Vol. 147, No. 2, 1987 September 15, 1987

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 772-777

PHOSPHOROTHIOATED BINARY COMPLEX OF EUKARYOTIC INITIATION FACTOR eIF-2 AND GDP INHIBITS PROTEIN SYNTHESIS IN HEMIN-SUPPLEMENTED

RETICULOCYTE LYSATES

Robin Hurst and Robert L. Matts

Department of Biochemistry, Oklahoma State University,

Stillwater, OK 74078-0454

Received August 3, 1987

Summary: The rabbit reticulocyte heme-regulated eIF-2a kinase (HRI) utilizes adenosine-S-0-(3- thiotriphosphate) (ATP-y-S) as a substrate for its autophosphorylation and activation, and for the phosphorylation of eIF-2. The phosphorothioated binary complex [eIF-2(a-[3sS]P)*GDP], interacted with the reticulocyte reversing factor (RF) in in vitro assays, and inhibited the ability of RF to catalyze GDP exchange from (eIF-2$H]GDP) complexes. The phosphorothioate residue in the binary complex was resistant to phosphatase action under protein synthesis conditions. eIF- 2(a-[%]P)*GDP inhibited protein synthesis in hemin-supplemented lysates with biphasic kinetics, but had no effect on protein synthesis in heme-deficient lysates. The data reported here indicate that phosphorylation of eIF-2*GDP alone, through the ability of eIF-2(a-P)*GDP to bind and sequester RF, is sufficient to inhibit protein chain initiation in the reticulocyte lysate. 0 1987

Academic Press, Inc.

Introduction: In the reticulocyte lysate, the normal recycling of the eukaryotic initiation factor

eIl-2 is blocked under conditions of heme deficiency or in the presence of low levels of double

stranded RNA (dsRNA) due to the activation of the heme-regulated (HRI) or the dsRNA-activated

(ds1) eJF2a kinase, respectively (1, 2). These kinases specifically phosphorylate the 38 KDa a-

subunit of eIF-2. The inhibition of protein chain initiation occurs due to the binding of the

reticulocyte reversing factor (RF) to phosphorylated binary complex [eIF-2(a-P)*GDP] to give a

FW*eIF-2(a-P) complex that is not readily dissociable (l-5). RF is then unavailable to catalyze the

exchange of GDP in the binary complex for GTP and the formation of the eIF-2*Met-tRNAi*GTP

ternary complex. Hence the recycling of eIF-2 and the initiation of protein synthesis are inhibited

(8-10).

Abbreviau: ATP-y-S, adenosine-5’-O-(3-thiotriphosphate); eIF-2, e&uyotic initiation factor 2; eIF-2a, a(38,OOO dalton) subunit of eIF-2; eIF-2(a-P), eIF-2 phosphorylated on a-subunit; eIF-2 (a-SP), eIF-2 with phosphorothioate residue on the a-subunit; eIF-2*GDP, binary complex of eIF- 2 with GDP; eIF-2(a-P)*GDP or eIF-2(a-SP)*GDP, binary complex of eIF-2 with GDP, phosphorylated or phosphorothioated on the a-subunit of eIF-2; RF, reversing factor (also referred to in the literature as GEF and eIF-2B); RF*eIF-2(a-P) or RFeeIF-2(a-SP), complex of RF with eIF-2 either phosphorylated or phosphorothioated on its a-subunit; HRI, heme-regulated eIF-2a kinase; dsRNA, double stranded RNA; ds1, dsRNA-activated eIF-2a kinase, NaDodSOq, sodium dodecyl sulfate.

