phosphorylation of translation initiation factor eif-4e is induced in

MOLECULAR AND CELLULAR BIOLOGY, Mar. 1992, p. 1239-12470270-7306/92/031239-09$02.00/0Copyright © 1992, American Society for Microbiology

Phosphorylation of Translation Initiation Factor eIF-4E Is Inducedin a ras-Dependent Manner during Nerve Growth

Factor-Mediated PC12 Cell DifferentiationROBERT M. FREDERICKSON,1'2 WALTER E. MUSHYNSKI,1 AND NAHUM SONENBERGl.2*

Department ofBiochemistry' and McGill Cancer Centre, 2 McGill University,Montreal, Quebec, Canada H3G 1Y6

Received 29 August 1991/Accepted 15 December 1991

Translation initiation factor eIF-4E, which binds to the 5' cap structure of eukaryotic mRNAs, is believed toplay an important role in the control of cell growth. Consistent with this, overexpression ofeIF4E in fibroblastsresults in their malignant transformation. The activity of eIF-4E is thought to be regulated by phosphorylationon a single serine residue (Ser-53). Treatment of rat pheochromocytoma (PC12) cells with nerve growth factor(NGF) strongly curtails their growth and causes their differentiation into cells that resemble sympatheticneurons. The present study shows that eIF-4E is rapidly phosphorylated in PC12 cells upon NGF treatment,resulting in a significant increase in the steady-state levels of the phosphorylated protein. In contrast, epidermalgrowth factor, a factor which elicits a weak mitogenic response in PC12 cells, did not significantly enhanceeIF-4E phosphorylation. We also show that although the mitogen and tumor promoter, phorbol 12-myristate-13-acetate, is able to induce phosphorylation of eIF-4E in PC12 cells, the NGF-mediated increase is primarilya protein kinase C-independent response. The NGF-induced enhancement of eiF4E phosphorylation isabrogated in PC12 cells expressing a dominant inhibitory ras mutant (Ser-17 replaced by Asn), indicating thateIF-4E phosphorylation is dependent on a ras signalling pathway. As phosphorylation of eIF-4E effectstranslation initiation, these results suggest that NGF-mediated and ras-dependent eIF-4E phosphorylation mayplay a role in switching the pattern of gene expression during the differentiation of PC12 cells.

Changes in translation rates play a significant role in thecontrol of cell growth (for a recent review, see reference 31).Enhanced protein synthesis is obligatory for entry into andprogression through the cell cycle (4, 5). Modulation of therate of protein synthesis also occurs during heat shock (14,51) and mitosis (17) and in response to cell growth modula-tors (60, 63). These changes are effected primarily at thelevel of initiation, which is rate-limiting under most circum-stances (34), and are thought to be governed by the phos-phorylation state of key initiation factors (for a review, seereference 30).

Regulation of the activity of eukaryotic initiation factoreIF-4F is believed to be an important means of controllingtranslation rates. eIF-4F is a three-subunit complex, whichbinds the 5' cap structure (m7GpppX, where X is anynucleotide) present on all eukaryotic cellular mRNAs (16,26, 62). eIF-4F is believed to facilitate ribosome binding tomRNA, in conjunction with eIF-4B, by unwinding the sec-ondary structure in the mRNA 5' noncoding region (54; forreviews, see references 53 and 58). The m7G cap-binding siteis contained within the 24-kDa subunit termed eIF-4E (59).eIF-4E is the limiting component of the 4F complex (15, 32)and is thus thought to be an important target for regulation ofmRNA binding to ribosomes (for reviews, see references 53and 58). The significance of the limiting nature of eIF-4E ismanifested most dramatically by its ability to transformprimary and established fibroblast cells (39, 40) and toperturb the growth of HeLa cells (11), when overexpressed.These findings clearly document the importance of eIF-4E inthe control of cell growth.eIF-4E contains one major site of phosphorylation, Ser-53

* Corresponding author.

(35, 55). The state of eIF-4E phosphorylation correlatesdirectly with its activity (45). Decreased phosphorylation ofeIF-4E is accompanied by a reduction in protein synthesisduring heat shock (14) and mitosis (2), while enhancedphosphorylation has been observed in a variety of cell linestreated with growth-promoting agents (6, 13, 18, 37, 43, 46)and in response to expression of tyrosine kinase oncogenes,such as src (18) or Ick (20). Mutation of Ser-53 to an Alaresidue prevents the association of eIF-4E with the 48Sinitiation complex (35) and blocks the ability of overex-pressed eIF-4E to transform fibroblast cells in culture (39),highlighting the importance of phosphorylation of Ser-53 forcontrol of cell growth.There are strong indications for an overlap in the growth

factor-mediated cellular signalling pathways controlling pro-liferation and differentiation (1, 3, 49). Tyrosine phosphory-lation, induced by binding of growth factors to specific cellsurface receptors, is believed to be a common initiatingsignal in these pathways. Enhanced phosphorylation andconcomitant activation of downstream serine/threonine pro-tein kinases are thought to further transmit both mitogenicand differentiation-inducing signals to relevant effector mol-ecules. Given the important roles played by eIF-4E and itsphosphorylation in the control of cellular proliferation, weexamined the possibility that regulation of eIF-4E phosphor-ylation accompanies the process of cellular differentiation.

Rat pheochromocytoma (PC12) cells provide a powerfultool for the analysis of growth factor-mediated cellulardifferentiation in vitro. PC12 cells are an established chro-maffin cell-like line which respond to exposure to nervegrowth factor (NGF) with a cessation of growth and differ-entiation into cells resembling sympathetic neurons (25).This process is accompanied by the extension of neuritesand by the induction of a variety of neuronal enzyme and

1239

Vol. 12, No. 3

on April 6, 2018 by guest

http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/

1240 FREDERICKSON ET AL.

protein markers (24, 25). In the present study, we show thatthere is a rapid increase in steady-state levels of phosphor-ylated eIF-4E upon exposure of PC12 cells to NGF. Inaddition, we demonstrate that this increase in eIF-4E phos-phorylation is mediated by a ras-dependent and proteinkinase C-independent signal transduction pathway(s).

MATERIALS AND METHODS

Cell culture. PC12 cells were obtained from the AmericanType Culture Collection (Rockville, Md.) and J. A. Wagner(Cornell University), and maintained in Dulbecco modifiedEagle medium (DMEM; GIBCO) containing 10% heat-inac-tivated horse serum (GIBCO), 5% fetal bovine serum(GIBCO), and antibiotics (41). The M7 and M-M17-26 PC12cell lines were kindly provided by G. M. Cooper (HarvardMedical School, Boston, Mass.) (61) and maintained asdescribed above, with the addition of 0.4 mg of G418 per ml.The BALB/c-MuSV-transformed cell line, 1274-8-1, wasderived from Rat 1 cells, by transfection with a plasmidcontaining the Ha-v-ras coding sequence (66; kindly pro-vided by Paul Jolicoeur, Montreal Clinical Research Insti-tute, Montreal, Quebec, Canada). These cells were main-tained in DMEM containing 10% fetal bovine serum andantibiotics (18).

