Phosphorylation of Eukaryotic Initiation Factor (eIF) 4E Is Not Required for de Novo Protein Synthesis following Recovery from Hypertonic Stress in Human Kidney Cells*

Previous work has suggested that increased phosphorylation of eukaryotic initiation factor (eIF) 4E at Ser-209 in the C-terminal loop of the protein often correlates with increased translation rates. However, the functional consequences of phosphorylation have remained contentious with our understanding of the role of eIF4E phosphorylation in translational control far from complete. To investigate the role for eIF4E phosphorylation in de novo translation, we studied the recovery of human kidney cells from hypertonic stress. Results show that hypertonic shock caused a rapid inhibition of protein synthesis and the disaggregation of polysomes. These changes were associated with the dephosphorylation of eIF4G, eIF4E, 4E-binding protein 1 (4E-BP1), and ribosomal protein S6. In addition, decreased levels of the eIF4F complex and increased association of 4E-BP1 with eIF4E were observed over a similar time course. The return of cells to isotonic medium rapidly promoted the phosphorylation of these initiation factors, increased levels of eIF4F complexes, promoted polysome assembly, and increased rates of translation. However, by using a cell-permeable, specific inhibitor of eIF4E kinase, Mnk1 (CGP57380), we show that de novoinitiation of translation and eIF4F complex assembly during this recovery phase did not require eIF4E phosphorylation.

Previous work has suggested that increased phosphorylation of eukaryotic initiation factor (eIF) 4E at Ser-209 in the C-terminal loop of the protein often correlates with increased translation rates. However, the functional consequences of phosphorylation have remained contentious with our understanding of the role of eIF4E phosphorylation in translational control far from complete. To investigate the role for eIF4E phosphorylation in de novo translation, we studied the recovery of human kidney cells from hypertonic stress. Results show that hypertonic shock caused a rapid inhibition of protein synthesis and the disaggregation of polysomes. These changes were associated with the dephosphorylation of eIF4G, eIF4E, 4E-binding protein 1 (4E-BP1), and ribosomal protein S6. In addition, decreased levels of the eIF4F complex and increased association of 4E-BP1 with eIF4E were observed over a similar time course. The return of cells to isotonic medium rapidly promoted the phosphorylation of these initiation factors, increased levels of eIF4F complexes, promoted polysome assembly, and increased rates of translation. However, by using a cell-permeable, specific inhibitor of eIF4E kinase, Mnk1 (CGP57380), we show that de novo initiation of translation and eIF4F complex assembly during this recovery phase did not require eIF4E phosphorylation.
Nuclear-encoded mRNAs have a unique cap structure at their 5Ј terminus with general composition m 7 G(5Ј)ppp(5Ј)N (where N is any nucleotide). The presence of this cap structure has a strong stimulatory effect on the translation of mRNA, facilitating the recruitment of translation initiation factors (eIFs) 1 to allow ribosome binding and initiation at the correct start site (for review, see Refs. [1][2][3][4][5]. eIF4E, a protein whose three-dimensional structure resembles a cupped hand, specifically interacts directly with the cap via its concave surface (6,7). It also forms a complex with the scaffold protein eIF4G (8) on its convex surface. eIF4G in turn recruits other initiation factors, such as eIF3, eIF4A, and poly(A)-binding protein (PABP) to the 5Ј end of the mRNA, allowing for efficient un-winding of secondary structure in the 5Ј untranslated region (9) and the functional circularization of mRNA believed to be necessary to promote efficient translation (2,4,5,10,11). In addition, eIF4E binds a family of regulatory proteins, 4E-binding proteins (4E-BPs), which compete with eIF4G for sites on the convex surface of eIF4E (12,13) and inhibit formation of initiation complexes (14). Association of 4E-BPs with eIF4E is modulated by phosphorylation events controlled via the mammalian target of rapamycin signaling pathway (3,5,15,16).
