©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Phosphorylation of Eukaryotic Protein Synthesis Initiation Factor 4E by Insulin-stimulated Protamine Kinase (*)

Anthony Makkinje (§) , Haishan Xiong (§) , Mei Li , Zahi Damuni (¶)

From the (1)Department of Cellular and Molecular Physiology, The Pennsylvania State University, College of Medicine, Milton S. Hershey Medical Center, Hershey, Pennsylvania 17033

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Insulin-stimulated protamine kinase (cPK) and protein kinase C (PKC) phosphorylated eukaryotic protein synthesis initiation factor 4E (eIF-4E) on serine and threonine residues located on an identical tryptic fragment as judged by two-dimensional phosphopeptide mapping. With cPK and PKC, the apparent K for eIF-4E was about 1.2 and 50 µM, respectively. Relative to recombinant human eIF-4E, cPK exhibited about 100% and 5% activity with eIF-4E and eIF-4E, respectively, and eIF-4E was phosphorylated exclusively on threonines. Bovine kidney eIF-4E enhanced up to 1.8-fold globin synthesis in mGTP-Sepharose-treated reticulocyte lysates. In contrast, following incubation with cPK, these eIF-4E preparations stimulated globin synthesis up to 6-fold. Compared to the dephosphorylation of the cPK-modified serine on eIF-4E, reticulocyte lysates and highly purified protein phosphatase 2A exhibited marked preference for the cPK-modified threonine. The results indicate that cPK phosphorylates eIF-4E on Ser and Thr, that the hydroxyl group or phosphorylation of Thr is necessary for cPK to act on Ser, and that Ser phosphorylation activates reticulocyte globin synthesis. The results suggest that cPK could contribute to the insulin-stimulated phosphorylation of eIF-4E, but that protein phosphatase 2A may confer the site specificity of this response.


INTRODUCTION

Evidence has accumulated that the capped-mRNA binding protein, eukaryotic protein synthesis initiation factor 4E (eIF-4E),()undergoes phosphorylation on serines coincident with the stimulation of protein synthesis in response to insulin (1, 2, 3) and numerous other extracellular stimuli (4-14). Conversely, dephosphorylation of eIF-4E correlates with decreased rates of protein synthesis (e.g. 15, 16). However, to date, a direct effect of eIF-4E phosphorylation on protein synthesis has not been established. In addition, the protein kinases and phosphatases responsible for controlling this phosphorylation have not been identified. Nonetheless, two-dimensional phosphopeptide mapping and phosphoamino acid analysis indicated that PKC phosphorylates eIF-4E at the site(s) modified in response to insulin(1) . The exact location of the modified site(s) was not determined. However, based on an earlier report(17) , which appears to be in error,()it was suggested that eIF-4E may be phosphorylated on Ser in response to insulin(1) . In addition, because insulin-stimulated phosphorylation of eIF-4E was abolished following prolonged incubation with phorbol esters(1) , a treatment which inactivated PKC(1) , it was suggested that this kinase may contribute to the insulin-stimulated phosphorylation of eIF-4E(1) . However, in cells, eIF-4E is present largely in the soluble fraction (18), and PKC appeared to be a reasonable eIF-4E kinase only when eIF-4E was purified from ribosomes(19) . From this fraction, eIF-4E was isolated as part of a complex termed eIF-4 which also contains eIF-4 of apparent M 46,000 and eIF-4 of apparent M 220,000(18) .

We have purified to apparent homogeneity an insulin-stimulated cytosolic Protamine Kinase (cPK) which was differentiated from other protein kinases including known PKC isoforms based on its catalytic, regulatory, chromatographic, and immunological properties(20, 21, 22) . In addition, in a preliminary report we showed that up to 1 mol of phosphoryl groups was incorporated per mol of purified preparations of eIF-4E following incubation with cPK (23). By contrast, all the other insulin-stimulated protein kinases examined displayed little or no activity with eIF-4E(23, 24, 25) , raising the possibility that cPK may contribute to the insulin-stimulated phosphorylation of eIF-4E(23) . However, neither the functional significance nor location of the cPK-catalyzed phosphorylation of eIF-4E were examined.