Metabolic labeling of cells and immunoprecipitation ofeIF-4E. Cells were harvested bv trypsinization and plated atdensities of 1 x 106 to 2 x 10 , on 60-mm-diameter culturedishes (Nunc) coated with rat tail collagen (PC12 cells only).Cells were incubated with 1.5 ml of either 32Pi (0.33 mCi/ml;NEN) in phosphate-free DMEM (Flow Laboratories), or[35S]methionine (0.17 mCi/ml; NEN) in methionine-freeDMEM (Flow) for 2 h. NGF (2.5S, 10-,ug/ml stock incomplete medium [Boehringer-Mannheim, Canada]), epider-mal growth factor (EGF) (100-,ug/ml stock in water [Boehr-inger-Mannheim]), and phorbol-12-myristate-13 acetate(PMA) (5-mg/ml stock in dimethyl sulfoxide [Pharmacia])were subsequently added for the times and concentrationsindicated in the figure legends. Cell lysates were prepared,and eIF-4E was immunoprecipitated with the rabbit anti-eIF-4E polyclonal antiserum, 5853, which recognizes bothrat and murine eIF-4E, as described previously (18). Im-mune complexes were collected and subjected to electropho-resis on sodium dodecyl sulfate-12.5% polyacrylamide gels(SDS-12.5% PAGs), which were dried prior to autoradiog-raphy or fluorography. Incorporation of 32Pi was quantifiedby excision of the bands corresponding to eIF-4E for Ceren-kov counting in a Beckman LS 3801 beta counter. Incorpo-ration of 32p or 35S into total cellular protein was monitoredby scintillation counting of trichloroacetic acid precipitates.

Analysis of ribosomal protein S6 phosphorylation. PC12cells were grown and 32p labeled as described above. Ribo-somal proteins were subsequently isolated as describedpreviously (12). Briefly, monolayers were washed twice with5 ml of ice-cold TMK buffer (10 mM Tris HCl, 10 mMMgCl2, 80 mM KCl, pH 7.4), and lysed in 700 ,ul of a solutioncontaining TMK buffer, 1% deoxycholate, 1% Triton X-100,plus phosphatase inhibitors as described previously (18).The extracts were centrifuged at 30,000 x g for 10 min, andsupernatants were layered on 4.5-ml cushions consisting of1.5 M sucrose in TMK buffer and ceintrifuged at 100,000 x gfor 4.5 h at 20C. Crude ribosomal pellets were rinsed twicewith TMK buffer, dissolved in sample buffer, and subjectedto electrophoresis through SDS-12.5% PAGs.

Two-dimensional gel electiophoresis. NGF-treated and un-treated PC12 cells were washed with 1 x phosphate-buffered

saline, pH 7.4, and lysed in 400 ,ul of urea lysis buffer (9.5 Murea, 2% Nonidet P-40, 1.6% pH 5-7 ampholines [Pharma-cia], 0.4% pH 3-10 ampholines, 5% 3-mercaptoethanol).Aliquots (50 ,u1) of each lysate were loaded immediately onisoelectric focusing tube gels (Bio-Rad) and subjected toelectrophoresis overnight, second-dimension electrophore-sis on SDS-12.5% PAGs, and transfer to nitrocellulose forimmunoblotting with eIF-4E antiserum and 1251I-labeled pro-tein A (Amersham) as described previously (39). eIF-4E wasvisualized by autoradiography.

Phosphopeptide analysis. PC12, Rat 1, and BALB/c-MuSV-transformed Rat 1 cells were plated at densities of 2x 106 cells per 60-mm-diameter plate and 32p labeled asdescribed above. Cell lysates were prepared, and 32P-labeledeIF-4E was immunoprecipitated and subjected to SDS-12.5% PAG analysis. Proteins were transferred to nitrocel-lulose, which was exposed to X-ray film to localize eIF-4E.Portions of the filter containing eIF-4E protein were excisedand subjected to trypsin digestion in situ by the procedure ofLuo et al. (42). Phosphopeptides were subjected to first-dimension electrophoresis and second-dimension chroma-tography as previously described (64) and visualized byautoradiography.

RESULTS

NGF induces eIF-4E phosphorylation in PC12 cells. Toexamine the effect of NGF on eIF-4E phosphorylation, PC12cells were equilibrated in 32p; and incubated with NGF for 30or 60 min. SDS-PAG electrophoresis analysis of eIF-4Eimmunoprecipitated from these cells revealed a significantincrease (four- and sixfold increase at 30 and 60 min,respectively) in incorporation of 32p, into eIF-4E in NGF-treated cells (Fig. 1A, compare lanes 2 and 4 [NGF-treated]with lanes 1 and 3 [untreated]). Quantitation of 32p; incorpo-ration into total cellular protein by trichloroacetic acidprecipitation revealed only a slight increase (up to 1.5-fold)in NGF-treated cells relative to that of untreated cells (datanot shown). Thus, the enhanced incorporation of radiolabelinto eIF-4E is specific and not simply due to stimulation byNGF of 32p; uptake by the cells. Furthermore, the increasedamount of phosphorylated eIF-4E in NGF-treated cellscannot be attributed to an elevation of levels of the protein,as metabolic labeling with [35S]methionine revealed nochange in the rate of eIF-4E synthesis under the conditionsused for the 32P radiolabeling experiment (Fig. 1B).

