effect of oxidizing agents and haemin on the phosphorylation of eukaryotic elongation factor 2 in...

ELSEVIER Biochimica et Biophysica Acta 1260 (1995) 200-206

Btt Biochi~ic~a et Biophysica A~ta

Effect of oxidizing agents and haemin on the phosphorylation of eukaryotic elongation factor 2 in rabbit reticulocyte lysates

Anders Nilsson, Odd Nyg~rd *

Department of Zoological Cell Biology, Arrhenius Laboratories E5, Stockholm University, S-106 91 Stockholm, Sweden

Received 30 May 1994; accepted 6 September 1994

Abstract

Incubation of rabbit reticulocyte lysates in the absence of added haemin resulted in the phosphorylation of a 95 kDa protein. This protein was suggested to be elongation factor 2 (eEF-2) based on the following observations, (i) phosphorylation of the 95 kDa protein was Ca 2÷ and CaM-dependent. (ii) eEF-2 supplemented to the lysates became phosphorylated and co-migrated with the endogenous 95 kDa phosphoprotein upon electrophoresis in SDS gels. (iii) The tryptophane specific cleavage pattern obtained from the isolated 95 kDa phosphoprotein was identical to that of phosphorylated eEF-2. Phosphorylation of the 95 kDa protein was stimulated by oxidizing agents such as oxidized glutathione and NAD + and inhibited by addition of haemin. The haemin concentration needed for 50% inhibition (IC5o) was 2.5/xM. Haemin also had an inhibitory effect on eEF-2 phosphorylation in a system containing highly purified components (IC5o = 2 /zM). In this system haemin inhibited phosphorylation of eEF-2 even in the presence of a 100-fold excess of fl-mercaptoethanol. Oxidizing agents had no effect on the kinase activity in the purified system.

Keywords: CaM PKIII; Elongation factor 2; Haemin; Phosphorylation

I. Introduct ion

Alterations in the phosphorylation status of protein synthesis initiation factors has traditionally been consid- ered responsible for regulating the overall efficiency and selectivity of the translation process (for reviews see Refs. [1,2]). The classical example is the haemin regulated phosphorylation of the a-subunit of initiation factor elF-2. It is now clear that the catalytic activities of the protein synthesis elongation factors are also subjected to regulation through phosphorylations (for a review see [3]).

Eukaryotic elongation factor 2, eEF-2, is a protein with a molecular mass of approx. 95 kDa [3]. The factor, in complex with GTP, promotes translocation of peptidyl- tRNA from the so called A-site to the P-site on the ribosome [3]. Elongation factor 2 is phosphorylated by a

Abbreviations: CaM, calmodulin; CaM PKIII, calcium and calmodulin-dependent protein kinase III; D'vr, dithiotreitol; eEF-2, eukaryotic elongation factor 2; elF-2, eukaryotic initiation factor 2; GSSG, oxidized glutathione; NEM, N-ethylmaleimide; OA, okadaic acid; SDS, sodium dodecylsulfate; SucNCI, N-chlorosuccinimide, TFP, trifluoperazine.

* Corresponding author. Fax: +46 8 159837.

0167-4781/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 1 6 7 - 4 7 8 1 ( 9 4 ) 0 0 1 9 8 - 7

Ca 2+ and calmodulin dependent protein kinase designated CaM PKIII [4-6]. The kinase rapidly phosphorylates eEF-2 at Thr-56 located near the N-terminus close to the putative GTP-binding site of the factor [7]. The kinase also phosphorylates Thr-58 albeit at a much slower rate [7]. The phosphorylated eEF-2 is unable to stimulate the translocation reaction and thereby to sustain protein synthesis [8- 11]. The malfunction is caused by a reduced ability of the phosphorylated factor to bind to the pre-translocation type of ribosome [8]. Phospho eEF-2 can be reactivated through dephosphorylation. In reticulocytes reactivation is carried out by phosphoprotein phosphatase PP2A but in other types of cells PP2C may also be involved in the reactivation [12-14].

The activity of the eEF-2 kinase is increased by phosphorylation [15] and by drugs that raise the intracellular concentration of Ca 2÷ [16,17] or stimulates cellular prolif- eration [17-19]. Decreased kinase activity is seen in the presence of drugs that increase the concentration of cAMP [20]. Furthermore the kinase activity can be inhibited by CaM antagonists such as cyclosporines [21,22] and by rottlerin [23]. In this report we have studied regulation of the eEF-2 kinase activity in rabbit reticulocyte lysates under conditions of in vitro protein synthesis.