While phosphorylation of eIF4E has been reported to increase its interaction with the cap structure (34), recent studies have suggested that our understanding of the role of eIF4E phosphorylation in translational control is far from complete (5). First, increased phosphorylation of eIF4E observed during cell stress is sometimes associated with a global inhibition of protein synthesis (5,21,22,25). In addition, phosphorylation of eIF4E is not required for restoration of translation in an eIF4Edependent system in vitro, and both wild-type and phosphorylation site variants are able to rescue the lethal phenotype of eIF4E deletion in Saccharomyces cerevisiae (35). Also it appears that phosphorylation of eIF4E is not a prerequisite for mitogen-induced cap-dependent translation in cultured cells (28). Furthermore, in adult cardiocytes, eIF4E phosphorylation had no effect on eIF4F complex formation or total rates of protein synthesis (36). However, eIF4E phosphorylation does appear to be important for cell growth in Drosophila; transgenic Drosophila expressing a non-phosphorylatable form of eIF4E in a null background show reduced viability, smaller cell size, and developmental delays (37). Despite this, no strong phenotype was observed by expressing a form of eIF4E that mimicked eIF4E phosphorylation (37). Biophysical studies by Scheper et al. (33) have demonstrated that phosphorylation of eIF4E actually reduces its binding to mRNA caps by promoting its rate of dissociation. These data are consistent with an earlier prediction (19) that, with analogy to the transcriptional machinery, phosphorylation may be required for the release of eIF4F from the cap complex during the initiation process (33). Alternatively, phosphorylation of eIF4E may be involved in reprogramming (de novo) translation by promoting the rate of release of factors from existing initiation complexes to give under-represented mRNAs a chance to compete for ribosome binding (5,19,33).
Exposure of mammalian cells to hypertonic stress is a very effective means of inducing a transient fall in protein synthesis, which is rapidly reversible upon restoration of isotonic medium (38 -40). This system has been used as a tool to study the function of the eIF4F complex during de novo recruitment of mRNA in intact cells (41). In this study we used hypertonic stress to investigate the role for eIF4E phosphorylation in de novo translation using a cell-permeable inhibitor of Mnk, CGP57380 (28), to prevent the rephosphorylation of eIF4E during recovery from such stress.
Cell Culture-Human 293 kidney cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. When used, CGP57380 or Me 2 SO alone was added to cultures and was present in all subsequent incubations as indicated.
Protein Synthesis Measurements-Cells were cultured in six-well plates, and [ 35 S]methionine was added to the complete growth medium at the specific activity and for the times indicated in the individual figure legends. Cells were harvested, and the incorporation of methionine into protein was determined by trichloroacetic acid precipitation as described previously (22).
Preparation of Cell Extracts-Following treatment, cells were scraped into PBS containing 40 mM ␤-glycerophosphate and 2 mM benzamidine and isolated in a cooled microfuge. Cell pellets were resuspended in 200 l of ice-cold Buffer A (50 mM Mops-KOH, pH 7.2, 2.5 mM EGTA, 1 mM EDTA, 40 mM sodium fluoride, 80 mM KCl, 7 mM 2-mercaptoethanol, 2 mM benzamidine, 0.1 mM GTP, 2 mM Na 3 VO 4 ) and lysed by the addition of 0.5% (by volume) each of Igepal and deoxycholate and vortexing. Cell debris and nuclei were removed by centrifugation in a microcentrifuge for 5 min at 4°C, and the resultant supernatants were frozen in liquid N 2 .
SDS-PAGE, Isoelectric Focusing, and Immunoblotting-Samples containing equal amounts of protein were resolved by SDS-PAGE and processed as described previously (21,22). Antiserum specific for the C-terminal domain of eIF4GI and those specific to eIF4E, eIF2␣, and phospho-eIF2␣ were as described previously (22); total levels of eIF4G, eIF4E, eIF2␣, PABP, eIF4A, and 4E-BP1 were visualized with alkaline phosphatase-conjugated secondary antibodies, while the determinations using commercial phosphospecific antisera and those for p38 MAP kinase used enhanced chemiluminescence for detection. In all cases, care was taken to ensure that detection was within the linear range of the response. One-dimensional vertical isoelectric focusing gels used for the analysis of eIF4E phosphorylation were performed as described previously (21,22).
Isolation of eIF4E and Associated Factors by m 7 GTP-Sepharose Chromatography-eIF4E and associated proteins were isolated by  3 and 7), or 20 M (lanes 4 and 8) CGP57380 for 60 min before the addition of PBS (lanes 1-4) or 10% fetal calf serum (lanes 5-8) for 60 min. Extracts were subjected to m 7 GTP-Sepharose chromatography, and eIF4E and associated factors were resolved by SDS-PAGE and visualized by immunoblotting. Results are representative of those obtained in three separate experiments. FCS, fetal calf serum. m 7 GTP-Sepharose chromatography as described previously (21,22), and recovered proteins were visualized by immunoblotting.