Steady-state level of phosphorylated eIF-4E is increased inNGF-treated PC12 cells. The increased incorporation of 32p;into eIF-4E in response to NGF might also be explained byan NGF-stimulated increase in phosphate turnover on eIF-4E. This would allow the specific activity of the eIF-4Ephosphate to approach that of the ATP pool more quickly inNGF-treated cells, without a significant change in the ratioof the phosphorylated and unphosphorylated isoforms. Toexamine this possibility directly, we performed two-dimen-sional gel electrophoresis on cell lysates from NGF-treatedversus untreated PC12 cells (Fig. 2). There was a significantshift toward the phosphorylated varian't, in NGF-treatedPC12 cells compared with untreated cells (Fig. 2, comparecontrol and NGF-treated cells). Phosphorylated and unphos-phorylated forms of eIF-4E are indicated by their isoelectricpoints (pls), 5.9 and 6.3, respectively. Quantitation of radio-activity associated with each isoform revealed ratios ofphosphorylated to unphosphorylated eIF-4E of 4:1 in NGF-treated cells and 1:1 in control PC12 cells. These resultsindicate that the increased incorporation of 32p; into eIF-4E

MOL. CELL. BIOL.


http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/

ras-DEPENDENT eIF-4E PHOSPHORYLATION IN PC12 CELLS 1241

A kDa 1 2 3

110

84 -

4

0l)(I)

47 -

33 >

IEF - a

v * Control

NGFtreated

24 -

NGF - + - +

B kDa 1 2

110 * -

84-

47- ii__

33-

4 45.9 6.3

FIG. 2. Isoelectric focusing analysis of eIF-4E in control andNGF-treated PC12 cells. PC12 cells were treated with 50 ng of NGFper ml for 1 hour, and two-dimensional immunoblot analysis wasperformed as described in Materials and Methods. Unphosphory-lated (pl 6.3) and phosphorylated (pl 5.9) isoforms of eIF-4E areindicated. IEF, isoelectric focusing.

incorporation of radiolabel into eIF-4E were already detect-able after 3.5 min (Fig. 3), and incorporation of radiolabelincreased linearly thereafter, reaching a maximum inductionof sixfold at 60 minutes (not shown). Thus, elevated phos-phorylation of eIF-4E is a relatively early response to NGFinduction of PC12 differentiation.

This rapid increase in the phosphorylation of eIF-4E ispresumably due to activation of an NGF-mediated cell

24 _ _ -l 4E

16-

NGF -

FIG. 1. Induction of eIF-4E phosphorylation in PC12 cells inresponse to NGF. Cells were treated with NGF (50 ng/ml) follow-ing labeling with 32p; or [35S]methionine, and eIF-4E was immuno-precipitated and analyzed on SDS-12.5% PAGs as described inMaterials and Methods. Volumes of lysates containing equivalenttrichloroacetic acid-precipitable counts were used for immunopre-cipitation. (A) 32p; labeling of eIF-4E. Cells were incubated with 32p;for 2 h and then treated with NGF for 30 (lane 2) or 60 (lane 4) min.Lanes 1 and 3, untreated cells. (B) Analysis of eIF-4E synthesisrates. Cells were labeled with [35S]methionine for 2 h and thentreated with NGF for 60 min. Migration of Bio-Rad low-molecular-size prestained protein standards are indicated at the left of eachautoradiograph.

in NGF-treated PC12 cells is due to an elevated steady-statelevel of the phosphorylated variant.

Phosphorylation of eIF-4E is an early response to NGFtreatment of PC12 cells. PC12 cells were treated for varioustimes with NGF, and phosphorylation of eIF-4E was ana-lyzed by immunoprecipitation. Specific changes in incorpo-ration of 32p; into eIF-4E in NGF-treated cells were calcu-lated, relative to incorporation in untreated cells for eachtime point, and plotted. Significant changes (60% increase) in

w

U-0

.0 Wo-0,0 0

D X

0,0

-6 >-

. -_ S

0

0C:0a

4

3

2

1

00 10 20

time of NGF treatment(minutes)

FIG. 3. Time course analysis of eIF-4E phosphorylation in PC12cells treated with 50 ng of NGF per ml. eIF-4E, immunoprecipitatedfrom 32Pi-labeled PC12 cells prepared as described in the legend toFig. 1A, was analyzed by SDS-12.5% PAGs. Incorporation ofradiolabel into eIF-4E at each time point was measured as describedin Materials and Methods. Fold incorporation was then calculatedrelative to untreated cells and plotted. Plotted data represent aver-age values from two separate experiments performed in duplicate.

VOL. 12, 1992

om --4E


http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/


AMOL. CELL. BIOL.

:-a

r~~~~~~~~~~~~~~~~~~~~~~~

e

O.

;~~~

o ;;.0

.4- i

:*

PM;tAi|ngi,-n_'..ve Or t.:4~~~~~~~~4 T;-

:o r c e .r r at i Q r

FIG. 4. NGF, EGF, and PMA dose-response analysis of eIF-4E phosphorylation. (A) 32Pi-labeled PC12 cells were treated with increasingconcentrations of NGF, EGF, or PMA, as described in Materials and Methods. Fold increase in incorporation of radiolabel was calculatedrelative to untreated cells as described in the legend to Fig. 3. Note the difference in scale of the abcissa for each agent; concentrations ofEGF and PMA used are obtained by multiplying the plotted values by 10. Plotted data represent average values from three separateexperiments, performed in duplicate. (B) EGF and NGF both induce phosphorylation of ribosomal protein S6 in PC12 cells. Isolation ofribosomal protein S6 from 32Pi-labeled PC12 cells and analysis on an SDS-12.5% PAG was performed as described in Materials and Methods.Cells were treated with EGF or NGF for 45 min prior to isolation of ribosomes.

signalling cascade. The NGF receptor kinase is the trkproto-oncogene, which is activated by tyrosine phosphory-lation upon binding the growth factor (36, 38). The phosphor-ylated receptor kinase then triggers activation of otherdownstream protein kinase cascades, such as those resultingin increased phosphorylation and activity of the mitogen-activated protein kinase (MAPK) (3, 22) and the 40S ribo-somal subunit protein, S6 (48). Similarly to NGF, EGFinduces tyrosine phosphorylation through a specific receptoron PC12 cells (33). However, EGF does not cause neuriteoutgrowth but rather exerts a mitogenic effect on PC12 cells.To determine whether the phosphorylation of eIF-4E wasspecific to induction of differentiation, we compared theeffects of the two growth factors on eIF-4E phosphorylationin a dose-response analysis. To this end, the experimentdescribed in the legend to Fig. 1A was repeated with a rangeof NGF and EGF concentrations. The levels of incorpora-tion of 32p; into eIF-4E for each concentration of NGF orEGF, relative to the incorporation in untreated cells, arecompared graphically in Fig. 4A. The results show a dose-dependent phosphorylation of eIF-4E in response to physi-ologically relevant levels of NGF, between 1 and 10 ng/ml(24). In contrast, EGF treatment did not result in a compa-rable increase in eIF-4E phosphorylation; there was only a50% increase at 500 ng of EGF per ml compared with sixfoldmaximal increase for NGF. EGF and NGF both inducemultiple phosphorylation of ribosomal protein S6 in PC12cells, though to different extents and with distinct kinetics(48). We also detect a significant increase in incorporation of32p; into ribosomal protein S6 in PC12 cells treated witheither NGF or EGF (Fig. 4B). This result demonstrates that