0006-291X/87 $1.50 CoDright 0 1987 by Academic Press, Inc. AN righb of reproduction in any form reserved. 772

Vol. 147, No. 2, 1987 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

While this mechanism for the regulation of protein chain initiation is generally agreed upon (1,

2), an alternative model has been proposed (6). The existence of highly active eIF-2a phosphatase

activity in the reticulocyte lysate (7) has made it impossible for investigators to unambiguously test the consequences of elF-20: phosphorylation by simply examining the activity of phosphorylated

eIF-2 upon its addition to normal or inhibited lysates. Adenosine-S-0-(3-thiotriphosphate) (ATP-

r-S) is a good substrate for many protein kinases (8) and the phosphorothioate residue transferred

to protein has been found to be resistant to the action of phosphatases. Hence one can study the

effect of protein phosphorylation under conditions where dephosphorylation would also be

occurring. We report here the use of ATP-YP in the phosphorothioation of elF-2 and the effects of

phosphorothioated binary complex [eIF-2(a-SP)*GDP] on protein synthesis in the reticulocyte

lysate.

Matera and Methods: Adenosine-5’-0-(3-thiotriphosphate) was obtained from Boehringer Mannheim and adenosine-5-y-[3?S] thiotriphosphate (1159 Ci/mmol) was from Amersham. L- [4,5-3Hj Leucine (60 Ci/mmol), [8-3H]GDP (5 Ci/mmol) and [y-32P] ATP (1000-3000 Ci/mmol) were purchased from New England Nuclear. GDP and CM-Sephadex were from Sigma Chemical. HRI was purified as described previously (3, 9). RF (>90% pure) was prepared as an RF*eIF-2 complex as described (3). eIF-2, eluted with buffer containing 350 mM KC1 from the CM-Sephadex step of the RF purification (10) was further purified by phosphocellulose chromatography and glycerol gradient centrifugation (11). This yields a preparation of eIF-2 from the post-ribosomal supernatant of rabbit reticulocyte lysate which is approximately 90% pure. Reticulocyte lysate was prepared from anemic rabbits as described (12). Prenaration of the elF-2la-135SlP)*GDP Complex: Binary complex was preformed by incubating approximately 250 pmol of eIF-2 in the presence of 12.5 J.&I GDP in buffer containing 10 mM Tris-HCl (pH 7.6), 50 mM KCl, 0.5 mM dithiothreitol, 0.1 mM EDTA and 5% glycerol for 10 min at 3O’C. The elF-2*GDP binary complex was stabilized on ice by the addition of Mg(OAc)z to a concentration of 2 mM. The binary complex was then phosphorylated at 3O’C for 60 min in an incubation mixture (200 l.tl final volume) containing 20 n&I Tris-HCl (pH 7.6), 50 mM KCl, 0.5 mM dithiothreitol, 2 mM Mg(OAc)z, 0.05 mM EDTA, 2.5% glycerol, 5 lg HRI, 50 l.tM adenosine-5’-O-(3-thiotriphosphate) and 125 uCi of adenosine-5-@S] thiotriphosphate (final specific activity - 12.5 Ci/mmol). The incubation was cooled on ice and applied to a 0.1 ml CM- Sephadex column equilibrated with buffer containing 20 mM Tris-HCl (pH 7.6), 50 mM KCl, 1.5 mM Mg(OAc)s, 1 mM dithiothreitol, 0.2 n-&f EDTA and 10% glycerol (Buffer A + 50 mM KCl). The column was washed with 10 column volumes of the above buffer and the eIF-2(a-[35S]P)*GDP complex was eluted with buffer A + 250 mM KCl. Single drop fractions of approximately 25 1.11 were monitored for radioactivity. Approximately 70% of the complex was found in a single fraction (fourth fraction eluted). The yield (-15 to 20%) of the eIF-2(a- [35S]P)*GDP complex was poor, but the eIF-2 was found to be free of HRI, which passes through the column in the flow through (Buffer A + 50 mM KCl). Unphosphorylated eIF-2 elutes from CM-Sephadex at 350 mM KC1 (10). The time course of the phosphorylation of eIF-2*GDP by I-IRI in the presence of ATP-Y[~~S] was determined by analyzing aliquots (4 p.l) on 10% NaDodSOq/polyacrylamide gels (37.5:1 acrylamide:bis) as described (13). Bands were excised and counted in 5 ml of Econoflour (New England Nuclear) to qua&ate the [35S] incorporated into the proteins. . . P otein Svnthesls Ret iculocyte LvsaQ: Protein synthesis was measured by the incorporation of $H]leucine (- 6O~mCilmmol final specific activity) into protein in standard reticulocyte lysate reaction mixtures (50 ~1) as described (12, 14). The dephosphorylation of eIF-2(a-[35S]PpGDP complex added to protein synthesis mixes was followed simultaneously by dual label determination. ufect of eTF-2(r&5SlP)*GDP on RF Activitv in vitro: Binary complex was preformed by incubating eIF-2 for 12 min at 30°C in the presence of buffer containing 20 mM Tris-HCl (pH 7.6), 100 mM KCl, 1 n&l dithiothreitol, 2 PM [IH]GDP, and 100 pg/ml creatine kinase as carrier

