Inhibition Rabbit Reticulocyte Lysate Protein Synthesis by ... · THE .JOURNAL OF BIOLOGICAL CHEMISTRY 0 1987 by The American Society for Biochemistry and Molecular Biology, Inc Vol

THE .JOURNAL OF BIOLOGICAL CHEMISTRY 0 1987 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 262, No. 33, Issue of November 25, pp. 1.5939-15945,1987 Printed in U.S.A.

Inhibition of Rabbit Reticulocyte Lysate Protein Synthesis by Heavy Metal Ions Involves the Phosphorylation of the cu-Subunit of the Eukaryotic Initiation Factor 2”

(Received for publication, May 12, 1987)

Robin Hurst, Jeffery R. Schatz, and Robert L. Matts$ From the Department of Biochemistry, Oklahoma State University, Stillwater, Oklahoma 74078-0454

The effect of heavy metal ions (in particular Cd”, Hg2+, and Pb”) on protein synthesis in hemin-supplemented reticulocyte lysates was investigated. Heavy metal ions were found to inhibit protein synthesis in hemin-supplemented lysates with biphasic kinetics. The shut off of protein synthesis occurred in conjunction with the phosphorylation of the a-subunit of the eukaryotic initiation factor (eIF) 2, the loss of reversing factor (RF) activity, and the disaggregation of polyribosomes. Addition of eIF-2 or RF to heavy metal ion-inhibited lysates restored protein synthesis to levels observed in hemin-supplemented controls. The stimulation of protein synthesis observed upon the addition of CAMP to heavy metal ion-inhibited lysates correlated with the inhibition of eIF-2a phosphorylation and the restoration of RF activity. The partial restoration of protein synthesis observed upon the addition of MgGTP to heavy metal ion-inhibited lysates correlated with a partial inhibition of eIF-2a phosphorylation. Addition of glucose 6-phosphate was found to have no effect on protein synthesis or eIF-2a phosphorylation under these conditions. Antiserum raised to the reticulocyte heme-regulated eIF-2a kinase inhibited the phosphorylation of eIF-2a catalyzed by Hg*+- inhibited lysate. The inhibition of protein synthesis observed in the presence of heavy metal ions correlated with the relative biological toxicity of the ions. Highly toxic ions (AsO;, Cd2+, Hg”, Pb2+) inhibited protein synthesis by 50% at concentrations of 2.5-10 PM. Cu2+, Fe3+, and Zn2+, which are moderately to slightly toxic ions, inhibited protein synthesis by 50% at concentrations of 40, 250, and 300 PM, respectively. The data presented here indicate that heavy metal ions inhibit protein chain initiation in hemin-supplemented lysates by stimulating the phosphorylation of eIF-2a apparently through the activation of the heme-regulated eIF-2a kinase rather than through inhibition of the rate of eIF-2a dephosphorylation.

The rabbit reticulocyte lysate has been commonly used as a model system for studying the regulation of protein synthesis at the level of translation because of its lack of interfering transcriptional effects (reviewed in Refs. 1-4). The initiation of protein synthesis in reticulocytes and their lysates is inhib-

* This work was supported by National Institutes of Health Grant R29 ES-04299 and the Oklahoma State University Agricultural Ex- periment Station of which this is journal article 5-5202. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adver- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed.

ited by heme deficiency or in the presence of low levels of double-stranded RNA (dsRNA).’ In lysates, the inhibitions are characterized by a brief period of control linear synthesis, followed by an abrupt decline in this rate and disaggregation of polyribosomes. The inhibition is due to the activation of a heme-regulated (HRI) or a dsRNA-activated protein kinase, respectively, which specifically phosphorylates the 38-kDa a- subunit of the eukaryotic initiation factor eIF-2. The principal effect of the activation of these eIF-2a kinases is the impair- ment of the recycling of eIF-2 (1-4). eIF-2 forms a ternary complex with initiator Met-tRNAi and GTP [eIF-2 .Met- tRNAi.GTP]. This complex subsequently binds to the 40 S ribosomal subunit to give a 43 S complex which is stabilized by eIF-3. The binding of mRNA to this 43 S complex occurs in the presence of a number of other initiation factors and is accompanied by the hydrolysis of ATP. Upon the joining of the 60 S ribosomal subunit to this 48 S initiation complex, the GTP present in the ternary complex is hydrolyzed in the presence of eIF-5. Recent studies have suggested that a ribo- some-bound eIF-2.GDP intermediate is generated at this stage (5-8) and that this complex may be associated with the 60 S ribosomal subunit of the completed 80 S initiation complex (5, 6). The binary complex formed between eIF-2 and GDP is highly stable, and the recycling of eIF-2 in initiation requires the replacement of GDP by GTP. This exchange is catalyzed by a multipolypeptide (5-subunit) factor, referred to here as the reversing factor (RF) (9-14) (also currently designated GEF, guanine nucleotide exchange factor (12, 13), and eIF-2B (14)). The inhibition of protein chain initiation which occurs upon the activation of eIF-2a kinases is due to the binding of RF to the phosphorylated binary complex (eIF-2(aP).GDP), which yields a RF.eIF-2(aP) complex that is not readily dissociable (8, 11, 15, 16). Since the amount of RF present in the lysate is much lower than the amount of eIF-2 present, phosphorylation of only 20-40% of the eIF-2a present has been shown to be sufficient to render RF unavailable to catalyze GTP/GDP exchange (16, 17). Hence, the recycling of eIF-2 and the initiation of protein synthesis are inhibited.