RESULTS AND DISCUSSION
CGP57380 Prevents the Serum-stimulated Phosphorylation of eIF4E without Affecting eIF4G Phosphorylation or Levels of the eIF4F Complex-In general, previous work has suggested that increased phosphorylation of eIF4E at Ser-209 in the C-terminal loop of the protein correlates with increased translation rates (1,5,19,23,42). These studies have prompted the view that eIF4E phosphorylation may have a role in modulating the cap binding activity of the protein, invoking elaborate models based on biochemical studies and three-dimensional structure analysis (2, 6, 7). However, the functional consequences of phosphorylation have remained elusive, and several exceptions have called into question the correlation with global rates of protein synthesis (for review, see Ref. 5). It has been suggested that phosphorylation of eIF4E, rather than modulating global protein synthesis, may be involved in reprogramming translation (5,19), possibly by promoting the rate of release of factors from existing initiation complexes to give underrepresented mRNAs a chance to compete for the 40 S ribosome (33). Such a mechanism could resolve apparently conflicting observations of increased phosphorylation of eIF4E following either mitogenic stimulation or the application of cell stress.
Recently a novel, non-toxic, specific inhibitor of Mnk1 (CGP57380) has been described that prevents eIF4E phosphorylation in response to mitogens (28). In our study we have used CGP57380 to investigate the role of eIF4E phosphorylation in de novo initiation of translation and eIF4F complex assembly. Fig. 1A shows that CGP57380 had no significant effect on the serum-stimulated up-regulation of translation rates in starved 293 cells. In addition, SDS-PAGE analysis of initiation factors shows that CGP57380 did not affect the serum-stimulated phosphorylation of eIF4G (Fig. 1B, lanes 1 and 6 versus lanes 5 and 10; quantified in Fig. 1C). However, although eIF4E phosphorylation was normally increased in response to serum (Fig. 1B, lane 1 versus 6), consistent with published data (28), both the basal level and the serum-stimulated phosphorylation of eIF4E were reduced by CGP57380 at concentrations greater than 10 -15 M. Isoelectric focusing and immunoblotting for eIF4E confirmed the dephosphorylation of eIF4E in response to CGP57380 (Fig. 1B); quantification of these data is shown in Fig. 1C. Fig. 1D shows that although CGP57380 could reduce eIF4F levels to a small extent in starved cells (lane 4 versus lane 1), treatment of cells with up to 20 M CGP57380 had no effect on the serum-stimulated recovery of eIF4G, PABP, or eIF4A associated with eIF4E (lane 8 versus lane 4). This finding is consistent with the observation that association of 4E-BP1 with eIF4E was unaffected by CGP57380 treatment alone (data not shown).

Exposure of Human Kidney Cells to Hypertonic Medium Results in the Dephosphorylation of Key Translation Initiation
Factors-To determine conditions for the investigation of the role of eIF4E phosphorylation in de novo initiation events, human kidney cells were incubated in the presence of NaCl in excess to that already present in the growth medium. Fig. 2 shows that 50 -200 mM added NaCl resulted in a concentration-(A) and time-dependent (B) inhibition of protein synthesis. To address whether hypertonic shock is associated with changes in the phosphorylation status of key initiation factors, extracts were prepared, and proteins were visualized by immunoblotting with phosphospecific antisera. Fig. 2C shows that dephosphorylation of a key phosphorylation site in 4E-BP1 (Ser-65) was complete within 15 min, while that of eIF4G (Ser-1108, Refs. 3 and 43) and S6 (Thr-421/Ser-424) occurred at a slower rate with no change in overall levels of initiation factors recovered in the extracts. eIF4E dephosphorylation (Ser-209) was evident at 15 min using a phosphospecific antibody for detection although hypertonic stress activates p38 MAP kinase, an upstream regulator of the physiological eIF4E kinase, Mnk1 (Refs. 26 and 29, and see Fig. 2D). However, isoelectric focusing of eIF4E showed that salt treatment for 60 min was required to reduce levels of the phosphorylated protein below 5% (Fig. 2B). In agreement with published data for HeLa cells (40), over the times used in this study there was little change in the phosphorylation status of eIF2␣ in response to hypertonic stress (Fig. 2C). We also investigated the effects of hypertonic shock on the eIF4F complex. Isolation of eIF4E and associated factors with m 7 GTP-Sepharose (Fig. 2E) indicated that hypertonic shock caused a dissociation of eIF4G, PABP, and eIF4A from eIF4E, indicative of a decrease in eIF4F complex levels. As predicted from accepted models (2,3,5), the dissociation of eIF4G from eIF4E occurred concomitantly with an increase in binding of the less phosphorylated forms of 4E-BP1 to eIF4E.