the lack of increased eIF-4E phosphorylation in response toEGF cannot be attributed to a defect in the EGF signallingpathway in the cells we used. Thus, in PC12 cells, significantincreases in eIF-4E phosphorylation occur only in responseto a differentiation-inducing growth factor.PMA induces eIF-4E phosphorylation in PC12 cells. PKC

has been implicated in mediating eIF-4E phosphorylation inNIH 3T3 and 3T3-L1 cells. PMA, a potent mitogen andactivator of PKC, has been shown to induce eIF-4E phos-phorylation in these fibroblasts (18, 45, 46). Furthermore,extended treatment of 3T3-L1 cells with high levels of PMAto down-regulate PKC abrogates the insulin-induced in-crease in eIF-4E phosphorylation, suggesting that a PKC-dependent signalling pathway mediates eIF-4E phosphory-lation in response to this growth factor in 3T3-L1 cells (46).We have examined the role of PKC-mediated pathways inNGF induction of eIF-4E phosphorylation in PC12 cells. Themaximum increase in eIF-4E phosphorylation due to expo-sure of cells to PMA is at least twofold lower than themaximum increase detected upon NGF treatment of the cells(Fig. 4A). Thus, phosphorylation of eIF-4E is also enhancedin PC12 cells in response to treatment with PMA; however,the increase is smaller than that observed upon treatmentwith NGF, even at very high concentrations of PMA. Theseresults demonstrate that PMA treatment can lead to in-creased eIF-4E phosphorylation in PC12 cells, presumablythrough a PKC-mediated phosphorylation cascade, yet leaveunresolved the role of this kinase in mediating the enhancedeIF-4E phosphorylation that accompanies PC12 cell differ-entiation.NGF induces eIF-4E phosphorylation in a manner indepen-


http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/


PMAcontrol pretreated

1 2 3 4 5 6

elF-4E_ _l

NGF: 0 5 50

<:LLc:

FIG. 5. Comparison of eIF-4E phosphorylation in control andPKC-down-regulated PC12 cells. PC12 cells were pretreated with 10pLM PMA for 18 h to down-regulate PKC. Cells were then labeled (2h) with 32p; alone (-) or in the presence of 1 p.M PMA or 50 ng ofNGF per ml for 45 min, as indicated under the lanes. eIF-4Eimmunoprecipitation and analysis on SDS-12.5% PAGs was done asdescribed in Materials and Methods.

dent of activation of PKC. To investigate more directly therole of PKC in mediating the NGF-induced enhancement ofeIF-4E phosphorylation in PC12 cells, we down-regulatedthe kinase by extended exposure of the cells to high levels ofPMA (10 p,M [65]). Long-term treatment of PC12 cells withPMA has been shown to result in loss of immunologicallydetectable PKC and 95% reduction of PKC activity in PC12cells (44). Subsequent exposure of PMA-down-regulatedcells to NGF resulted in an elevated level of eIF-4E phos-phorylation, which was similar in magnitude to that ob-served in cells which had not been previously exposed toPMA (Fig. 5, compare lane 3 [NGF-treated control cells]with lane 6 [NGF-treated down-regulated cells]). As ex-pected, long-term exposure to PMA abrogated the responseto subsequent PMA treatment (compare lane 5 [PMA-treateddown-regulated cells] with lane 2 [PMA-treated controlcells]), indicating that the PKC-dependent pathway(s) con-trolling eIF-4E phosphorylation was indeed down-regulated.Western blot (immunoblot) analyses using a-PKC antibodyM5C (Amersham) and a-PKC antiserum B (a kind gift of T.Hunter) revealed a loss of immunologically detectable PKC(a, 1, and -y isoforms), and a PKC-specific kinase assayverified the reduction of PKC activity (to 9%) in the down-regulated cells (data not shown). Thus, the NGF-inducedincrease in eIF-4E phosphorylation does not occur throughthe activation of a PKC-dependent signalling pathway(s).NGF-induced eIF4E phosphorylation occurs in a Ras-

dependent manner. It is conceivable that eIF-4E is a com-mon component of diverse cell signalling cascades, whichare activated in response to growth factors and mitogens,such as NGF and PMA, or tyrosine kinase oncogene expres-sion. Ras is a likely transducer of growth factor-mediatedsignals to the kinases and/or phosphatases regulating eIF-4Ephosphorylation, as the protein has been implicated in thesignal transduction pathways mediated by mitogen-activatedreceptor phosphotyrosine kinases, and tyrosine kinase on-cogenes such as src (56). Furthermore, expression of v-srcand transforming variants of c-src induce an increase in

0 5 50 ng/mI

FIG. 6. eIF-4E phosphorylation in response to NGF is ras de-pendent: analysis of eIF-4E phosphorylation in M7 (control) andM-M17-26 (expressing dominant negative mutant ras) PC12 celllines upon stimulation with NGF. Cells were treated for 45 min withthe indicated concentrations of NGF, following a 2-h labeling periodwith 32pi. eIF-4E immunoprecipitation and analysis on SDS-12.5%PAGs was done as described in Materials and Methods. Theautoradiograph of M-M17-26 cells was exposed to film twice as longas that of the M7 cells.

eIF-4E phosphorylation in fibroblast cells (18), and Ras isdistal to Src on the same signal transduction pathway (47,56).

In PC12 cells, expression of Ha-v-ras or v-src inducesNGF-independent differentiation (49). Expression of a trans-dominant negative ras gene (61) or injection of antibodiesagainst cellular p2lras (27) both inhibit NGF-mediated neu-rite outgrowth. These findings suggest that Ras proteinsmight mediate the NGF-induced signal that leads to in-creased eIF-4E phosphorylation in PC12 cells. To test thisidea, we made use of a PC12 cell line (M-M17-26) thatoverexpresses a cellular Ha-ras protein containing a substi-tution of Asn for Ser at position 17 (61). This mutant ras(Ha-ras Asn-17) acts as a dominant negative repressor ofwild-type ras and inhibits the action of mitogenic agents inNIH 3T3 cells (8). PC12 cells expressing Ha-ras Asn-17show a marked inhibition of the morphological differentia-tion induced by NGF (61). We examined the effect of NGFon eIF-4E phosphorylation in these cells. In the transfectedcontrol cell line (M-7) expressing only the G418 resistancegene, NGF treatment caused an increase in eIF-4E phos-phorylation in a dose-dependent manner (Fig. 6, lanes 1 to3), similar to the results obtained for parental PC12 cells(Fig. 4). However, strikingly, no increase in eIF-4E phos-phorylation was evident in the Ha-ras Asn-17-expressingM-M17-26 cells in response to NGF; instead, a decrease ineIF-4E phosphorylation was observed (Fig. 6, lanes 4 to 6).Time course analyses of eIF-4E phosphorylation upon ex-posure of M-M17-26 cells to NGF revealed no change by 15min and significant decreases at 45 and 60 min; no transientincrease in eIF-4E phosphorylation was evident (data notshown).The NGF-mediated decrease in eIF-4E phosphorylation in

cells expressing Ha-ras Asn-17 may be caused by either aconstitutive or NGF-induced eIF-4E phosphatase activity.Consistent with this idea is the finding that the potentphosphatase inhibitor, okadaic acid, enhances eIF-4E phos-phorylation, suggesting a role for serine/threonine phos-

M7

1 2 3

M-M17-26

4 5 6456

- m

elF-4E

VOL. 12, 1992


http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/


A $

4 4

C.