Inhibition of protein synthesis has also been shown to occur in hemin-supplemented lysates under a variety of other conditions. These include: gel filtration to remove glucose 6- phosphate (18-22); affinity chromatography on 2’,5’-ADP- Sepharose which removes glucose-6-phosphate dehydrogen-

The abbreviations used are: dsRNA, double-stranded RNA; eIF- 2, eukaryotic initiation factor 2; eIF-Pa, 38,000-dalton a-subunit of eIF-2; eIF-Z(aP), eIF-2 phosphorylated on a-subunit; RF, reversing factor (also referred to in the literature as GEF and eIF-2B); HRI, heme-regulated eIF-La kinase; Poly LC, polyinosinic polycytidylic acid; NaDodSO,, sodium dodecyl sulfate; Glu-6-P, glucose 6-phosphate.

15939

15940 Inhibition of Protein Synthesis by Heavy Metal Ions

ase, glutathione reductase, and thioredoxin reductase (21,22); addition of oxidized glutathione (GSSG) (18,23-25), N-ethyl- maleimide (26), or lipid peroxides (27); high partial pressures of O2 (PO*) (28); and heat shock (29, 30). The inhibition of protein synthesis under these conditions occurs due to the activation of an eIF-2a kinase, with properties similar to HRI. Many agents which commonly elicit the heat shock or stress response in eukaryotic cells are also capable of generating reactive oxygen species, reacting with reduced sulfhydryl groups, or inhibiting the ability of the cell to generate reducing equivalents (31-34). Among these agents are the heavy metal ions (AsO;, Cd*+, Cu2+, H$+, Pb", and Zn2+), which have been shown to inhibit protein synthesis and disaggregate polyribosomes (35-39) and which elicit the stress response in a variety of eukaryotic cell types and tissues (32-34). The initial phase of the stress response is typically characterized by a shutdown of normal protein synthesis due to an inhibition of initiation (32-34). Phosphorylation of eIF-2 has been reported to occur in HeLa cells in response to heat shock (40), apparently due to the activation of an eIF-2a kinase with antigenic properties similar to HRI (41). Therefore, the reticulocyte lysate was selected as the model system in which to initiate studies on the mechanism by which heavy metal ions inhibit protein synthesis in eukaryotic cells.

EXPERIMENTAL PROCEDURES

Materiak-GTP, CAMP, glucose 6-phosphate, oxidized glutathione, dithiothreitol, and Poly I:C were purchased from Sigma. L-["C] Leucine (340 mCi/mmol), [-y-3ZP]ATP (1000-3000 Ci/mmol), and [8- 3H]GDP (5 Ci/mmol) were obtained from Du Pont-New England Nuclear. RF (>go% pure) was prepared as an RF.eIF-2 complex as described (11). eIF-2, eluted with buffer containing 350 mM KC1 from the CM-Sephadex step of the RF preparation (10, l l ) , was further purified by phosphocellulose chromatography and glycerol gradient centrifugation (42). This yields a preparation of eIF-2 from the postribosomal supernatant of rabbit reticulocyte lysate which is approximately 90% pure. Anti-HRI antiserum was generously provided by Dr. Ray Petryshyn (SUNY-Health Sciences Center, Syracuse, NY).

Protein Synthesis in Reticulocyte Lysates-Reticulocyte lysates were prepared from anemic rabbits as described (43). Protein synthesis was measured by the incorporation of ['4C]leucine into protein at 30 "C in standard reticulocyte lysate reaction mixtures (50 pl) as described (18, 43). Where indicated, protein synthesis was inhibited by the omission of hemin or by the addition of metal ions. Solutions of metal ions Cd(OAc)p, Pb(OAc)p, Zn(OAc)*, CuC12, FeCl3, HgC12, and NaAs02 were prepared in deionized water. All ions were found to inhibit protein synthesis with biphasic kinetics. Several lysate preparations were used in the series of experiments reported here. Each lysate varied slightly in its sensitivity to inhibition by a given ion, but in general the order of potency was found to be H%+ > Pb2+> Cd2+.