In this paper, we present evidence that cPK and PKC phosphorylate eIF-4E at an identical tryptic peptide. By mutational analysis, we show that cPK phosphorylates eIF-4E at Ser and Thr, that phosphorylation at Ser activates eIF-4E, and that PP2A, a major cytoplasmic protein serine threonine phosphatase(26) , preferentially dephosphorylates Thr. The results are consistent with the idea that cPK contributes to the insulin-stimulated phosphorylation of eIF-4E and concomitant activation of protein synthesis, but indicate an important role for PP2A in this insulin response.


EXPERIMENTAL PROCEDURES

Materials

N-Tosyl-L-phenylalanine chloromethyl ketone-treated trypsin and Na-p-Tosyl-L-lysine chloromethyl ketone-treated -chymotrypsin were from Sigma. DH10B Escherichia coli containing cDNA encoding human eIF-4E in expression vector pMW19 (27) was provided by Dr. Rosemary Jagus, Center Marine Biotechnology, University of Maryland. TAQuence DNA sequencing kit was from United States Biochemicals. Rat brain PKC and mGTP-Sepharose were from Calbiochem and Pharmacia/LKB, respectively. Globin mRNA was from Life Technologies, Inc. Haemin-containing nuclease-treated rabbit reticulocyte lysates were from Promega. [S]Methionine (1100 Ci/mmol) was from Amersham Corp. Sources of other materials are given elsewhere(28, 29, 30) .

Enzyme Preparations

Myelin basic protein(29) , PP2A(28) , and cPK (20) were purified to apparent homogeneity, and the activities of cPK(20) , PKC(20) , and PP2A(29) were determined as described. Protein was determined as described(31) . Homogeneity of enzyme preparations was determined by SDS-PAGE(32) . Protein bands were detected with Coomassie Brilliant Blue.

Purification of eIF-4E and eIF-4 to near homogeneity from bovine kidney extracts was as described(23, 33) , except that gel permeation chromatography on Superose 12 was performed instead of ion-exchange chromatography on Mono Q. Following SDS-PAGE, fractions containing eIF-4E were identified by Coomassie Blue staining, pooled, and concentrated and washed with 50 mM Tris-chloride, pH 7.0, containing 10% glycerol, 0.1 mM EDTA, 0.1 mM phenylmethanesulfonyl fluoride, 1 mM benzamidine, and 14 mM -mercaptoethanol, using Centricon-10 microconcentrators (Amicon). The preparations were aliquoted and stored at -70 °C.

Phosphorylation of eIF-4E

The incubations (0.05 ml) were performed at 30 °C in 50 mM Tris-chloride, pH 7.0, containing 10% glycerol, 1 mM benzamidine, 0.1 mM phenylmethanesulfonyl fluoride, 14 mM -mercaptoethanol, 0.01 units of cPK, 2 mM MgCl, 0.2 mM [-P]ATP (1000 cpm/pmol), and 4 µg of eIF-4E. Incubations with PKC also contained 20 µg/ml phosphatidylserine and 0.25 mM CaCl. Reactions were initiated with ATP and MgCl. Control incubations were performed in the absence of cPK, PKC, or eIF-4E. Reactions were terminated with sample buffer(32) , and the mixtures were heated at 100 °C for 5 min. Following SDS-PAGE, proteins were electrophoretically transferred onto Immobilon-P transfer membranes. Membrane strips were stained with Ponceau S, washed extensively with HO, dried, and exposed to Kodak X-Omat AR-5 film. In some experiments, radiolabeled bands identified by autoradiography were cut out from membrane strips, immersed in scintillant, and counted.

Peptide Mapping and Phosphoamino Acid Analysis

Two-dimensional phosphopeptide mapping was performed essentially as described(1) . Briefly, P-labeled eIF-4E (4 µg) prepared by incubation with cPK or PKC was subjected to SDS-PAGE and then electrophoretically transferred onto Immobilon-P membranes. Membrane strips were stained with Ponceau S, washed extensively with HO, and dried. Bands corresponding to eIF-4E were excised and incubated at 37 °C for 16 h with trypsin (50:1 w/w) in 0.05 ml of 0.2 M ammonium bicarbonate, pH 8.4. An additional aliquot of trypsin was added, and the mixture was incubated for a further 6 h. About 80-90% of the P-label was released following incubation with trypsin. The solution was then dried under vacuum, redissolved in a solution containing 10% pyridine and 0.4% acetic acid, spotted onto a thin layer chromatography plate, and subjected to electrophoresis in the same solution at 600 V for 1.5 h, and then to ascending chromatography in a solution containing 60% n-butanol and 20% acetic acid. After drying, the plates were exposed to x-ray film. Phosphoamino acid analysis was performed at pH 3.5 as described(33) . Phosphoserine, phosphothreonine, and phosphotyrosine standards were identified by staining with ninhydrin.