4 44

FIG. 7. Analysis of eIF-4E tryptic phosphopeptides from NGF-treated PC12 cells. Analysis was performed as described in Materials andMethods. (A) Control PC12 cells, (B) serum-starved Rat 1 cells, (C) NGF-treated PC12 cells, (D) serum-starved ras-transformed Rat 1 cells,(E) mixing of peptides from panels C and D, containing equivalent amounts of radioactive material. Arrows indicate the point of sampleapplication. Electrophoresis was done in the horizontal direction, and the subsequent chromatography was done in the vertical direction.

phatases (ppl and/or pp2A) in the regulation of eIF-4Ephosphorylation (13). In summary, these results stronglysuggest that the increase in eIF-4E phosphorylation in re-sponse to NGF-treatment of PC12 cells is mediated by aras-dependent signalling pathway. Moreover, the findingthat NGF-mediated eIF-4E phosphorylation in PC12 cellsoccurs as a result of activation of a PKC-independent (Fig.5), but ras-dependent, signal pathway (Fig. 6), providesfurther evidence favoring the notion that ras function can beindependent of PKC in PC12 cells.

Phosphorylation of PC12 cell eIF-4E in response to NGFoccurs on the identical serine that is phosphorylated in fibro-blast cells transformed by Ha-v-ras. Peptide maps of eIF-4Efrom cells treated with growth-promoting agents, PMA, ortransformed by viral oncogenes have revealed a single majorsite of phosphorylation (6, 18, 35, 46). To determine whetherNGF promotes phosphorylation on novel sites, we per-formed tryptic map analysis of 32P-radiolabeled eIF-4E fromcontrol and NGF-treated PC12 cells. We compared thesemaps to those from Rat 1 cells and from a p2lHa-v-ras-transformed Rat 1 cell line, 1274-8-1 (66). The latter cell linemaintains elevated levels of phosphorylated eIF-4E uponserum deprivation (57). Analysis of the tryptic digests bytwo-dimensional electrophoresis revealed a single majorphosphopeptide in each sample (Fig. 7A through D); phos-phorylation was increased upon NGF treatment of PC12cells (compare Fig. 7C [NGF-treated] and A [untreated]) orexpression of p21Ha-v-ra (compare Fig. 7B [Rat 1] and D[p21Ha-v-ras-transformed Rat 1 cells]). These peptides comi-grated when mixed and subjected to the same analysis,indicating their identity (Fig. 7E). A single spot was alsoobtained when peptides from NGF- or PMA-treated PC12cells were mixed (data not shown). Thus, the enhancedeIF-4E phosphorylation detected upon NGF or PMA treat-ment of PC12 cells occurs at the same single major site thatis phosphorylated upon transformation of Rat 1 fibroblastcells by Ha-v-ras.

DISCUSSION

The PC12 pheochromocytoma cell line responds to NGFby shifting from a chromaffin cell-like phenotype to a neu-rite-bearing neuronal phenotype (25, 28). The NGF receptoris phosphorylated on tyrosine in response to NGF anddemonstrates intrinsic tyrosine kinase activity which maymediate the downstream effects of NGF in PC12 cells (36,38). However, both the proximal and distal components ofthe signal transduction pathways that transmit the NGF-induced signals are not well understood. Many of the down-stream events resulting from activation of the NGF receptorphosphotyrosine kinase in PC12 cells also occur upon acti-vation of mitogen-activated receptor phosphotyrosine ki-nases in fibroblasts. These effects include transient inductionof early response genes, such as fos and jun, and thephosphorylation of a number of cytoplasmic and nuclearproteins (23, 28). Some of these phosphorylations also occurin mitogenically stimulated fibroblasts, such as phosphory-lation and activation of both mitogen-activated protein andS6 kinases (3, 48). eIF-4E phosphorylation has been shownto be induced by a wide variety of growth factors andmitogenic agents in fibroblast cells (19). We show here thatan increase in the steady-state level of the major phosphor-ylated variant of eIF-4E is an early response to treatment ofPC12 cells with concentrations of NGF that induce differen-tiation. This contrasts with the minor change in eIF-4Ephosphorylation mediated by EGF, a weak mitogen in PC12cells. eIF-4E activity appears to correlate directly with itsphosphorylation state (35, 45). eIF-4E phosphorylation andthe concomitant phosphorylation of ribosomal protein S6correlate with an overall stimulation of protein synthesis(19). Consistent with this, NGF treatment of PC12 cellsinduces a modest general increase in translation rates (21,33). More interestingly, translation of subsets of the proteinsexpressed in PC12 cells is differentially modulated uponinduction of differentiation by NGF (21, 25).

MOL. CELL. BIOL.


http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/


We have shown previously that eIF-4E causes transfor-mation when overexpressed in fibroblast cells and havesuggested that this effect is due to alleviation of the transla-tional repression mediated by the 5' noncoding region ofgrowth-related mRNAs (39). Our current results suggest thatphosphorylation of eIF-4E and the concomitant increase inits activity may play a similar role in gene regulation duringdifferentiation of PC12 cells and by extension during devel-opment of the neural system. One interesting possibility isthat some targets of enhanced eIF-4E activity could differbetween NGF-treated PC12 cells and mitogenically stimu-lated NIH 3T3 cells, depending on the particular repertoireof translationally regulated mRNAs expressed in each celltype. Alternatively, it is also conceivable that increasedeIF-4E activity could stimulate the translation of a commonset of proteins in both fibroblasts and pheochromocytomacells, whose induction is a general and perhaps necc.ssarycomponent of the activation of signal transduction pathwaysby a diverse set of mitogens and growth factors, regardlessof the ultimate effects on cell growth. Both models couldreconcile a role for induction of eIF-4E phosphorylation inresponse to disparate factors which engender such opposingoutcomes as fibroblast cell proliferation and PC12 cell dif-ferentiation. Potential targets of eIF-4E activity in differen-tiating PC12 cells could also include genes involved in theexpression of the neuronal phenotype, such as the geneencoding the largest neurofilament subunit, NF-H, whichappears to be posttranscriptionally regulated (41). The iden-tification of such translationally regulated genes in PC12cells may prove instrumental in gaining a better understand-ing of the process of cellular differentiation.Ras proteins (49, 61) and PKC (7, 9, 29) have been