Phosphoprotein Profiles-Protein-synthesizing lysates were pulsed with [-y-3ZP]ATP (0.5 pCi/pl protein synthesis mix) at 4-8 min or 16- 20 min (before or after shutoff). Aliquots (5 pl) were added to 0.5 ml of 50 mM NaF, 5 mM EDTA and adjusted to pH 5 with acetic acid (8). The pH 5 precipitates were recovered by centrifugation, dissolved in NaDodS04 sample buffer, and analyzed by electrophoresis on 8% NaDodS04-polyacrylamide gels (37.5:l acry1amide:bis) followed by autoradiography as described (8).

Polyribosome Gradients-Protein synthesis mixes (75 pl) were placed on ice and immediately diluted with 125 pl of ice-cold buffer containing 25 mM Tris-HC1 (pH 7.6), 2 mM Mg(OAc)z, 0.1 mM dithiothreitol, 25 mM KCI, and 25 mM KF. Samples were layered over a 5-ml linear 15-40% (w/v) sucrose gradient containing the above buffer and centrifuged at 189,000 X g,, (45,000 rpm) at 2 "C in a Beckman SW 50.1 rotor for the times indicated in the figure. Ribo- some profiles were monitored at 254 nm by upward displacement of the gradient with an Isco (model 640) density gradient fractionator. Since no polyribosomes were found to be present, He-inhibited lysates were centrifuged for a longer period of time than the control jn order to resolve the 40,60, and 80 S ribosomes.

Assays for RF and elF-Sa Phosphatase Actioity-RF activity was

assayed by determining the rate at which [3H]GDP is chased from preformed eIF-2. [3H]GDP complexes added to reticulocyte lysates incubated under conditions for protein synthesis, as described previously (16, 17). Phosphatase activity was measured by the ability of lysates to dephosphorylate exogenously added ~ I F - ~ ( C Y - ~ ' P ) . GDP under conditions for protein synthesis. Approximately 5 pmol of eIF-2 was incubated in buffer containing 20 mM Tris-HC1 (pH 7.4), 50 mM KC1,O.l mM dithiothreitol, and 25 p h ~ GDP for 10 min at 30 "C. The eIF-2.GDP complex was stabilized by the addition of Mg(0Ac)' to a concentration of 1 mM, and the eIF-2.GDP complex was phosphorylated by the addition of 50 p~ [T-~'P]ATP (10 Ci/mmol) and 0.2 pg of purified HRI (11). 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 eIF-2(w3'P) .GDP complex (10 pl final volume) was added to 40 pl of hemin-supplemented reticulocyte lysates which had been preincubated at 30 "C for 15 min under conditions for protein synthesis with or without the addition of 50 pM H e or Cd2+. Aliquots (6 pl) were removed at 0, 2.5, 5, 10, and 15 min and denatured in NaDodSO, sample buffer. Proteins were separated by electrophoresis in 8% NaDodS04-polyacrylamide gels (8). The protein bands containing eIF-2(m3'P) were visualized by autoradiography, and the 32P-labeled eIF-201 remaining was quantitated by excising the band from the gel and counting the gel slice in 5 ml of Econofluor (Du Pont-New England Nuclear) in a scintillation counter.

RESULTS

Effect of Heavy Metal Ions on Protein Synthesis in Hemin- supplemented Reticulocyte Lysates-Using the protein-synthesizing rabbit reticulocyte lysate system, model studies were initiated to determine the mechanism by which heavy metal ions affect protein synthesis in eukaryotic cells. While a range of metal ions was examined, we chose to concentrate these studies on the effects of mercury, lead, and cadmium ions, as these are considered to be the most important and toxic heavy metal ions present in the environment to which living orga- nisms are currently exposed. Protein synthesis in heme-supplemented reticulocyte lysates was found to be inhibited in the presence of H F , Pb", or Cd2+ (Fig. la) with biphasic kinetics, and this inhibition was accompanied by the disaggregation of polyribosomes (Fig. lb, +h+Hg). These properties are characteristic of protein synthesis being inhibited at the level of protein chain initiation. The data in Fig. la indicate that heavy metal ions differ in their relative abilities to inhibit protein synthesis in the reticulocyte lysate. Various heavy metal ions were titrated into hemin-supplemented reticulocyte lysates (Fig. 2) to determine the concentration of metal ion required to inhibit protein synthesis by 50% in a 30-min incubation. This relative value for the potency ,of a given metal ion in inhibiting protein synthesis was found to correlate generally with the known toxicity of the ions to eukaryotic cells. Highly toxic ions (AsO;, Cd2+, H e , and Pb") inhibited protein synthesis by 50% at concentrations between 2.5 and 10 p ~ . In comparison, Cu2+, which is moderately toxic, and Fe3+ and Zn2+, which are slightly toxic, inhibited protein synthesis by 50% a t concentrations of 40, 250, and 300 p ~ , respectively. The concentration of oxidized glutathione (GSSG) required to inhibit protein synthesis by 50% in these lysates was approximately 250 pM.