Construction and Expression of eIF-4E and eIF-4E

Expression vector pMW19 containing cDNA encoding human eIF-4E (27) was used as template to change to Ala the codons for Ser and Thr by inverse polymerase chain reaction site-directed mutagenesis(35) . Mutagenic primers used to independently introduce these changes in the sense strand of eIF-4E cDNA were pGCGGCGCTACCACTAAAAATAGG (Ala) and pGCGGCTCCGCTACTAAAAATAGG (Ala) (changed nucleotides are underlined.) A single primer, pTCTTAGTAGCTGTGTCTGCGTGG, was used for antisense strand amplification. The reaction mixtures (0.1 ml) contained 100 pmol each of sense and antisense primer, 10 fmol of pMW19 DNA in 20 mM Tris-chloride, pH 8.7, 10 mM KCl, 10 mM ammonium sulfate, 2 mM MgCl, 0.1% Triton X-100, 0.1 mg/ml bovine serum albumin, 200 µM of each dNTP, and 2.5 units of Pfu DNA polymerase (Strategene). The mixtures were subjected to an initial 3 min denaturation at 94 °C, followed by 30 cycles of denaturation at 94 °C (1 min), annealing at 50 °C (1 min), and extension at 72 °C (12 min). Reaction products with relative electrophoretic motility of about 6.2 kilobase pairs were excised from 1.8% low melting agarose gels and recovered using glass milk (GeneClean II, BIO 101). Approximately 200 ng of this DNA was incubated overnight at 12 °C in 0.01 ml of 20 mM Tris-chloride, pH 7.6, containing, 1 mM ATP, 5 mM MgCl, 5 mM dithiothreitol, 50 µg/ml bovine serum albumin, and 4 units of T4 DNA ligase (New England Biolabs) as recommended(36) . The mixture was then used to transform One ShotE. coli (Invitrogen), and plasmids were isolated and analyzed by restriction mapping and by sequencing both cDNA strands to verify for mutations and the integrity of the remaining coding region. Plasmids containing wild type and mutant cDNA were then separately used to transform BL21(DE3)pLysS E. coli (Novagen), and expression was induced as described(27) . Purification of recombinant wild type and mutant proteins was also as described(27) , except that, chromatography on mGTP-Sepharose was omitted because >90% of the eIF-4E preparations did not bind to this resin and are therefore considered inactive in capped mRNA binding.

Reticulocyte Protein Synthesis

Reticulocyte lysates (0.1 ml of 90 mg/ml) were subjected to chromatography on a 1-ml column of mGTP-Sepharose equilibrated and developed in 50 mM HEPES-KOH, pH 7.6, containing 0.1 M KCl, 10% glycerol, 0.1 mM phenylmethanesulfonyl fluoride, 1 mM benzamidine, and 14 mM -mercaptoethanol. Fractions (0.2 ml) were collected and those exhibiting the highest absorbance at 280 nm employed. Protein synthesis in these fractions was then measured with globin mRNA and [S]methionine essentially as recommended by the manufacturer (Promega). Reactions contained 0.015 ml of the mGTP-Sepharose-treated lysates, 0.004 ml of HO, 0.001 ml of RNasin inhibitor (40 units), 0.001 ml of 1 mM amino acid mixture lacking methionine, 0.001 ml of a 25 µg/ml solution of globin mRNA, 0.002 ml of [S]methionine (final concentration 500 µCi/ml), and a 0.005-ml aliquot of highly purified bovine kidney eIF-4E as indicated. After 5 min at 30 °C, 0.25 ml of a solution containing 1 N NaOH and 2% HO was added. After 10 min at 37 °C, 1 ml of 25% trichloroacetic acid (w/v) was added, and the mixture was centrifuged at 12,000 g for 5 min in a microcentrifuge. The supernatant was discarded, and pellets were washed five times with 1 ml of 10% trichloroacetic. To the final pellets was added 1 ml of scintillation fluid, and the radioactivity was determined. Control incubations were performed in the absence of globin mRNA. Chromatography on mGTP-Sepharose reduced the rate of reticulocyte lysate globin synthesis by about 7.2-fold.