A. Nilsson, O. Nygdrd / Biochimica et Biophysica Acta 1260 (1995) 200-206 201

2. Materials and methods

2.1. Chemicals

['y-32p]ATP and [14C]NAD+ were from Amersham In- ternational (UK). Creatine phosphokinase, creatine phosphate, NAD +, cobalt protoporphyrin IX, DTr, NADH, NEM and GSSG were from Sigma (St. Louis, MO, USA). TFP and CaM was from Boehringer-Mannheim, (Germany). Okadaic acid was a gift from Dr. Y. Tsukitani (Fujisawa Pharmaceuticals, Tokyo, Japan).

trophoresis. The stained protein bands were excised from the gel and the gel pieces were washed with water for two times 10 min and water/acetic acid/urea (1 ml /1 ml /1 g) for two times 10 min. Thereafter the gel pieces were treated with 15 mM SucNCI in water/acetic acid/urea for 30 min, washed with water for an additional two times 10 min and equilibrated with SDS gel electrophoresis sample buffer. The SucNC1 generated cleavage pattern was analyzed on a second dimension SDS polyacrylamide gradient gel.

2.2. Preparation of proteins

Translation factors eEF-2 and elF-2 were purified from rat liver as previously described [24,25]. The eEF-2 kinase (CaM PKIII) was purified from rabbit reticulocyte lysates essentially as previously described [6]. Rabbit reticulocyte lysates were prepared as described by Nyg~rd and Hultin [26].

For treatment of eEF-2 and the purified eEF-2 kinase by NEM, the proteins were dialysed against a buffer containing 100 mM KCI, 20 mM Tris-HC1 (pH 7.6), 0.07 mM EDTA, 7% (by vol.) glycerol and 6 mM thiodiethanol. The proteins were then treated with 5 mM NEM for 10 min at 20°C and the excess of NEM was removed by addition of /3-mercaptoethanol to a final concentration of 10 mM.

2.3. Determination of kinase activity

To allow protein synthesis, the reticulocyte lysates were supplemented with a mixture of amino acids and an energy regenerating system consisting of creatine phosphate and creatine phosphokinase [27]. The capacity of the lysates to phosphorylate eEF-2 was analyzed after addition of [y- 32 P]ATP (specific activity 280 Ci/mol) to a final concentration of 0.25 mM.

In the purified system the phosphorylation reactions contained, if not otherwise indicated, 100 mM KCI, 20 mM Tris-HC1 (pH 7.6), 6 mM 2-mercaptoethanol, 10 mM MgCI z, 1.0 mM EGTA, 1.5 mM Ca 2÷, 0.07 mM EDTA, 7% (by vol.) glycerol, 0.25 mM [y-32p]ATP (specific activity 280 Ci/mol), 90 pmol CaM, 1 /xmol eEF-2 and 1 pmol kinase. The reaction mixtures were, if not otherwise indicated, incubated at 35°C for 5 min.

2.4. Gel electrophoresis

3. Results and discussion

3.1. Effect of red-ox agents on the phosphorylation of eEF-2

The rate of protein synthesis in rabbit reticulocyte lysates is regulated by haemin [1,2]. Incubation of haemin deficient lysates, supplemented with [ y-32 P]ATP, resulted in a rapid phosphorylation of a 95 kDa protein, as seen by SDS gel electrophoresis and autoradiography (Fig. 1A). After prolonged exposure of the autoradiogram an additional phosphorylated 40 kDa protein appeared (not shown). Addition of 20 /zM haemin to the reticulocyte lysates inhibited phosphorylation of both the 95 and the 40 kDa proteins (Fig. 1B).

The molecular size and the inhibitory effect of haemin on the phosphorylation of the 40 kDa protein suggested that this phosphoprotein was identical to elF-2 a , a protein known to be phosphorylated in haemin deficient reticulocyte lysates ([1,2] and references therein). This sugges-

A B

I 2 I 2

9 7 . 4 - ~ ~ 0

66.2-

42.7-

SDS gel electrophoresis using poly acrylamide gradient slab gels was according to Laemmli [28]. The gels were stained with Coommassie Brilliant Blue and destained. The dried gels were exposed to X-ray films at - 80°C using intensifying screens.