Addition of GSSG to hemin-supplemented reticulocyte lysates apparently brings about the activation of HRI, which leads to the phosphorylation of eIF-2 and the inhibition of protein synthesis (18, 23-25). Addition of 10 mM CAMP, 2 mM MgGTP, eIF-2, or RF is known to stimulate protein synthesis in lysates where protein chain initiation is inhibited due to the activation of eIF-2a kinases (9-11, 44). Glucose 6- phosphate (Glu-6-P) also stimulates protein synthesis in GSSG-inhibited lysates (18). The inhibition of protein synthesis brought about by the addition of H$+, Pb2+, or Cd2+ to hemin-supplemented lysates is prevented (addition at 0 min)

Inhibition of Protein Synthesis by Heavy Metal Ions 15941

FIG. 1. Effect of Cd2+, H d + , and Pb2+ on protein synthesis in hemin- supplemented reticulocyte lysates. a, protein synthesis in hemin-supplemented reticulocyte lysates. Protein synthesis in standard reticulocyte lysate incubation mixtures (50 pl) containing 20 p~ hemin and no additions (+h, W); 100 ng of Poly I:C (+dsRNA,

0); 25 p~ HgC1, (+Hg, X-X); or 25 PM Pb(OAc), (+Pb, A-A). b, ribo- some profiles in sucrose gradients. Pro- tein-synthesizing lysates containing 20 PM hemin were incubated for 15 min with no additions (+h, upper panel) or with 25 PM HgCl, (+h+Hg, lowerpanel). Samples were diluted and centrifuged for 1.25 h (+h) or 2.25 h (+h+Hg) in sucrose gradients as described under "Experi- mental Procedures."

A-A); 25 FM Cd(0Ac)z (+Cd, b

40000

30000

E 8 9 F ; 20000 s C

a# c - - -I

I

10000

0 0

a. Protein Synthesis

0 +Cd * +Hg -t +dsRNA + +hemin

* + P b 1 10 20 30

Time (min)

120 0- + C d

100 -

80 - 60 - 40 - 20 -

Zn Pb cu Fe

As GSSG

Hg

0 -7 -6 - 5 - 4 - 3 -2

log (molar concentration) FIG. 2. Effect of heavy metal ion concentrations on protein

synthesis in hemin-supplemented reticulocyte lysates. Reticu- locyte lysates containing 20 p~ hemin were incubated for 30 min at 30 "C in the presence of increasing concentrations of Cd(0Ac)Z

FeC13(C"-.); HgCl2(=); NaAsO, (A-A); or GSSG (0-0); Pb(OAc), (X-X); Zn(OAc):! (W); CuC12(+-+);

(A-A). Data is plotted as the log of the heavy metal ion concentration uersus the percent of ['4C]leucine incorporated into protein compared to the hemin-supplemented control (100%). Addition of 100 ng/ml Poly I C was found to reduce protein synthesis to 20% of that observed in the hemin-supplemented control.

and reversed (addition a t 10 min) by the addition of CAMP, eIF-2, or RF (Fig. 3 and Table I). Addition of MgGTP brought about a partial stimulation of protein synthesis in Hg2+-, Pb'+-, or Cd2+-inhibited lysates, while the addition of Glu-6- P was found to have no effect.

Effect of Heavy Metal Ions on eZF-2a Phosphorylation- The properties of the inhibition of protein synthesis in the

b. Ribor- Proflles

0 10 20

40 0 10 Fraction

20

30000

20000

10000

0

1

0 10 20 30 40 0 10 20 30 40

time (mln) time (rnin)

FIG. 3. Stimulation of protein synthesis in Hgz+ ion-inhibited reticulocyte lysates. Protein synthesis in standard reticulocyte lysate incubation mixtures (50 pl) containing 20 p~ hemin: Panel A , no additions (X-X); 25 p~ HgCl, (0-0); 25 PM HgC1, with 2 mM MgGTP added at 0 min (U) or 10 min (W); 25 p~ HgC1, with 10 mM CAMP added at 0 min (A-A) or 10 min (A-A); 25 FM HgCl, with 1 mM Glu-6-P added at 0 min (+) or 10 min (+); Panel B , no additions (X-X); 25 PM HgClz (0-0); 25 PM HgC12 with approximately 10 pmol of purified eIF-2 added at 0 min (U) or 10 min (W); 25 pM HgCl, with approximately 2.5 pmol of purified RF added at 0 min (A--A) or 10 min (A- A).

reticulocyte lysate in response to the addition of heavy metal ions are characteristic of inhibitions of protein synthesis brought about through the activation of eIF-Sa kinases (44). Therefore, we examined the effect of heavy metal ions on the [32P]phosphoprotein profiles in hemin-supplemented reticulocyte lysates. The addition of 50 p~ Hg+, Pb", or Cd2+ stimulated the phosphorylation of the a-subunit of eIF-2 (Fig.