773

Vol. 147, No. 2, 1987 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

protein. The binary complex was stabilized on ice by the addition of Mg(OAc)z to 1 mM concentration. Incubations contained either 7.5 or 12.5 pmol eIF-2 per 50 J incubation. RF (1.25 pmol) was preincubated for 12 min at 30°C in 50 ul of buffer containing 20 mM Tris*HCl (pH 7.6), 100 mM KCl, 1 mM dithiothreitol, 1 mM Mg(OAc)z, 40 J.&I GDP, 100 pg/mI creatine kinase as carrier and either 1.25 pmol eIF-2, 1.25 pmol eIF-2(a-[35S]P), 4.25 pmol eIF-2 or 4.25 pmol eIF-2(c@S]P). Binary complex (50 ~1) was then added and 20 ul aliquots were withdrawn at 0,4, 8 and 12 min, and undissociated eIF-2*[3H]GDP remaining was bound to nitrocellulose filters as described previously (3, 5). To prevent isotopic dilution from being mistaken for inhibition of RF catalyzed chase of [3H]GDP from eIF-2, 7.5 pmol and 12.5 pm01 of eIF-2*[3H]GDP were added to incubations containing 1.25 pmol eIF-2*GDP or eIF-2(c@S]P)*GDP, and 4.25 pmol eIF-2*GDP or eIF-2(cx-[35S]P)*GDP, respectively. This maintained the specific activity of the binary complex in the final incubations within about 14% of one another. Assays eIF-2cc phosphatase activib: Phosphatase activity was measured by the ability of lysates to dephosphorylate exogenously added eW2(&2P])*GDP or eIF-2(a-[35S]P)*GDP under conditions for protein synthesis (7). Approximately 5 pm01 of eIF-2 were incubated in buffer containing 20 mM Tris-HCl (pH 7.4), 50 mM KCl, 0.1 mM DTT and 25 pM GDP for 10 min at 30°C. The eIF-2*GDP complex was stabilized by the addition of Mg(OAc)z to a concentration of 1 mM and the eIF-2*GDP complex was phosphorylated by the addition of 50 @4 [+P]ATP (10 Wmmol) or [35S]ATP-y-S (10 Ci/mmol) and 0.2 ug of purified HRL After incubation for 20 min at 30°C the specific activity of the ATP was diluted 20 fold by the addition of unlabeled ATP. The labeled complexes (10 ul final volume) were added to 40 ul of hemin-supplemented reticulocyte lysates which were then incubated at 30°C under conditions for protein synthesis. Aliquots (6 p.l) were removed at 0, 2.5, 5, 10, 15, 20 and 30 min, and denatured in NaDodSO4 sample buffer. Proteins were separated by electrophoresis in 8% NaDodS04/polyacrylamide gels (13). The protein bands containing eIF-2(a-[32P]) or eIF-2(a-[35S]P) were visualized by autoradiography and the labeled-eIF-2a remaining was quantitated by excising bands from the gel and counting the gel slices in 5 ml of Econoflour.

