the 43 S initiation complex already containing ~220 and other eIF-4 group ini- tiation factors. Another is that eIF-4E is present in a complex with eIF-4A and ~220 (termed eIF-4F) when it first encoun- ters mRNA. A third is that the eIF-4F complex is already bound to the 43 S initiation complex and that mRNA binds directly to this assembly. Previously, we have made arguments favoring the first of these (Hiremath et al., 1989). However, the implications of finding that only phosphorylated eIF-4E is present on 48 S initiation complexes can be accommodated as a regulatory mechanism into any of the three models. The implications of such a mechanism are different for each model and present testable hypotheses which should aid in deline- ating the steps of initiation.

Acknowledgments-We are indebted to Drs. Kortylewicz and Gal- ardy for synthesis of phosphoseryl-lysine, Carrie Rinker-Schaeffer for growing HeLa cells, and Jana De Benedetti for assistance with preparing the manuscript. Also, we would like to thank Bhavesh Joshi for helpful suggestions regarding experimental design.

REFERENCES Bonneau, A-M., and Sonenberg, N. (1987) J. Biol. Chem. 262,11134-

11139 Buckley, B., and Ehrenfeld, E. (1986) Virology 152, 497-501 Duncan, R., Milburn, S. C., and Hershey, J. W. B. (1987) J. Biol.

Chen. 262.380-388 Hiremath, L. S., Hiremath, S. T., Rychlik, W., Joshi, S., Domier, L.

L.. and Rhoads. R. E. (19891 J. Biol. C&m. 264, 1132-1138 Kaspar, R. L., Rychlik, W., White, M. W., Rhoads, k. E., and Morris,

D. R. (1990) J. Biol. Chem. 265, in press Kunkel, T. A., Roberts, J. D., and Zakour, R. A. (1987) M&hods

Enzymol. 154,367-382 Laemmli, U. K. (1970) Nature 227,680-685 Morley, S. J., and Traugh, J. A. (1988) J. Cell Biol. 107, 109a Morley, S. J., and Traugh, J. A. (1989) J. Biol. Chem. 264, 2401-

2404 Palmiter, R. D. (1977) J. Biol. Chem. 252,8781-8783 Rhoads, R. E. (1988) Trends Biochem. Sci. 13, 52-56 Rychlik, W., Gardner, P. R., Vanaman, T. C., and Rhoads, R. E.

(1986) J. Biol. Chem. 261, 71-75 Rychlik, W., Domier, L. L., Gardner, P. R., Hellmann, G. M., and

Rhoads, R. E. (1987a) Proc. Natl. Acad. Sci. U. S. A. 84,945-949 Rychlik, W., Russ, M. A., and Rhoads, R. E. (198713) J. Biol. Chem.

262,10434-10437 Sonenbere, N. (1988) Prog. Nucleic Acid Rex Mol. Biol. 35. 173-207 Waltz, K.;and Lopo, A. C: (1987) in Translational Control (Mathews,

M., Hershey, J., and Safer, B., eds) p. 157, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

Webb, N. R., Chari, R. V. J., DePillis, G., Kozarich, J. W., and Rhoads, R. E. (1984) Biochemistry 23, 177-181

by guest on April 16, 2018

http://ww

w.jbc.org/

Dow

nloaded from

S Joshi-Barve, W Rychlik and R E Rhoadsinitiation factor 4E prevents its association with the 48 S initiation complex.Alteration of the major phosphorylation site of eukaryotic protein synthesis

1990, 265:2979-2983.J. Biol. Chem. 

  http://www.jbc.org/content/265/5/2979Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/265/5/2979.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on April 16, 2018

http://ww

w.jbc.org/

Dow

nloaded from