2.5. SucNCl cleavage

For the cleavage at tryptophan residues [29], proteins were isolated by SDS polyacrylamide gradient gel elec-

31.0-

Fig. 1. Protein phosphorylation in reticulocyte lysates. Lysate were incubated under protein synthesis conditions as described in Materials and methods. (A) Lysates incubated in the absence of haemin for 1 min (lane 1) and 2 min (lane 2). (B) Effect of haemin on protein phosphorylation in reticulocyte lysates. Lysate with 20 /xM haemin added (lane 1); control without haemin (lane 2).

202 A. Nilsson, O. Nyg~rd / Biochimica et Biophysica Acta 1260 (1995) 200-206

A B C

I 2 3 I 2 I 2

97.4- O

6 6 . 2 - o O

31.0-

Fig. 2. Identification of the 95 kDa protein phosphorylated in haemin deficient reticulocyte lysates. Rabbit reticulocyte lysates were incubated as described in Materials and methods. (A) Haemin deficient lysates (lane 1) were incubated in the presence of 10 mM EGTA (lane 2) or 0.I mM TFP (lane 3). (B) SucNCl-cleavage of the 32 P-labelled 95 kDa phosphoprotein isolated from reticulocyte lysates as described in Materials and methods (lane 1). SucNCl-cleavage of purified eEF-2 phosphorylated in the presence of purified eEF-2 kinase and [)'-3:p]ATP as described in Materials and methods (lane 2). (C) Co-migration of eEF-2 and the 95 kDa protein. Lysates incubated in the absence (lane 1) and presence (lane 2) of added eEF-2.

tion was also confirmed by the observed co-migration of added purified elF-2 a and the 40 kDa phosphoprotein in SDS-polyacrylamide gels (results not shown). The identity of the 95 kDa protein was less obvious. Phosphorylation of elF-2 a is known to require activation of a specific protein kinase, HRI [30,31]. Activation of this kinase is accompa- nied by phosphorylation of a 95 kDa protein which has been suggested to be HRI it self or a regulatory protein [32-34]. However, the amount of phosphorylation seen in the 95 kDa region suggested that the phosphoprotein was more abundant than could be expected for a protein kinase such as HRI. One other 95 kDa protein that can be phosphorylated in reticulocyte lysates is elongation factor eEF-2 [12]. Phosphorylation of eEF-2 is dependent on Ca 2÷ and CaM [4-6]. If the endogenous 95 kDa phosphoprotein was identical to eEF-2 removal of Ca 2+ from the lysates by addition of EGTA or inactivation of CaM by TFP should reduce the extent of phosphorylation in the 95 kDa region [9]. As seen in Fig. 2A, addition of either EGTA or TFP resulted in a total inhibition of the phosphorylation of the 95 kDa protein. Furthermore, purified eEF-2 added to lysates supplemented with [32p]ATP became phosphorylated and co-migrated with the endogenous 95 kDa protein (Fig. 2C). These results suggest that the 95 kDa protein was identical to eEF-2.

This hypothesis was further investigated by comparing the tryptophane specific cleavage pattern of the endoge-

nous 95 kDa protein with that of purified phosphorylated eEF-2. For this purpose the endogenous [32p]phosphate- labelled 95 kDa protein was isolated by gel electrophoresis and specifically cleaved at tryptophan residues using Suc- NC1 [29]. As seen in Fig. 2B, the cleavage pattern was closely similar to that obtained from purified phosphorylated eEF-2.

If the endogenous 95 kDa protein was identical to eEF-2 then haemin must have an inhibitory effect on the phosphorylation of eEF-2. This assumption was confirmed by the observation that haemin blocked phosphorylation of purified eEF-2 added to the lysates (see Fig. 7A). Thus, the accumulated experimental data suggested that the 95 kDa protein phosphorylated in haemin deficient rabbit reticulocytes was eEF-2.

In the reticulocytes, phosphorylation of eEF-2 is coun- terbalanced by a dephosphorylation catalyzed by phosphoprotein phosphatase PP2A [13,14]. To omit the possibility that haemin stimulated dephosphorylation of eEF-2 rather than inhibited phosphorylation of the factor, the activity of PP2A was inhibited by addition of OA. As seen in Fig. 3, haemin blocked eEF-2 phosphorylation even after inhibition of the PP2A activity, suggesting that the effect was due to a reduced phosphorylation of eEF-2 and not to an increased dephosphorylation activity.