Results and Discussion

Time course of the nhosnhorothioation of HRT and eTF-2o: Figure 1 shows that ATP-7-[35S] was

utilized as a substrate for both the autophosphorylation and activation of HRI, and the

phosphorylation of eIF-2. As with ATP (15), an increase in eIF2a kinase activity, as reflected in

the rate of elF-2a phosphorylation, was observed with increased phosphorylation of HRI.

Preparation of the eTF-2(a-T35SlPl*GDP complex: HRI and eIF-2*GDP were incubated for 60

min, and the eIF-2(a-[35S]P)*GDP complex was then purified by CM-Sephadex chromatography.

The product was analyzed by NaDodS04/polyacrylamide gel electrophoresis, followed by

autoradiography. Greater than 95% of the radioactivity present in the eIF-2(a-[35S]P)*GDP

preparation was found to be incorporated into the a-subunit of eIF-2. Two other minor bands

were also visible. One band corresponded to the g-subunit of eIF-2, indicating that the eIF-2g

(casein) kinase (16, 17), which is present in the eIF-2 preparation as a minor contaminant can also

use ATP-YS as a substrate. The second minor band corresponded to a 35 KDa protein, which is a

minor contaminant present in the eIF-2 preparation. No [35S] labeled-HRI was observed to be

present in the eIF-2(a-[35S]P)*GDP preparation.

Inhibition of RF catalvzed dissociation of GDP from unohosnhorvla&d eIF-2*[3HlGDP comm

by eIF 2fa - - 1 35S1PI*GDP in vitro: In in vitro assays where the ratios of eIF-2(c@S]P)*GDP to RF were 1: 1 and 3.4: 1, the initial rates of RF catalyzed GDP exchange were decreased by 67% and

80%, respectively (Fig. 2). Previous studies (3, 4) have demonstrated that incubating RF in the

presence of increasing levels of eIF-2(c(-P)*GDP leads to increased formation of RF*eIF-2(a-P)

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0 1

10 - -a- HRI

8-

a- control * 12.5 pmollml -W 42.5 pmollml + control

-&

0 10 20 30 40 50 0 5 10 15

time (min) 0 2 time (min)

Figure 1. Time course of the incorporation of [35S] labeled phosphorothioate into H.N ( 13 - 0)) and-a-subunit of elF-2( m - I).

Figure 2. Effect of eIF-2(a-[35S]P)*GDP on RF catalyzed GDP exchange in vitro. The rate of RF catalyzed GDP exchange was assayed in the presence of 1.25 pm01 of elF2aGDP (A - A), 1.25 pm01 of elF-2(a-[35S]P)*GDP (A - A), 4.25 pmol of eIF-2*GDP([7 -n), or 4.25 pm01 of @- 2(a-[35S]P)*GDP (H-I), and either 7.5 pmol of eIF-2*[3H]GDP [(A - A) and (A - A)] or 12.5 pm01 of eIF-2*[3H]GDP [( 0 - 0) and m - R)] as substrate, as described under “Experimental Procedures”.

and inhibition of RF mediated guanine nucleotide exchange. The present data indicated that the

eW2(c@S]P)*GDP complex can interact normally with RF and sequester it in an inactive

complex.

The effect of eIF-2(o-l35SIP)*GDP on protein synthesis: Protein synthesis in hemin-supplemented

and heme-deficient lysates was examined, while simultaneously monitoring for the loss of protein

bound [35S]. Addition of 1.8 or 2.7 pmol of eIF-2(o-[35S]P)*GDP complex to hemin

supplemented lysates (40 ~1) was found to inhibit protein synthesis by 35 and 55%, respectively

(Fig. 3); the inhibition occurred with biphasic kinetics. Addition of the eIF-2(a-[35S]P) complex

(2.7 pmol) to heme-deficient lysate caused no stimulation of protein synthesis. A loss of

approximately 10% of the protein bound [35S] was observed during the 30 min time period of the experiment. To examine more closely the dephosphorylation of eIF-2, eIF-2(a-[32P])*GDP or

eIF-2(a-[35S]P)*GDP were added to hemin-supplemented lysates and the label remaining bound to

eIF-2a after various times of incubation was analyzed by NaDodSO4lpolyacrylamide gel

electrophoresis (Fig. 4). The [35S] phosphorothioate residue transferred to the a-subunit of eIF-2

by HRI was found to be completely resistant to the action of phosphatases. In comparison,

approximately 50% of the bound [32P] phosphate was hydrolyzed from the eIF-2(a-[32P])*GDP

complex within 2.5 min of incubation, and 95% was removed by 15 min.