RESULTS AND DISCUSSION

Phosphopeptide Mapping

As a first step to examine the sites modified on eIF-4E by cPK, and to relate the results to insulin action, we examined the tryptic phosphopeptides derived from P-labeled eIF-4E prepared by incubation with cPK or PKC. The results presented in Fig. 1show that a single co-migrating tryptic phosphopeptide was obtained from recombinant human eIF-4E incubated with either cPK or PKC. Identical results also were obtained with bovine kidney eIF-4E, and when eIF-4E was isolated as part of eIF-4 from this tissue source (not shown). Phosphoamino acid analysis showed that both cPK (Fig. 2) and PKC (Fig. 3) phosphorylated eIF-4E on serines and threonines. Assuming that a single serine and threonine residue was modified, we estimate that cPK incorporated up to 0.45 and 0.55 mol, and PKC incorporated up to 0.04 and 0.06 mol of phosphoryl groups on serine and threonine residues, respectively. Little or no difference was detected in the rates of phosphorylation of these residues by cPK (Fig. 2) or PKC (not shown).


Figure 1: Phosphopeptide mapping. Recombinant human eIF-4E was incubated for 30 min with cPK (1 unit/ml) or PKC (22 units/ml) in the presence of [-P]ATP, and the mixtures were electrophoretically transferred onto Immobilon-P membranes as described under ``Experimental Procedures.'' Membrane strips were exposed to x-ray film (10 min with cPK, 3 days with PKC), and bands corresponding to eIF-4E were cut out, treated with trypsin, and two-dimensional phosphopeptide mapping was performed as described under ``Experimental Procedures.'' The figure shows the autoradiograms of the dried thin layer chromatography plates indicating the pattern derived from the eIF-4E incubations with cPK (panel A), PKC (panel B), and a mixture from each of the incubations (panel C). The arrows denote the position the samples were applied to the plates. The radioactivity applied to the plates was panel A, 6,000 cpm, panel B, 3,400 cpm, and panel C, 5,200 cpm made up of 2,600 cpm from the incubation with cPK and 2,600 cpm from the incubation with PKC.




Figure 2: Phosphorylation of eIF-4E by cPK. Recombinant human eIF-4E was incubated with cPK in the presence of [-P]ATP, and, at the indicated times, reactions were terminated, and electrophoretically transferred onto Immobilon-P membranes as described under ``Experimental Procedures.'' Bands corresponding to eIF-4E were cut out, incubated with trypsin, and phosphoamino acid analysis was performed as described under ``Experimental Procedures.'' After drying, the thin layer plate was exposed to x-ray film. The figure shows an autoradiogram of the dried plate. The position of phosphoserine (S), phosphothreonine (T), and phosphotyrosine (Y) standards is indicated. Control incubations from which eIF-4E or cPK was omitted displayed little or no phosphorylation (not shown).




Figure 3: Phosphorylation site analysis of eIF-4E. Recombinant human eIF-4E (lane 1), eIF-4E (lane 2), and eIF-4E (lane 3) each at 4 µg were incubated with 1 unit/ml cPK (panels A and C) or 25 units/ml PKC (panels B and D) in the presence of [-P]ATP for 30 min, and the mixtures were then electrophoretically transferred onto Immobilon-P membranes as described under ``Experimental Procedures.'' The membrane strips were exposed for 10 min (panel A) and 3 days (panel B) to x-ray film. An autoradiogram of each membrane is shown. The position of eIF-4E is denoted by the arrow. Control incubations from which cPK or PKC were omitted displayed little or no phosphorylation (not shown). Control incubations from which eIF-4E, eIF-4E, and eIF-4E were omitted from the reactions with cPK showed little or no phosphorylation. In panel B, the band corresponding to autophosphorylated PKC is denoted by an arrow. Autophosphorylation of PKC was also noted in incubations from which eIF-4E, eIF-4E, and eIF-4E were omitted were omitted (not shown). In panels C and D, the eIF-4E bands were subjected to phosphoamino acid analysis after trypsinolysis as described under ``Experimental Procedures.'' The autoradiograms of the dried thin layer plates are shown. The position of phosphoserine (S), phosphothreonine (T), and phosphotyrosine (Y) standards is indicated.