implicated as effectors of NGF action. In PC12 cells, expres-sion of a dominant inhibitory Ha-ras (Asn-17) gene specifi-cally interferes with the signal transduction pathway leadingto growth factor-induced differentiation in PC12 cells (61).We have shown here that expression of this Ha-ras mutantalso results in the repression of NGF-stimulated eIF-4Ephosphorylation, demonstrating that eIF-4E phosphoryla-tion is mediated by a ras-dependent signalling pathway.These results demonstrate a link between regulation oftranslation initiation and the ras signalling system and areconsistent with the findings that the mitogenic activity ofeIF-4E in fibroblasts is also dependent upon ras: induction ofDNA synthesis by microinjection of eIF-4E into quiescentNIH 3T3 cells is inhibited when coinjected with the domi-nant negative p21(Asn-17)Haras (57). Thus, the results pre-sented here provide a possible explanation for the depen-dence of eIF-4E mitogenic activity on p2lras; the dominantnegative p21(Asn-17) la-ra may interfere with the regulationof eIF-4E phosphorylation in the injected cells, resulting inunderphosphorylated and less-active eIF-4E. It is equallyplausible, however, that p21(Asn-17)Ha-ras may interferewith a parallel, eIF-4E-independent signalling pathwaywhich is also necessary for induction of DNA synthesis.Taken together, these findings strongly support the hypoth-esis that eIF-4E is an important component of mitogen- andgrowth factor-activated signal transduction pathways.A pertinent question is the role of PKC in the NGF-

mediated increase in eIF-4E phosphorylation. The involve-ment of PKC in neurite formation and differentiation of PC12cells is controversial. Whereas PMA treatment of PC12 cellscan partially mimic a number of the effects of NGF, suchtreatment does not elicit differentiation or neurite outgrowth(7). Exposure of PC12 cells to NGF results in early transientactivation of PKC (29), and persistent activation of PKC by

phorbol ester induces a pattern of protein phosphorylationthat is similar to that resulting from NGF treatment (9).However, down-regulation of PKC via long-term exposureof PC12 cells to high doses of PMA does not interfere withsubsequent NGF-mediated differentiation or neurite out-growth (10, 52). We show here that PMA treatment of PC12cells results in an increase in eIF-4E phosphorylation, yet toa significantly lesser extent than the increase observed uponexposure of the cells to NGF. Nevertheless, PKC-depletedPC12 cells still responded to NGF treatment with a stronginduction of eIF-4E phosphorylation. These results offerevidence supporting the existence both PKC-independentand -dependent signaling pathways controlling eIF-4E phos-phorylation in PC12 cells. The NGF-mediated increase ineIF-4E phosphorylation occurs in a primarily PKC-indepen-dent manner. This conclusion is consistent with the findingthat NGF-promoted neuritogenesis and PC12 differentiationalso occur in a PKC-independent manner (10, 52) and servesto strengthen the correlation between PC12 differentiationand eIF-4E phosphorylation. Interestingly, these resultsdiffer from those reported by Morley and Traugh (46), whohave demonstrated PKC dependence of eIF-4E phosphory-lation in 3T3-L1 cells mitogenically stimulated with insulin.Thus, the signalling pathways controlling eIF-4E phosphor-ylation appear to be cell and/or growth factor dependent. Wecannot, however, rule out the possibility that NGF treatmentof PC12 cells results in induction of a PMA-independentPKC isoform, which could phosphorylate eIF-4E directly, ortransduce the NGF-mediated signal to an eIF-4E kinaseand/or phosphatase. Several novel isoforms of PKC haverecently been described, one of which, PKC-t, is not acti-vated by treatment with phorbol esters (50). Nevertheless, itis clear from our results that the NGF-stimulated response isnot dependent on PKC (a, I, and -y isoforms).The important role which eIF-4E plays in the regulation of

cell growth in fibroblast cells points to a possible regulatoryfunction in the process of cellular differentiation in PC12cells. The rapid and significant enhancement of eIF-4Ephosphorylation in PC12 cells treated with a differentiation-inducing agent lends support to this idea. Thus, it is impor-tant to identify the kinase(s) and phosphatase(s) whichcontrol eIF-4E phosphorylation in PC12 cells and the com-ponents of the signalling pathways which regulate theiractivity. Such studies may help identify signal transductionpathways and components which mediate PC12 cell differ-entiation.

ACKNOWLEDGMENTS

We thank P. Jolicoeur for Rat 1 and 1274-8-1 cell lines, G. Cooperand J. Szeberenyi for the M7 and M-M17-26 PC12 cell lines, TonyHunter for the serum against PKC, and Ullrich Bommer and AndreVeillette for helpful comments on the manuscript.

This research was supported by grants from the Medical ResearchCouncil of Canada to N.S. and W.E.M., and the National CancerInstitute of Canada to N.S. R.M.F. is a recipient of a StudentshipAward from the Medical Research Council of Canada.

REFERENCES1. Alema, S., P. Casalbore, E. Agostini, and F. Tato. 1985. Differ-

entiation of PC12 pheochromocytoma cells induced by v-srconcogene. Nature (London) 316:557-559.

2. Bonneau, A. M., and N. Sonenberg. 1987. Involvement of the24-kDa cap binding protein in regulation of protein synthesis inmitosis. J. Biol. Chem. 262:11134-11139.

3. Boulton, T. G., S. H. Nye, D. J. Robbins, N. Y. Ip, E.Radziejewska, S. D. Morganbesser, R. A. DePinho, N. Papaya-tatos, M. H. Cobb, and G. D. Yancopoulos. 1991. ERKs: a family

VOL. 12, 1992


http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/


of protein-serine/threonine kinases that are activated and ty-rosine phosphorylated in response to insulin and NGF. Cell65:663-665.

4. Brooks, R. F. 1976. Regulation of the fibroblast cell cycle byserum. Nature (London) 260:248-250.

5. Brooks, R. F. 1977. Continuous protein synthesis is required tomaintain the probability of entry into S phase. Cell 12:311-317.

6. Bu, X., and C. H. Hagedorn. 1991. Platelet-derived growthfactor stimulates phosphorylation of the 25 kDa mRNA capbinding protein (eIF-4E) in human lung fibroblasts. FEBS Lett.283:219-222.

7. Burstein, D. E., P. M. Blumberg, and L. A. Greene. 1982. Nervegrowth factor induced neuronal differentiation of PC12 pheo-chromocytoma cells: lack of inhibition by a tumour promoter.Brain Res. 247:115-119.