De Novo Initiation of Protein Synthesis Does Not Require Increases in the Level of eIF4E Phosphorylation-Using the conditions determined above, we investigated the requirement for eIF4E phosphorylation in de novo protein synthesis following recovery from hypertonic shock, a process requiring the integrity of the eIF4F complex (41). Incubation of cells with 200 mM NaCl for 60 min resulted in an inhibition of the rate of protein synthesis (Fig. 3A) and disaggregation of polysomes (Fig. 3B). Together these events are characteristic of a defect in the initiation stage of translation, a finding confirmed by the observation that polysomes were stabilized by the inclusion of cycloheximide in the medium during salt treatment (data not shown). On return to isotonic medium, protein synthesis rates recovered to control levels within 30 -45 min (Fig. 3A) and polysomes reformed (Fig. 3B). In the presence of 20 M CGP57380, polysomes reformed on return to isotonic conditions, but there was a distinct shift toward smaller polysomes, suggesting that eIF4E phosphorylation may have a subtle role in the initiation of translation. Further work will be required to address this possibility.
Consistent with the data presented in Fig. 2, hypertonic shock for 60 min caused a decrease in the phosphorylation of eIF4G, eIF4E, 4E-BP1, and S6, an increase in the phosphoryl- ation of p38 MAP kinase (Fig. 2C, lane 2 versus lane 1), and a decrease in the recovery of eIF4G associated with eIF4E. Conversely, there was an increase in association of 4E-BP1 with eIF4E following salt stress (Fig. 2D, lane 2 versus lane 1). Return of the cells to isotonic medium promoted a time-dependent phosphorylation of total eIF4G, which occurred with similar kinetics in the absence or presence of CGP57380 (Fig. 3C). Similarly, recovery was also associated with phosphorylation of 4E-BP1 on Ser-65 as visualized with the phosphospecific antiserum and a characteristic mobility shift of the total 4E-BP1 on SDS-PAGE using antiserum that recognizes the protein irrespective of its phosphorylation status (Fig. 3C). Furthermore, these events and the dephosphorylation (and inactivation) of p38 MAP kinase were seen to occur with essentially the same kinetics in the absence (Fig. 3C, lanes 3-6) or presence of CGP57380 (lanes 9 -12). In contrast, although the phosphorylation of total eIF4E increased from very low levels in the absence of inhibitor on return to isotonic conditions (Fig. 3C,  lanes 3-6), CGP57380 completely prevented this increase (lanes 9 -12). These data were confirmed with isoelectric focusing/immunoblotting analysis of eIF4E (data not shown) and were not due to changes in the levels of eIF4E recovered in the extracts (Figs. 3C). The phosphorylation of S6 lagged behind that of eIF4E and 4E-BP1 and the increase in the rate of protein synthesis during the recovery period. These data are in agreement with previous findings with MPC11 cells in showing that changes in S6 phosphorylation are too slow to account for rapid changes in protein synthesis following tonicity shifts (39).
We also examined the phosphorylation status of the eIF4F complex during recovery from salt stress. Following the return of cells to isotonic conditions, the assembly of the eIF4F complex occurred rapidly both in the absence and presence of CGP57380. In each instance, this was associated with the release of 4E-BP1 from eIF4E and enhanced binding of eIF4G to eIF4E (Fig. 3D). The increase in levels of eIF4F complex was confirmed by the finding that there was enhanced association of PABP and eIF4A with eIF4E under such conditions (data not shown). The phosphorylation of the population of eIF4G recovered in the eIF4F complex also increased with similar kinetics to that observed for the total eIF4G in the absence or presence of CGP57380 (Fig. 3D). In contrast, the phosphorylation of eIF4E was completely prevented by the presence of CGP57380 during the recovery period (Fig. 3D, lanes 9 -12). Therefore, although the integrity of eIF4G is critical for the reprogramming (de novo) translation during recovery of cells from hypertonic stress (41), eIF4E phosphorylation was not a prerequisite for increased rates of de novo translation or enhanced levels of eIF4F complexes. Further work will be needed to resolve the role of eIF4E phosphorylation in growth control in cultured cells.