Purified preparations of cPK displayed an apparent K of about 1.4 and 1.2 µM with recombinant human and bovine kidney eIF-4E, respectively. Similarly, with the eIF-4E component of bovine kidney eIF-4, cPK exhibited an apparent K of about 1.0 µM, and up to 1 mol of phosphoryl groups was incorporated per mol of the eIF-4E component of eIF-4 following incubation with cPK indicating that eIF-4 and eIF-4, the other subunits of eIF-4, may have little or no effect on cPK activity with eIF-4E.

By contrast to cPK, PKC displayed an apparent K of about 40 and 52 µM with recombinant human and bovine kidney eIF-4E, respectively. In addition, consistent with earlier observations(19, 23, 37, 38) , PKC incorporated up to 0.1 mol of phosphoryl groups/mol of these eIF-4E preparations. Highly purified PKC also displayed an apparent K of about 29 µM with the eIF-4E component of bovine kidney eIF-4. With eIF-4 as substrate, PKC incorporated up to 0.3 mol of phosphoryl groups/mol of the eIF-4E component. These observations appear to be at variance with an earlier study which indicated that 1 mol of phosphoryl groups could be incorporated per mol of the eIF-4E component of eIF-4(19) . The reason for this apparent discrepancy is unclear.

Identification of Target Sites

Because there was little or no difference in the tryptic phosphopeptide patterns (Fig. 1) and kinetics of phosphorylation, the results indicated that information on the sites modified by cPK on native eIF-4E could be derived from the recombinant eIF-4E preparations even though these preparations appeared to be largely inactive in capped mRNA binding. In addition, because only one tryptic phosphopeptide was derived from the eIF-4E preparations incubated with either cPK or PKC, the results suggested that the modified serines and threonines (Fig. 1) were located on the same tryptic fragment. Examination of the deduced amino acid sequence suggested that this tryptic fragment may be SGSTTK located at the C terminus of eIF-4E, residues 207-212. To test this idea, eIF-4E cDNA in which Ala replaced Ser or Thr (SGSTTK, underlined residues) was prepared, and each cDNA was expressed separately in BL21(DE3)pLysS E. coli. Mutant proteins were then purified as described(27) , and the activities of cPK and PKC with these proteins examined and compared to wild type eIF-4E.

The results presented in Fig. 3A show that cPK phosphorylated eIF-4E to an extent similar to wild type eIF-4E, but that, relative to these preparations, the kinase exhibited 5% activity with eIF-4E. The purified preparations of cPK displayed an apparent K of about 1.2 µM with eIF-4E, also similar to that observed with wild type eIF-4E. However, in contrast to wild type eIF-4E, phosphoamino acid analysis showed that cPK phosphorylated eIF-4E exclusively on threonines (Fig. 3C). Together, these results indicate that Ser and Thr are target sites for cPK. They also indicate that either the hydroxyl group or phosphorylation of Thr may be required for cPK to phosphorylate Ser. Arguing against the latter possibility is that there were no discernable differences in the rates that cPK phosphorylated these residues on wild type eIF-4E (Fig. 2).

Interestingly, relative to wild type eIF-4E, threonine phosphorylation of eIF-4E was enhanced by about 1.8-fold (Fig. 3C). Because with wild type eIF-4E, cPK incorporated up to 0.45 mol and 0.55 mol of phosphoryl groups/mol of the modified serine and threonine residues, respectively, these observations suggest that phosphorylation of eIF-4E on Ser may inhibit Thr phosphorylation and vice versa. However, the exact location of the threonine(s) modified by cPK on eIF-4E remains to be determined. Consistent with the possibility that cPK phosphorylated eIF-4E in the tryptic fragment SGATTK containing Thr (underlined residue) is that two-dimensional peptide mapping of P-labeled eIF-4E showed a single tryptic phosphopeptide which comigrated with the one derived from P-labeled wild type eIF-4E (not shown, cf. Fig. 1). However, in this tryptic fragment, cPK could also have phosphorylated eIF-4E on Thr.