15942 Inhibition of Protein Synthesis by Heavy Metal Ions TABLE I

Stimulation of protein synthesis in Cd2+ and Pb2+-inhibited reticulocyte lysates

Reticulocyte lysates (20 p1) containing 20 p~ hemin were incubated for 30 min at 30 "C as follows: with no additions (None); with 25 p~ Cd(OAc)z (+Cd); with 25 p~ Pb(OAc)z (+Pb); with 1 mM Glu-6-P, 2 mM MgGTP, 10 mM CAMP, 2 pmol of purified eIF-2, or 0.5 pmol of purified RF added either at the beginning of the incubation (0 min) or after 10 min of incubation (10 min). Data shows counts/min of ["Clleucine incorporated into acid-precipitable protein from a 5-p1 aliquot of the incubation mixture.

Additions Other Time of ["CILeucine a t 0 min additions addition incorporated

I. -None +Cd +Cd +Cd +Cd +Cd +Cd +Cd +Pb +Pb +Pb +Pb +Pb +Pb +Pb

11. -None +Cd +Cd +Cd +Cd +Cd +Pb +Pb +Pb +Pb +Pb

mM

Glu-6-P (1.0) Glu-6-P (1.0) MgGTP (2.0) MgGTP (2.0) cAMP (10) cAMP (10)

Glu-6-P (1.0) Glu-6-P (1.0) MgGTP (2.0) MgGTP (2.0) cAMP (10) cAMP (IO)

eIF-2 eIF-2 RF RF

eIF-2 eIF-2 RF

min

0 10 0

10 0

10

0 10 0

10 0

10

0 10 0

10

0 10 0

cpm X

32 16 15 17 21 18 36 31 15 16 14 20 18 32 28

31 15 30 27 31 24 14 31 26 30

RF 10 27

" " elF-2 a

1 2 3 4 5 6 7 8 FIG. 4. Effect of heavy metal ions on eIF-2a phosphoryla-

tion. Reticulocyte lysates (20 pl) were incubated under conditions for protein synthesis as follows: lane 1 , without hemin; lane 2, with 20 p~ hemin; lune 3, with 20 p~ hemin plus 500 p~ GSSG; lane 4 , with 20 p~ hemin plus 50 p~ HgClz; lane 5, with 20 pM hemin plus 50 p~ Pb(OAc),; lane 6, with 20 p~ hemin plus 50 p~ Cd(0Ac)z; lane 7, with 20 p~ hemin plus 50 pM Zn(0Ac)Z; lane 8, with 20 pM hemin plus 500 p~ Zn(0Ac)z. Lysates were pulsed with [y-32P]ATP from 4- 8 min (before shutoff) and aliquots (5 pl) were analyzed as described under "Experimental Procedures." Lysates which were pulsed with [Y-'~P]ATP from 16-20 min (after shutoff) showed essentially the same phosphorylation profiles. The figure is an autoradiogram.

4). The relative abilities of the metal ions in stimulating eIF- 2a phosphorylation (H$+ L Pb2+ > Cd2+) correlate with the relative potencies of the ions in inhibiting protein synthesis in this lysate ( H e L Pb2+ > Cd") (see Fig. 1). Zn", a t a concentration of 50 PM, did not inhibit protein synthesis and had no effect on eIF-2a phosphorylation, while 500 PM Zn2+, which inhibits protein synthesis (Fig. 2), was found to stimulate the phosphorylation of eIF-2a (Fig. 4).

The effects of CAMP, MgGTP, and Glu-6-P on eIF-2a phosphorylation in H$+-inhibited lysates were then examined. Addition of 10 mM CAMP, which completely restores protein synthesis in heavy metal ion-inhibited lysates, totally prevented Hg2+-stimulated phosphorylation of eIF-2a (Fig. 5 , lane 6); MgGTP, which partially restores protein synthesis in He- inhib i ted lysates, partially inhibited the phosphorylation of eIF-2a (Fig. 5, lane 4 ) ; and the addition of Glu-6-P, which has no effect on protein synthesis in H$+-inhibited lysates, had no effect on eIF-2a phosphorylation (Fig. 5, lane 5 ) . Similar results were observed in Pb2+- and Cd2+-inhibited lysates (data not shown). Therefore, the ability of an agent to stimulate protein synthesis in heavy metal ion-inhibited reticulocyte lysates was found to correlate with their ability to inhibit eIF-2a phosphorylation.