To see if the effect of haemin was only demonstrable in the crude lysate or if haemin had a direct effect on the eEF-2 kinase we replaced the reticulocyte lysate by a phosphorylation system containing purified components [6,8]. As seen in Fig. 7B, haemin inhibited the eEF-2 phosphorylation even in this purified system. The effect was not restricted to haemin as other protoporphyrin com- pounds such as cobalt protoporphyrin IX inhibited eEF-2

I 2 3 4

97.4- ~ 4 1 D

66.2-

42.7-

31.0-

Fig. 3. The effect of OA on protein phosphorylation in reticulocyte lysates. Reticulocyte lysates were incubated in the presence of 4 pmol eEF-2 as described in Materials and methods. Lysates without (lanes 1 and 2) or with (lanes 3 and 4) 10 ~M haemin. Incubation without (lanes 1 and 3) and with (lanes 2 and 4) 0.5 /xM OA.

A. Nilsson, O. Nyg?trd / Biochimica et Biophysica Acta 1260 (1995) 200-206 203

I 2 3

97.4- 0

66.2-

42.7-

31.0-

Fig. 4. Effect of protoporphyrines on the phosphorylation of eEF-2. Purified eEF-2 was phosphorylated in the presence of isolated eEF-2 kinase as described in Materials and methods. Control incubated in the absence of protoporphyrines (lane 1); addition of 20/~M haemin, (lane 2) and addition of 20 /xM cobalt protoporphyrin IX (lane 3).

phosphorylation (Fig. 4). The concentration of cobalt protoporphyrin IX needed for inhibition was comparable to that used to inhibit the phosphorylation of elF-2 o~ in rabbit reticulocytes [35]. This suggests that haemin, i.e., metallo- protoporphyrin groups regulates the phosphorylation of elF-2 a and eEF-2 by a similar molecular mechanism.

Analysis of the effect of haemin on the biological activity of eEF-2 showed that haemin did not affect the ribosome-dependent GTP hydrolase activity nor did haemin have any effect on the diphtheria toxin catalyzed ADP- ribosylation of the elongation factor (not illustrated). These results suggest that haemin had a direct effect on the eEF-2 kinase activity rather than on the substrate, eEF-2.

Haemin is known to control the phosphorylation of elF-2 by regulating the activity of the specific elF-2 kinase HRI [31]. Activation of the kinase could also be influenced by sulfhydryl reagents and by alterations in the redox potential [36-38]. Based on these observations it has been suggested that haemin controls the kinase activity by pro- moting disulfide bridge formation in HRI [36]. To see if this type of alterations had a similar effect on eEF-2 phosphorylation, reticulocyte lysates were incubated in the presence of oxidizing agents such as GSSG and NAD +. As seen in Fig. 5A, both reagents stimulated eEF-2 phosphorylation. However, the reducing agents NADH, D q T did not alter the extent of eEF-2 phosphorylation in the lysates (Fig. 5A). Furthermore, analysis of the effects of reducing and oxidizing agents on the eEF-2 phosphorylation using a purified system showed that neither the reducing nor the oxidizing agents affected the kinase activity (Fig. 5B). These results suggest that the effect of oxidizing agents observed in the crude lysates required additional proteins and was not mediated by a direct effect on the kinase.

Addition of sulfhydryl reagents such as NEM to rabbit reticulocyte lysates increases the extent of phosphorylation

of elF-2 a [35]. As seen in Fig. 6A, 5 mM NEM totally inhibited eEF-2 phosphorylation in the reticulocyte lysates. NEM also inhibited eEF-2 phosphorylation in the purified system (Fig. 6B, lane 2). However, pre-treatment of the eEF-2 kinase with NEM followed by addition of an excess of /3-mercaptoethanol, had no effect on the kinase activity (Fig. 6B, lane 4). Instead, pre-treatment of eEF-2 (Fig. 6B, lane 3) with NEM completely abolished the eEF-2 phosphorylation, suggesting that alkylation of the sulfllydryl groups in eEF-2 altered the structure of the factor such that the threonines, Thr-56 and Thr-58 [7], that normally func- tion as attachment points for the phosphate became un- available for phosphorylation.

Recently M6ndez et al. [39] suggested a model for the regulation of the elF-2 kinase activity by haemin and reducing agents. In this model the kinase is inactivated by formation of an -S-S- bridge between the kinase and heat shock protein 90 (HSP-90). The inactivation could be reversed by addition of fl-mercaptoethanol [39]. Although, haemin inhibits phosphorylation of eEF-2 at concentrations comparable to that needed for inhibition of elF-2 phosphorylation the haemin effect on the eEF-2 kinase can not be mediated via a mechanism similar to that proposed for HRI. The activity of the purified eEF-2 kinase is not influenced by sulfhydryl reagents or by oxidizing and reducing reagents. In fact, the haemin induced inhibition of eEF-2 phosphorylation was observed even in the presence of 6 mM /3-mercaptoethanol (Fig. 5B, lane 4), suggesting