In contrast to cPK, PKC phosphorylated eIF-4E and eIF-4E to approximately 50% of the level observed with wild type eIF-4E (Fig. 3B). The highly purified PKC preparations displayed an apparent K of about 70 and 80 µM with eIF-4E and eIF-4E, respectively. These values are about 1.8-fold higher than those displayed by PKC with wild type eIF-4E. Phosphoamino acid analysis showed that PKC phosphorylated eIF-4E on threonines and eIF-4E on serines and threonines (Fig. 3D). By contrast to cPK (Fig. 3C), threonine phosphorylation of eIF-4E by PKC was equivalent to that observed with wild type eIF-4E (Fig. 3D). These results indicate that, like cPK, PKC acts on Ser, but that, unlike cPK, PKC phosphorylated a threonine other than Thr. The location of this threonine, which does not appear to affect PKC activity with Ser, remains to be determined.

Effect of eIF-4E Phosphorylation on Protein Synthesis

As a first step to assess the functional consequence of the cPK-catalyzed phosphorylation of eIF-4E, bovine kidney eIF-4E was incubated with and without cPK for various times. An aliquot of the incubations was then added to reticulocyte lysates treated with mGTP-Sepharose to deplete them of endogenous eIF-4E, and the initial rate of globin mRNA translation was measured by the incorporation in 5 min of [S]methionine into protein as described under ``Experimental Procedures.''

Addition of bovine kidney eIF-4E to the mGTP-Sepharose-treated lysates enhanced the rate of globin synthesis by about 1.8-fold (not shown). In contrast, following incubation with cPK, these eIF-4E preparations enhanced the rate of globin synthesis up to 6-fold (Fig. 4A). Control incubations, from which eIF-4E was omitted, showed that cPK (0.075 units) displayed little effect, if any, on the initial rate of globin synthesis (not shown). However, at 30-fold higher concentrations of cPK, globin synthesis was markedly inhibited (not shown). The molecular basis of this cPK effect remains to be determined.


Figure 4: Effect of eIF-4E on protein synthesis. In panel A, highly purified bovine kidney eIF-4E was incubated with 0.2 mM ATP instead of [-P]ATP in the absence () or presence () of cPK (30 units/ml) as described under ``Experimental Procedures.'' At the indicated times, EDTA to a final concentration of 4 mM was added. A 0.0025-ml aliquot of the mixtures (5 µg of eIF-4E) was then added to mGTP-Sepharose-treated reticulocyte lysates, and globin synthesis was measured after 5 min as described under ``Experimental Procedures.'' In panel B, parallel incubations of eIF-4E with cPK were performed in the presence of 0.2 mM [-P]ATP. After 30 min at 30 °C, a 0.005-ml aliquot of 30 mM EDTA was added, and a 0.005-ml sample of the mixture was then incubated with the reticulocyte lysates as described above. At the indicated times, reactions were terminated, and phosphoamino acid analysis was performed after electrophoretic transfer onto ImmobilonP membrane strips and trypsinolysis as described under ``Experimental Procedures.'' P-Labeled serine () and P-labeled threonine () residues were identified by autoradiography and comigration with ninhydrin-staining phosphoserine and phosphothreonine standards, and then scraped from the thin layer chromatography plate into a microcentrifuge tube. The radioactivity was determined after addition of 1 ml of scintillant. The figure shows the percent of P-label remaining at the indicated time points.



The dephosphorylation of eIF-4E by reticulocyte lysates was then examined. Phosphoamino acid analysis showed that, by contrast to the cPK-modified serine (t 15 min), the cPK-modified threonine was dephosphorylated rapidly (t 1 min) by the lysates (Fig. 4B). Because the rate of reticulocyte lysate globin synthesis was linear during the 5-min period of the measurements in the absence or presence of added eIF-4E, eIF-4E that had been incubated with cPK up to 30 min, or 0.075 units of cPK (not shown), the results indicate that phosphorylation of eIF-4E on Ser activated globin synthesis, and that Thr phosphorylation may have had little, or no, effect.

Dephosphorylation by PP2A

Earlier studies indicated that PP2A could dephosphorylate eIF-4E(38, 39) . Therefore, we examined whether this phosphatase exhibited preference for the cPK-modified serine and threonine residues. The results presented in Fig. 5show that PP2A preferentially dephosphorylated the cPK-modified threonine. The rate of this dephosphorylation was about 60-fold higher than the rate of Ser dephosphorylation. However, whether PP2A is a physiologically relevant eIF-4E Thr phosphatase remains to be determined. In this connection, it is pertinent that only in cells incubated with okadaic acid has the phosphorylation of eIF-4E on both serines and threonines been observed(40) . However, okadaic acid is not only a potent inhibitor of PP2A(26) , but also of protein phosphatase 1 (26) and protein phosphatase X(41) . Whether these protein phosphatases, and/or the recently described eIF-4E-binding protein, PHAS-1(42) , also termed 4E-BP1(43) , regulate the phosphorylation of eIF-4E remains to be determined. Phosphorylation of PHAS-1 by mitogen-activated protein kinase in response to insulin releases eIF-4E from the eIF-4EPHAS-1 complex present in the soluble fraction, and increases the levels of eIF-4E available to participate in protein synthesis(42, 43) .