Effect of Heavy Metal Ions on Reticulocyte Lysate RF Activ- ity-The phosphorylation of eIF-2 in heme-deficient, dsRNA- inhibited or GSSG-inhibited reticulocyte lysates leads to the inhibition of RF catalyzed GTP/GDP exchange through the sequestration of RF in an RF-eIF-S(aP) complex (16, 17). The loss of RF activity is time-dependent and correlates with the stimulation of eIF-2a kinase activity, the phosphorylation of eIF-2, and the shut off of protein synthesis (16, 17). Similarly, in hemin-supplemented lysates in which protein synthesis is inhibited by the addition of H$+, there is an inhibition of RF-catalyzed eIF-2. ['HH]GDP dissociation (Fig. 6A). The loss of RF activity was time-dependent and correlated with the shutoff of protein synthesis in the lysate (Fig. 6B and Fig. 3). Restoration of protein synthesis in H$+- inhibited lysates upon the addition of 10 mM cAMP occurred

A. B.

- - "elF-2 a

1 2 3 4 5 6 1 2 3 4 5 6 FIG. 5. Effect of MgGTP, Glu-6-P, and CAMP on eIF-2a

phosphorylation in Hg2+-inhibited lysates. Reticulocyte lysates (20 pl) were incubated under conditions for protein synthesis as follows: lane 1, without hemin; lane 2, with 20 p~ hemin; lane 3, with 20 p M hemin plus 25 pM HgC1,; lane 4, with 20 p~ hemin plus 25 p~ HgC12 and 2 mM MgGTP; lane 5, with 20 p~ hemin plus 25 p~ HgCl, and 1 mM Glu-6-P; lane 6, with 20 pM hemin plus 25 p~ HgC12 and 10 mM CAMP. Panel A , MgGTP, Glu-6-P, and cAMP were added at the beginning of the incubation, and the lysates were pulsed with [y- "PIATP from 4-8 min. Panel B, MgGTP, Glu-6-P, and cAMP were added at 10 min, and lysates were pulsed with [y-"PIATP from 16- 20 min. Aliquots (5 pl) were analyzed as described under "Experi- mental Procedures." The figure is an autoradiogram.


0 2 4 6 8 1 0 1 2

A +hemin

0 -hemin 0.0 , I . ,

0 10 20 30 time fminl

FIG. 6. Effect of Hg2+ on RF activity in hemin-supplemented lysates. A , protein synthesis mixtures (200 pl, 30 "C) were diluted with an equal volume of dilution buffer (17) followed by the immediate addition of 20 p1 containing 10 pmol of preformed eIF-2.['H]GDP complex. 100-pl aliquots were taken at the times indicated in the figure, and the amount of [3H]GDP dissociated from eIF-2 was determined as described previously (16, 17). Lysates were preincubated as follows: without hemin for 15 min (X-X); with 20 p~ hemin for 15 min (0-0); with 20 p M hemin plus 10 p M HgClz for 0 min (A-A), 5 min (+-+), 10 min (U), and 15 min (M); with 20 pM hemin plus 10 pM HgC12 for 15 min, with 10 mM cAMP added a t 10 min (A-A). B, protein synthesis mixtures (50 pl) were preincubated at 30 "C. At the times indicated in the figure, samples were diluted with an equal volume of dilution buffer (17), and 2 pmol of preformed eIF-2.['H]GDP was added. The amount of [3H]GDP dissociated from eIF-2 in 5 min was determined as previously described (16, 17). Lysates were incubated as follows: without hemin (0); with 20 p~ hemin (A); with 20 p~ hemin plus 10 p~ HgC12 (U); with 20 pM hemin plus 10 pM HgCIZ with 10 mM cAMP added a t 10 min (A).

in conjunction with the restoration of RF activity (Fig. 6). The data indicate that the inhibition of RF-catalyzed GDP exchange correlates with eIF-2a phosphorylation in H$+- inhibited lysates and is not a result of direct inactivation of RF activity by the Hg2+ itself.

Heavy Metal Ion-induced Phosphorylation of eIF-2a Is Cat- alyzed by HRI-An eIF-2a kinase, whose activity is inhibited by an anti-HRI antiserum, is activated in hemin-supplemented reticulocyte lysates upon gel filtration (19), addition of GSSG (45), or heat shock (30). We, therefore, examined the ability of anti-HRI antiserum (46) to inhibit H$+-stimulated eIF-2a phosphorylation in hemin-supplemented lysates. The addition of anti-HRI antiserum markedly inhibited the ability of H$+-inhibited lysate to phosphorylate eIF-2a compared to control assays to which preimmune chicken antiserum was added or to which there were no additions (Fig. 7). The anti-HRI antiserum was found to inhibit the phosphorylation of eIF-2a by heme-deficient lysate to a similar extent compared to the controls. The effect of preimmune and anti-HRI antiserum on the phosphorylation of eIF-2a in uninhibited hemin-supplemented reticulocyte lysate is shown as a control for conditions in which no HRI is active.