A B

1 2 3 4 5 1 2 3 4 5 6

97.4- ~ - " ~

66.2-

42.7-

31.0-

Fig. 5. Effects of different oxidizing and reducing agents on eEF-2 phosphorylation. (A) Haemin deficient reticulocyte lysates were incubated in the presence of 4 pmol eEF-2 as described in Materials and methods. Control (lane 1); addition of 1 mM NAD + (lane 2); 1 mM GSSG (lane 3); 1 mM NADH (lane 4) and 1 mM DTT (lane 5). (B) Phosphorylation of eEF-2 in the purified system (see Materials and methods). Control (lane 1); addition of 1 mM NAD + (lane 2); l mM GSSG (lane 3); 20 /xM haemin; (lane 4); l mM NADH (lane 5) and 1 mM DTT (lane 6).

204 A. Nilsson, O. Nygdrd / Biochimica et Biophysica Acta 1260 (1995) 200-206

A B

I 2 I 2 3 4

97.4- ~ ~ O

66.2-

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31.0-

Fig. 6. The effect of NEM on the phosphorylation of eEF-2. (A) Reticulocyte lysates were incubated in the absence of haemin as described in Materials and methods. Control (lane 1); addition of 5 mM NEM (lane 2). (B) Effect of NEM on eEF-2 phosphorylation in the purified system. Control (lane 1); addition of 5 mM NEM (lane 2). eEF-2 (lane 3) and eEF-2 kinase (lane 4) pre-treated with 5 mM NEM as described in Materials and methods.

that the effect of haemin was not due to haemin acting as an oxidant.

Moreover, eEF-2 kinase preparations that did not contain any HSP-90, as detected by immunoblotting using HSP 90 specific antibodies, still responded to haemin at micromolar concentrations (not shown). Thus, our results suggest that the eEF-2 kinase activity is regulated by haemin via a mechanism different from that proposed for regulation of the elF-2 kinase activity.

3.2. Effect of haemin on the eEF-2 kinase

It has previously been observed that haemin inhibits the activity of other protein kinases than the elF-2 kinase. Thus, the activity of both cyclic AMP-dependent kinases and phosphorylase kinase are inhibited by haemin [40]. However, the effective haemin concentrations were considerably higher than that normally found to be optimal for translation in reticulocyte lysates [27] and 100 /zM only inhibited the activity of these kinases by 50 and 80%, respectively. These observations prompted us to determine the concentration of haemin needed for inhibition of the eEF-2 phosphorylation. As seen in Fig. 7A, 10/~M haemin was sufficient to completely inhibit eEF-2 phosphorylation in the reticulocyte lysates. The concentration needed for 50% inhibition (IC50) was calculated to 2.5 ~M haemin. Thus, the haemin concentration needed to inhibit the eEF-2 kinase was in the same range as that reported for inhibition of 6-aminolevulinat synthase in hepatocytes [40].

It has previously been noted that the haemin concentra-

tion needed to inhibit the phosphorylation of elF-2a in reticulocyte lysates are influenced by the extent of dilution of the lysate, i.e., the concentration of haemin needed to inhibit phosphorylation of eIF-2 a is decreased upon dilution [41]. This was also the case for the haemin induced inhibition of the eEF-2 kinase (not illustrated), showing that the effect of haemin is stoichiometric, i.e., the number of haemin molecules required for inhibition is proportional to the number of molecules in the phosphorylating system capable of interacting with haemin. As all our experiments were performed in lysates that had been optimized for maximum protein synthesis capacity the various lysate preparations showed an identical response at the same concentrations of added haemin.

Analysis of the haemin concentrations needed to inhibit eEF-2 phosphorylation in the purified system showed that 10 /xM haemin was sufficient to inhibit the reaction by more than 90% (Fig. 7B). The ICs0 value for the purified system was calculated to 2 ~M. These results show that the concentration of haemin needed to inhibit eEF-2 phosphorylation was considerably lower than that required for the general inhibition of proteinkinases such as the cyclic AMP-dependent kinases and phosphorylase b kinase [40].

Recently Leclerc et al. [42] showed that the reduced form of haemin, haem containing Fe 2+, i.e., haem-CO, but not the oxidized form containing Fe 3+, can bind to

1oo

80

"~- 60

40

20

A

5 10 15 Haemin (pM)

,00t B

40

20

• I , I , i . I • I , I . I ,

00 2 4 6 8 10 12 14

H a e m i r l ( g M )

Fig. 7. Inhibition of the eEF-2 phosphorylation by increasing concentrations of haemin, eEF-2 was phosphorylated in reticulocyte lysates (A) or in a system containing purified components (B). Phosphorylation was as described in Materials and methods.