8. Cai, H., J. Szeberenyi, and G. M. Cooper. 1990. Effect of adominant inhibitory Ha-ras mutation on mitogenic signal trans-duction in NIH 3T3 cells. Mol. Cell. Biol. 10:5314-5323.

9. Cremins, J., J. A. Wagner, and S. Halegoua. 1986. Nerve growthfactor action is mediated by cyclic AMP- and Ca+2/phospho-lipid-dependent protein kinases. J. Cell Biol. 103:887-893.

10. Damon, D. H., P. D'Amore, and J. Wagner. 1990. NGF andFGF regulate neurite outgrowth and gene expression in PC12cells via both PKC- and cAMP-independent mechanisms. J. CellBiol. 110:1333-1339.

11. de Benedetti, A., and R. E. Rhoads. 1990. Overexpression ofeukaryotic protein synthesis factor 4E in HeLa cells results inaberrant growth and morphology. Proc. Natl. Acad. Sci. USA87:8212-8216.

12. Decker, S. 1981. Phosphorylation of ribosomal protein S6 inavian sarcoma virus-transformed chicken embryo fibroblasts.Proc. Natl. Acad. Sci. USA 78:4112-4115.

13. Donaldson, R. W., C. H. Hagedorn, and S. Cohen. 1991.Epidermal growth factor or okadaic acid stimulates phosphory-lation of eukaryotic initiation factor 4F. J. Biol. Chem. 266:3162-3166.

14. Duncan, R., and J. W. B. Hershey. 1984. Heat shock-inducedtranslational alterations in HeLa cells. J. Biol. Chem. 259:11882-11889.

15. Duncan, R., S. C. Milburn, and J. W. B. Hershey. 1987.Regulated phosphorylation and low abundance of HeLa cellinitiation factor eIF-4F suggest a role in translational control. J.Biol. Chem. 262:380-388.

16. Edery, I., M. Humbelin, A. Darveau, K. A. W. Lee, S. Milburn,J. W. B. Hershey, H. Trachsel, and N. Sonenberg. 1983. Involve-ment of eukaryotic initiation factor 4A in the cap recognitionprocess. J. Biol. Chem. 258:11398-11403.

17. Fan, H., and S. Penman. 1970. Regulation of protein synthesis inmammalian cells. J. Mol. Biol. 50:665-670.

18. Frederickson, R. M., K. S. Montine, and N. Sonenberg. 1991.Phosphorylation of eukaryotic translation initiation factor 4E isincreased in Src-transformed cell lines. Mol. Cell. Biol. 11:2896-2900.

19. Frederickson, R. M., and N. Sonenberg. Signal transduction andregulation of translation initiation. Semin. Cell Biol., in press.

20. Frederickson, R. M., A. Veillette, and N. Sonenberg. Unpub-lished results.

21. Garrels, J. I., and D. Schubert. 1979. Modulation of proteinsynthesis by nerve growth factor. J. Biol. Chem. 254:7978-7985.

22. Gomez, N., and P. Cohen. 1991. Dissection of the protein kinasecascade by which nerve growth factor activates MAP kinase.Nature (London) 353:170-173.

23. Greenberg, M. E., L. A. Greene, and E. B. Ziff. 1985. Nervegrowth factor and epidermal growth factor induce rapid tran-sient changes in proto-oncogene transcription in PC12 cells. J.Biol. Chem. 260:14101-14110.

24. Greene, L. A., and E. M. Shooter. 1980. The nerve growthfactor. Annu. Rev. Neurosci. 3:353-402.

25. Greene, L. A., and A. S. Tischler. 1976. Establishment of anoradrenergic clonal line of rat adrenal pheochromocytomacells which respond to nerve growth factor. Proc. Natl. Acad.Sci. USA 73:2424-2428.

26. Grifo, J. A., S. M. Tahara, M. A. Morgan, A. J. Shatkin, and

W. C. Merrick. 1983. New initiation factor activity required forglobin mRNA translation. J. Biol. Chem. 258:5804-5810.

27. Hagag, N., S. Halegoua, and M. Viola. 1986. Inhibition of growthfactor induced differentiation of PC12 cells by microinjection ofantibody to ras p21. Nature (London) 319:680-682.

28. Halegoua, S., and J. Patrick. 1980. Nerve growth factor medi-ates phosphorylation of specific proteins. Cell 22:571-581.

29. Heasley, L. E., and G. L. Johnson. 1989. Regulation of proteinkinase C by nerve growth factor, epidermal growth factor, andphorbol esters in PC12 pheochromocytoma cells. J. Biol. Chem.264:8646-8652.

30. Hershey, J. W. B. 1989. Protein phosphorylation controls trans-lation rates. J. Biol. Chem. 264:20823-20826.

31. Hershey, J. W. B. 1991. Translational control in mammaliancells. Annu. Rev. Biochem. 60:717-756.

32. Hiremath, L. S., N. R. Webb, and R. E. Rhoads. 1985. Immu-nological detection of the messenger RNA cap-binding protein.J. Biol. Chem. 260:7843-7849.

33. Huff, K., D. End, and G. Guroff. 1981. Nerve growth factor-induced alteration in the response of PC12 pheochromocytomacells to epidermal growth factor. J. Cell Biol. 88:189-198.

34. Jagus, R., W. F. Anderson, and B. Safer. 1981. Initiation ofmammalian protein biosynthesis. Prog. Nucleic Acid Res. Mol.Biol. 25:127-185.

35. Joshi-Barve, S., W. Rychlik, and R. E. Rhoads. 1990. Alterationof the major phosphorylation site of eukaryotic protein synthe-sis initiation factor 4E prevents its association with the 48Sinitiation complex. J. Biol. Chem. 265:2979-2983.

36. Kaplan, D., D. Martin-Zanca, and L. F. Parada. 1991. Tyrosinephosphorylation and tyrosine kinase activity of the trk proto-oncogene product induced by NGF. Nature (London) 350:158-160.

37. Kaspar, R. L., W. Rychlik, M. W. White, R. E. Rhoads, andD. R. Morris. 1990. Simultaneous cytoplasmic redistribution ofribosomal protein L32 mRNA and phosphorylation of eukary-otic initiation factor 4E after mitogenic stimulation of Swiss 3T3cells. J. Biol. Chem. 265:3619-3622.

38. Klein, R., S. Jing, V. Nanduri, E. O'Rourke, and M. Barbacid.1991. The trk proto-oncogene encodes a receptor for nervegrowth factor. Cell 65:189-197.

39. Lazaris-Karatzas, A., K. S. Montine, and N. Sonenberg. 1990.Malignant transformation by a eukaryotic initiation factor sub-unit that binds to mRNA 5' cap. Nature (London) 345:544-547.