Increased eIF-20 phosphorylation and inhibition of protein synthesis has also been observed in hemin-supplemented reticulocyte lysates when phosphoprotein phosphatase I activity

elF-2 a

C I - G I - C I

-h +h +h +Hg

FIG. 7. Effect of anti-HRI antiserum on eIF-Sa phosphorylation catalyzed by Hg2+-inhibited lysates. Reticulocyte lysates (20 pl) were incubated for 20 min under the following conditions: without hemin ( 4 ) ; with 20 p~ hemin (+h); and with 20 p~ hemin plus 50 p~ HgCl2 (+h+Hg). Aliquots (5 pl) were diluted with buffer containing 20 mM Tris-HC1 (pH 7.61, 50 mM KCl, and 2 mM Mg(OAc),. Samples were placed on ice for 20 min with no additions (-), with 1 pl of control antiserum ( C ) , or with 1 p1 of anti-HRI antiserum (I) (46). 6.5 pCi of [y3*P]ATP and 1 pmol of eIF-2 were then added to the samples (20 pl final volume), and the samples were incubated for 5 min a t 30 "C. Samples were denatured in NaDodSOl sample buffer and separated in 8% NaDodS04-polyacrylamide gels as described under "Experimental Procedures." The figure is an autoradiogram.

has been inhibited upon addition of phosphatase inhibitor I- 2 (47). Therefore, we examined the effects of H e and Cd2+ on the ability of hemin-supplemented lysate to dephosphorylate ~IF-~(CV~'P) -GDP. No change in the initial rate of dephosphorylation of exogenously added ~ I F - ~ ( c Y - ~ ' P ) . GDP substrate was observed in H$+- or Cd2+-inhibited lysates compared to the hemin-supplemented control; 50% of the substrate was dephosphorylated within 2 min in both the normal and the heavy metal ion-inhibited lysates (data not shown). However, it must be noted that while the dephosphorylation of exogenously added ~ I F - ~ ( o - ~ ' P ) has been used over the past several years as a tool for investigating whether changes in phosphatase activity have taken place (22,30,48), the results of such experiments are ambiguous. The reason for this is that the dephosphorylation of the RF.eIF-2(aP) complex, and not the dephosphorylation of free eIF-2(aP), is critical for the maintenance and restoration of protein synthesis in lysates (16). We noted in the above experiment that 5% of the eIF-2( L U - ~ ~ P ) . GDP complex added to H$+- or CdZ+- inhibited lysates was resistant to dephosphorylation at the longer incubation times compared to the uninhibited hemin- supplemented control. Therefore, the data indicate that the increased phosphorylation of eIF-2a observed in heavy metal ion-inhibited lysates occurs due to the activation of an eIF- 2a kinase, but it does not rule out the possibility that heavy metal ions might inhibit a phosphatase activity specific for the RF . eIF-2( aP) complex.

DISCUSSION

Maintenance of protein synthesis in hemin-supplemented reticulocyte lysates has been shown to require the presence of certain sugar phosphates (i.e. Glu-6-P), an NADPH-generating system, and a functional thioredoxin/thioredoxin reductase system (18-22). Metabolism of sugar phosphates by way of the pentose phosphate shunt leads to the generation of

15944 Inhibition of Protein Synthesis by Heavy Metal Ions

NADPH. In addition, a direct requirement for sugar phosphates as a stimulatory “cofactor” affecting the rate of protein chain initiation has been demonstrated (18-20). NADPH is also required for the reduction of thioredoxin by thioredoxin reductase (21, 22). The presence of a functional thioredoxin/ thioredoxin reductase system is thought to be required to prevent or reverse disulfide bond formation in cekain sensitive proteins in the lysate. I t is the oxidation of sulfhydryl groups in some “critical” protein(s) that has been proposed to lead to the activation of an eIF-2a kinase in the reticulocyte lysate; this kinase has physical and antigenic properties similar to HRI (19, 22,45).