A. Nilsson, O. Nygdrd / Biochimica et Biophysica Acta 1260 (1995) 200-206 205

lO0

80

• -~ 6O

4o

20

~ ---©

I . i , i , i . I i

2 4 6 8 10 Calmoaulin (.M)

Fig. 8. Influence of calmodulin on the eEF-2 kinase activity at increasing concentrations of haemin. Purified eEF-2 was phosphorylated in the presence of isolated eEF-2 kinase and increasing concentrations of calmodulin as described in Materials and methods. The extent of phosphorylation was analyzed by SDS-gel electrophoresis and autoradiography as described in Materials and methods. The autoradiographs were quantified using a computer assisted image processing system [58]. Controls incubated in the absence of haemin (©). Phosphorylation of eEF-2 in the presence of 5 (11) and 10 (A) /xM haemin.

calmodulin. To rule out the possibility that our haemin preparation was contaminated with haem we determined the amount of haemin by measuring the haemin dependent oxidation of NADH to NAD ÷ [43]. As the haemin preparations were able to oxidized equimolar amounts of NADH (data not shown) we conclude that the haemin preparations were free from haem.

We also determined the effect of calmodulin on the haemin induced inhibition of the eEF-2 kinase activity. As seen in Fig. 8, addition of increasing concentrations of calmodulin to a phosphorylation system containing purified components and either 5 /xM or 10 /xM haemin resulted in a slight increase in the eEF-2 kinase activity but were not able to restore the eEF-2 phosphorylation capacity. This result suggests that haemin did not inhibit the kinase activity by binding to calmodulin. This is in agree- ment with the observation that haem-CO but not haemin binds to calmodulin [42].

[47]. However, it is worth noting that addition of OA to reticulocyte lysates results in an increased phosphorylation of eEF-2, an accumulation of polysomes and a reduced rate of protein synthesis [12].

In reticulocyte lysates haemin deficiency is known to result in an inhibition of protein synthesis initiation rather than in a reduced rate of elongation [48]. However, unlike HRI, that only seems to exist in erythroid cells, the eEF-2 kinase is present in all cell types so far tested [2,49,50]. As haemin seems to interfere directly with the kinase, a haemin induced inhibition of the eEF-2 kinase in other cell types than in reticulocytes could be expected. Indeed, haemin induced inhibition of the eEF-2 phosphorylation has also been observed in homogenates from liver and myoblasts (not illustrated). This may indicate that regulation of the elongation rate by reduced intracellular concentrations of haemin plays a more important role in other cells than reticulocytes.

It has been observed that the extent of phosphorylation of eEF-2 and the activity of the eEF-2 kinase varies during the cell cycle [51,52] and that proliferating cells display higher eEF-2 kinase activity than quiescent cells [19,53]. This may suggests that eEF-2 phosphorylation is involved in fine tuning of the translational rate. A slowing down of the elongation cycle may favour translation of mRNAs that are bad competitors for the translational initiation machin- ery [54]. Such an effect has been observed in the presence of low concentrations of the elongation inhibitor cyclohex- imide [54-57].

Acknowledgements

We are indebted to Birgit Lundberg for skilful technical assistance. This work was supported by Grant B-Bu-8463- 307 from the Swedish Natural Science Research Council. Part of this work was presented at the conference on 'The translational apparatus' in Berlin 1992.

3.3. Phospho-eEF-2 and protein synthesis

The role of phosphorylation of eEF-2 in regulating the overall translational efficiency is not clear. The cell contains approximately 1.2 molecules of eEF-2/ribosome [44]. In reticulocytes about 20% of the ribosomes are able to interact with eEF-2, i.e., the amount of ribosomes being in the pre-translocation phase of the elongation cycle [45]. Thus, the cell contains more eEF-2 than is presently engaged in ribosomal interaction under steady state conditions. The cytoplasmic pool of eEF-2 can rapidly become phosphorylated while the ribosome-bound population of the factor is not immediately available as a substrate for the eEF-2 kinase [46]. This could explain the observation that almost 90% of the factor can be phosphorylated without any major effect on the translational efficiency

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effect of oxidizing agents and haemin on the phosphorylation of eukaryotic elongation factor 2 in...

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