40. Lazaris-Karatzas, A., and N. Sonenberg. The mRNA 5' Cap-binding protein, eIF-4E, cooperates with v-myc or ElA in thetransformation of primary rodent fibroblasts. Mol. Cell. Biol.12:1234-1238.

41. Lindenbaum, M. H., S. T. Carbonetto, and W. E. Mushynski.1987. Nerve growth factor enhances the synthesis, phosphory-lation, and metabolic stability of neurofilament proteins in PC12cells. J. Biol. Chem. 262:605-610.

42. Luo, K., T. R. Hurley, and B. M. Sefton. 1990. Transfer ofproteins to membranes facilitates both cyanogen bromide cleav-age and two-dimensional proteolytic mapping. Oncogene 5:921-923.

43. Marino, M. W., L. M. Pfeffer, P. T. Guidon, and D. B. Donner.1989. Tumor necrosis factor induces phosphorylation of a 28kDa mRNA cap-binding protein in human cervical carcinomacells. Proc. Natl. Acad. Sci. USA 86:8417-8421.

44. Matthies, H. J. G., H. C. Palfrey, C. D. Hirming, and R. J.Miller. 1987. Down-regulation of protein kinase C in neuronalcells: effects on neurotransmitter release. J. Neurosci. 7:1198-1206.

45. Morley, S. J., T. E. Dever, D. Etchison, and J. A. Traugh. 1991.Phosphorylation of eIF-4F by protein kinase C or multipotentialS6 kinase stimulates protein synthesis at initiation. J. Biol.Chem. 266:4669-4672.

46. Morley, S. J., and J. A. Traugh. 1990. Differential stimulation ofphosphorylation of initiation factors eIF-4F, eIF-4B, eIF-3, andribosomal protein S6 by insulin and phorbol esters. J. Biol.Chem. 265:10611-10616.

47. Mulcahy, L. S., M. R. Smith, and D. W. Stacey. 1985. Require-ment for ras proto-oncogene function during serum-stimulated

MOL. CELL. BIOL.


http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/


growth of NIH 3T3 cells. Nature (London) 313:241-243.48. Mutoh, T., B. B. Rudkin, S. Koizumi, and G. Guroff. 1988.

Nerve growth factor, a differentiating agent, and epidermalgrowth factor, a mitogen, increase the activities of different S6kinases in PC12 cells. J. Biol. Chem. 263:15853-15856.

49. Noda, M., M. Ko, A. Ogura, D. Liu, T. Amano, T. Takano, andY. Ikawa. 1985. Sarcoma viruses carring ras oncogenes inducedifferentiation associated properties in a neuronal cell line.Nature (London) 318:73-75.

50. Ono, Y., Y. Fujii, K. Ogita, U. Kikkawa, K. Igarashi, and Y.Nishizuka. 1989. Protein kinase C 4 subspecies from rat brain: itsstructure, expression, and properties. Proc. Natl. Acad. Sci.USA 86:3099-3103.

51. Panniers, R., and E. C. Henshaw. 1984. Mechanism of inhibitionof polypeptide chain initiation in heat-shocked Ehrlich ascitestumour cells. Eur. J. Biochem. 140:209-214.

52. Reinhold, D. S., and K. E. Neet. 1989. The lack of a role forprotein kinase C in neurite extension and in the induction ofornithine decarboxylase by nerve growth factor in PC12 cells. J.Biol. Chem. 264:3538-3544.

53. Rhoads, R. E. 1988. Cap recognition and the entry of mRNAinto the protein synthesis initiation cycle. Trends Biochem. Sci.13:52-56.

54. Rozen, F., I. Edery, K. Meerovitch, T. E. Dever, W. C. Merrick,and N. Sonenberg. 1990. Bidirectional RNA helicase activity ofeukaryotic translation initiation factors 4A and 4F. Mol. Cell.Biol. 10:1134-1144.

55. Rychlik, W., M. A. Russ, and R. E. Rhoads. 1987. Phosphory-lation site of eukaryotic initiation factor 4E. J. Biol. Chem.262:10434-10437.

56. Smith, M. R., S. J. DeGudicibus, and D. W. Stacey. 1986.Requirement for c-ras proteins during viral oncogene transfor-mation. Nature (London) 320:540-543.

57. Smith, M. R., R. M. Frederickson, M. L. Jaramillo, Y. Liu, H.

Kung, and N. Sonenberg. Unpublished results.58. Sonenberg, N. 1988. Cap binding proteins of eukaryotic messen-

ger RNA: functions in initiation and control of translation. Prog.Nucleic Acid Res. Mol. Biol. 35:174-207.

59. Sonenberg, N., M. A. Morgan, W. C. Merrick, and A. J.Shatkin. 1978. A polypeptide in eukaryotic initiation factors thatcrosslinks specifically to the 5'-terminal cap in mRNA. Proc.Natl. Acad. Sci. USA 75:4843-4847.

60. Stanners, C. P., and H. Becker. 1971. Control of macromolecu-lar synthesis in proliferation and resting Syrian hamster cells inmonolayer culture. 1. Ribosome function. J. Cell. Physiol.77:31-42.

61. Szeberenyi, J., H. Cai, and G. Cooper. 1990. Effect of adominant inhibitory Ha-ras mutation on neuronal differentiationof PC12 cells. Mol. Cell. Biol. 10:5324-5332.

62. Tahara, S., M. A. Morgan, and A. J. Shatkin. 1981. Two formsof purified m7G-cap binding protein with different effects oncapped mRNA translation in extracts of uninfected and polio-virus-infected HeLa cells. J. Biol. Chem. 256:7691-7694.

63. Thomas, G., J. Martin-Perez, M. Steigman, and A. M. Otto.1982. The effect of serum, EGF, PGF2<X, and insulin on S6phosphorylation and the initiation of protein and DNA synthe-sis. Cell 30:235-242.

64. Veillette, A., I. D. Horak, E. M. Horak, M. A. Bookman, andJ. B. Bolen. 1988. Alterations of the lymphocyte-specific proteintyrosine kinase during T-cell activation. Mol. Cell. Biol. 8:4353-4361.

65. Woodgett, J. R., and T. Hunter. 1987. Immunological evidencefor two physiological forms of protein kinase C. Mol. Cell. Biol.7:85-96.

66. Zarbl, H., J. Latreille, and P. Jolicoeur. 1987. Revertants ofv-fos-transformed fibroblasts have mutations in cellular genesessential for transformation by other oncogenes. Cell 51:357-369.

VOL. 12, 1992


http://mcb.asm

.org/D

ownloaded from

http://mcb.asm.org/

phosphorylation of translation initiation factor eif-4e is induced in

Documents