Uncharacterized cellular oxidation-reduction processes have been implicated in the mechanism by which agents, that induce the heat shock response, are cytotoxic to cells (49-51). Heavy metal ions can mediate oxidation either by direct sulfhydryl oxidation or by generation of active oxygen species (52-54). In addition, heavy metal ions are known to inhibit glutathione peroxidase, an enzyme essential for destroying lipid peroxides and endogenous hydrogen peroxide (55, 56). Cd’+ has also been reported to be a potent inhibitor of a thio1:protein disulfide oxidoreductase (57). This is an enzyme which is capable of catalyzing protein disulfide bond formation or reduction depending on whether net oxidizing or reducing conditions are present (58) and whose function is proposed to be similar to that of thioredoxin (59). Therefore, heavy metal ions could bring about the inhibition of protein synthesis in the reticulocyte lysate both through the direct inhibition of some critical enzymatic activity(s) needed to regenerate reducing equivalents or reduce disulfide bonds or through the depletion of Glu-6-P, NADPH, or GSH by pro- moting hydroperoxide and disulfide bond formation.

Protein synthesis in GSSG-inhibited lysates is stimulated by the addition of Glu-6-P (18), as is protein synthesis in lysates inhibited by preincubation at high PO, (28). Addition of Glu-6-P was found to have no effect on protein synthesis in heavy metal ion-inhibited lysates indicating that heavy metal ions do not inhibit protein synthesis simply by generating an oxidative stress which depletes the lysate of Glu-6- P. Dithiothreitol has been shown to satisfy the lysates requirement for reducing power in the absence of a functional thioredoxin/thioredoxin reductase system or the ability to regenerate NADPH (21, 22). The addition of 1 mM dithiothreitol to hemin-supplemented lysates both prevented and reversed the inhibition of protein synthesis brought about by the addition of Cd2+, H$+, or Pb*+.’ Heavy metal ions may act to inhibit the ability of thioredoxin to maintain sulfhydryl groups in certain sensitive proteins in a reduced state in the reticulocyte lysate. Dithiothreitol could be acting to maintain or restore protein synthesis in heavy metal ion-inhibited lysates by directly providing the reducing power required to prevent or reverse the disulfide bond formation which has been proposed to be responsible for HRI activation (22). In addition, dithiothreitol may act directly, by chelating heavy metal ions, thereby preventing or reversing the effects of the ions on protein chain initiation. Currently we are correlating the oxidation-reduction potential and the metal ion chelating strength of a series of agents, with the ability of these agents to stimulate protein synthesis in heavy metal ion-inhibited lysates. This approach should help us determine whether one or possibly both of these mechanisms are responsible for the ability of an agent to maintain or restore protein synthesis in the reticulocyte lysate in the presence of heavy metal ions.

In this study, the concentrations of heavy metal ions required to affect protein synthesis in the reticulocyte lysate

* R. Hurst, J. R. Schatz, and R. L. Matts, unpublished observations.

can be considered to be within a physiologically relevant range, as these metal ion concentrations are comparable to levels used to induce the heat shock response in various model systems (32-34, 60, 61) and they are well within the range to which these ions have been found to accumulate in tissues upon experimental or environmental exposure (60-64). Heavy metal ions (e.g. Cd” and Hg2+) accumulate primarily in the liver and kidney as divalent ions bound to metallothionein (64,65). The synthesis of metallothionein is induced in many cell types upon exposure to heavy metal ions (65). Metallo- thionein’s primary function is thought to be detoxification through its ability to chelate heavy metal ions (65). Significant resistance to heavy metal toxicity is only observed in cells after the induction and synthesis of metallothionein (65). In the presence of metallothionein actual levels of free intracellular heavy metal ions present would be much lower than the total cellular concentration. The ability of metallothionein to protect cells against significant accumulation of free heavy metal ions is reflected in the observation that the critical organ concentration for Cd” in the kidney during a long term exposure has been estimated to be 200 pg/g (63); this is roughly equivalent to a concentration of 1-2 mM. Unfoku- nately, most data on tissue levels of metal ions are reported as total cellular concentrations, and good estimates of free intracellular ion concentrations are not readily available. However, it would not be unreasonable to assume that micro- molar levels of heavy metal ions are attained in cells prior to the induction of metallothionein synthesis and their subse- quent detoxification.

The data presented here indicate that heavy metal ions inhibit protein synthesis in hemin-supplemented reticulocyte lysates by stimulating eIF-Pa phosphorylation, which results in a loss of RF activity, the inhibition of initiation, and the disaggregation of polyribosomes. The mechanism by which the oxidative stress, generated by heavy metal ions, brings about the apparent activation of HRI is currently under investigation. In addition it will be of interest to determine whether a similar mechanism is responsible for the inhibition of protein synthesis observed in other nucleated eukaryotic cells, which undergo the heat shock or stress response upon exposure to heavy metals.

Acknowledgments-We would like to thank Dr. Ray Petryshyn (SUNY-HSC, Syracuse, NY) for kindly providing us with the anti- HRI antibody.

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