Figure 5: Dephosphorylation of eIF-4E by PP2A. Recombinant human eIF-4E was incubated with cPK (1 unit/ml) in the presence of [-P]ATP as described under ``Experimental Procedures.'' After 60 min, a 0.005-ml aliquot was incubated at 30 °C in a final volume of 0.025 ml in the presence of PP2A (50 units/ml) in 50 mM Tris-chloride, pH 7.0, containing 10% glycerol, 1 mM benzamidine, 0.1 mM phenylmethanesulfonyl fluoride, and 14 mM -mercaptoethanol. At the indicated times, reactions were stopped, and then electrophoretically transferred onto Immobilon-P membranes as described under ``Experimental Procedures.'' Panel A shows an autoradiogram of the membrane strips. The arrow denotes the bands corresponding to eIF-4E. These bands were cut out, incubated with trypsin, and phosphoamino acid analysis was performed as described under ``Experimental Procedures.'' Panel B shows an autoradiogram of the dried plate. The position of phosphoserine (S), phosphothreonine (T), and phosphotyrosine (Y) standards is indicated. Control incubations from which PP2A was omitted showed little or no dephosphorylation of the eIF-4E preparations (not shown).




CONCLUSION

Using a two-dimensional phosphopeptide mapping procedure identical to the one employed in this study, evidence was obtained earlier that PKC modifies eIF-4E at the serine residue phosphorylated in insulin-treated 3T3-L1 cells(1) . The results presented herein indicate that cPK phosphorylates eIF-4E at the serine residue modified by PKC ( Fig. 1and Fig. 3). In addition, the mutational analyses described in this paper indicate that this residue is Ser (Fig. 3). Thus, eIF-4E appears to be phosphorylated on Ser in insulin-treated cells. The results indicate that cPK, an insulin-stimulated enzyme(21) , may contribute directly to this phosphorylation, but that PKC is unlikely to do so because PKC is a relatively poor eIF-4E kinase.

The results presented also indicate that phosphorylation of Ser enhances the activity of eIF-4E in globin synthesis in reticulocyte lysates depleted of endogenous eIF-4E (Fig. 4A). They also establish that, whereas cPK phosphorylates serine and threonine residues on eIF-4E at equivalent rates (Fig. 2), reticulocyte lysates (Fig. 4B) and purified PP2A (Fig. 5) dephosphorylate the modified threonine preferentially. These observations suggest that PP2A may play an important role in controlling the site specificity of the insulin-stimulated phosphorylation of eIF-4E.

Earlier studies suggest that phosphorylation may alter the affinity of eIF-4E for capped mRNA(44, 45) . Alternatively, phosphorylation may trigger and/or stabilize the association of eIF-4E with the other subunits of eIF-4(4, 40) . The phosphorylation site mutant cDNAs described in this paper should facilitate studies on the functional significance of the insulin-stimulated phosphorylation of eIF-4E and provide further information on the role of cPK in this regulation.


FOOTNOTES

*
This work was supported in part by Grant MCB-9019882 from the National Science Foundation. 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.

§
The first two authors contributed equally to this study.

To whom correspondence and reprint requests should be addressed. Tel.: 717-531-4195; Fax: 717-531-7667.

The abbreviations used are: eIF-4E, eukaryotic protein synthesis initiation factor 4E; cPK, cytosolic protamine kinase; PKC, protein kinase C; PP2A, protein phosphatase 2A; PP2A, protein phosphatase 2A; PAGE, polyacrylamide gel electrophoresis; cpm, counts/min.

R. E. Rhoads, personal communication.


ACKNOWLEDGEMENTS

We are most grateful to Dr. Rosemary Jagus for the human eIF-4E cDNA. We also thank Dr. Leonard S. Jefferson for helpful comments.


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