The data reported here indicate that ATP-y-S can be used as a substrate by HRI for its

activation, and for the phosphorylation of eIF-2, and that the phosphorothioate residue, which is

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Vol. 147, No. 2, 1987 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

4 +h + 1.8 pmol + + h + 2.7 pmol

+ -h + 2.7 pmol

0 10 20 30 40

Time (min)

Figure 3. Effect of eIF-2(a-[%]P)*GDP on protein synthesis in hemin-supplemented and heme- aeftcrent lysates. Reticulocyte lysates (40 ul) were incubated under conditions for protein synthesis as follows: without hemin ( 0 - 0); pmol eIF-2(u-[%]P)*GDP (I - I);

with 20 u.M hemin (X - X); without hemin plus 2.7

of eIF-2(u-[%]P)*GDP. with 20 &l hemin plus 1.8 pmol (A - A) or 2.7 pmol (A - A)

transferred to the a-subunit of eIF-2, is metabolically stable and resistant to phosphatase action in

the reticulocyte lysate. Similar results were recently reported by Ranu (18). In addition, Ranu

reported that phosphorothioated eIF-2 [eIF-2(a-SP)] was less effective than phosphorylated eIF-2

[eIF-2(a-P)] in promoting protein synthesis in heme-deficient lysates, presumably due to its

resistance to dephosphorylation. However, interpretation of this result is ambiguous since free

eIF-2, and not eIF-2*GDP, was used as a substrate for phosphorothioation; and HRI and any

unphosphorylated eIF-2 possibly remaining were not removed from the eIF-2(u-SP) prior to its

addition to the protein synthesis assay. Free eIF-2(a-SP) could form both binary and ternary

complexes upon addition to the lysate, and therefore, cause a slight stimulation of protein

-elF-2( a-[ 32P])

- elF-2( a -I 35 SIP)

0 2.5 5 10 15 20 30

(min)

Figure 4. Rate of dephosphorylation of eIF-2(a-[3*P])*GDP (A) and eIF-2 (a-[%]P~GDP (E8) m the retxulocyte lysate. The figure is an autoradiogram.

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synthesis. The current view of protein chain initiation is that the recycling of eIF-2 requires RF

catalyzed exchange of GDP for GTP. When eIF-2a kinases are activated, inhibition of protein

chain initiation occurs due to the binding of RF to phosphorylated binary complex to give a

RF*eIF-2(a-P) complex which is not readily dissociable and inactive. This hypothesis predicts

that the utilization of the eIF-2(a-SP)*GDP complex in protein chain initiation requires its

interaction with RF, and that upon its interaction with RF, an inactive RF*eIl?-2(a-SP) complex

will be formed. Thus, if the phosphorothioate residue is metabolically stable, the addition of eIF-

2(a-SP)*GDP to hemin-supplemented lysates should inhibit protein synthesis with biphasic

kinetics and its addition to heme-deficient lysate should have no stimulatory effect on protein

synthesis. This is what was observed experimentally. Therefore, the modification of no other

protein, and no other event, other than the binding of RF to phosphorylated binary complex, is

necessary to explain how protein chain initiation is inhibited in the reticulocyte lysate upon

activation of an eIF-2a kinase.

ckno $pokuv&thene, Durvis Hulen and Yining Zhou, for carrying out the initial dual iabel proteii

edeements: We would like to thank the students of Biochemistry 5930 Jinhua An

synthesis experiment. This work has been supported by the Oklahoma Agricultural Experiment Station of which this is publication no. J-5209.

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