protein synthesis in reticulocyte lysates

Proc. Natl. Acad. Sci. USA Vol. 74, No. 4, pp. 1463-1467, April 1977

Biochemistry

Role of 3':5'-cyclic-AMP-dependent protein kinase in regulation of protein synthesis in reticulocyte lysates (translational control/inhibition of translation/protein phosphorylation/Met-tRNA; binding factor/ternary complex formation)

ASIS DATTA, CESAR DE HARO, JOSE M. SIERRA, AND SEVERO OCHOA Roche Institute of Molecular Biology, Nutley, New Jersey 07110

Contributed by Severo Ochoa, January 17, 1977

ABSTRACT The initiation inhibitor of reticulocyte lysates has been shown by others to be associated with a 3':5'-cyclicAMP-independent protein kinase that catalyzes the phosphorylation of the small (38,000 daltons) subunit of the polypeptide chain initiation factor eIF-2. This factor forms a ternary complex with Met-tRNAi and GTP which, on interaction with a 40S ribosome, gives rise to a 40S complex. Ternary complex formation is inhibited by prior incubation of partially purified eIF-2 with reticulocyte inhibitor and ATP. The relation between phosphorylation and inactivation of eIF-2 is indicated by the lack of inhibition when ATP is omitted. Translation in hemincontaining reticulocyte lysates is also inhibited by cyclicAMP-dependent protein kinases or their catalytic subunits. They act by converting proinhibitor (inactive eIF-2 kinase) present in lysates to inhibitor (active eIF-2 kinase). This reaction is analogous to the conversion of inactive phosphorylase kinase to active phosphorylase kinase.

Protein synthesis in reticulocyte lysates comes to a standstill within a few minutes unless the incubation medium contains hemin (1). Gross and Rabinovitz showed that, in the absence of hemin, an inhibitor of polypeptide chain initiation is formed from an inactive proinhibitor of similar molecular weight (2). Recent evidence (3-5) indicates that the translational inhibitor in reticulocyte lysates is a 3':5'-cyclic-AMP (cAMP)-independent protein kinase that catalyzes the transfer of the -y phosphate of ATP to the small (38,000 daltons) subunit of the initiation factor eIF-2 (6-8). This modification, directly or indirectly, renders the factor inactive in chain initiation. The mechanism of conversion of proinhibitor (inactive eIF-2 kinase) to inhibitor (active eIF-2 kinase) remained unknown. We show in this paper that cAMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37), or its catalytic subunit, can promote the conversion of proinhibitor to inhibitor. This observation is consistent with the view that, as in the case of phosphorylase kinase (ATP:phosphorylase-b phosphotransferase, EC 2.7.1.38) (9, 10), inactive eIF-2 kinase is activated by phosphorylation catalyzed by cAMP-dependent protein kinase. It would then appear that protein synthesis in reticulocytes, and probably in other cells, is under cAMP control. Abbreviations: Met-tRNA1 designates the initiator species of eukaryotic cytoplasmic Met-tRNA. The A. salina initiator species used here is not formylated by E. coli transformylase and should not be designated as Met-tRNAf. eIF-2 (6), for eukaryotic initiation factor 2, designates the initiation factor that forms a ternary complex with Met-tRNA1 and GTP. This term has been accepted at the International Symposium on Protein Synthesis, October 18-20, 1976, National Institutes of Health, Bethesda, MD. The factor was formerly referred to as IF-MP by Anderson's group (7) and as IF-E2 by Staehelin's (8). Remaining abbreviations: cAMP, 3':5'-cyclic AMP; BHK, cAMP-dependent bovine heart muscle protein kinase; Hepes, N-2-hydroxyethylpiperazine-N'-2ethanesulfonic acid. 1463

MATERIALS AND METHODS Preparation and Assay of Inhibitor and Proinhibitor. A partially purified preparation of inhibitor was obtained by a procedure patterned after that of Clemens et al. (1) and Beuzard and London (11) by pH 5.0 and ammonium sulfate precipitation followed by chromatography on DEAE-cellulose. The rabbit reticulocyte lysate used for this preparation, made by the procedure of Gilbert and Anderson (12), was purchased from Gibco Diagnostics, Madison, WI. The inhibitor solution, containing about 1 mg of protein per ml, was stored in small aliquots in liquid nitrogen. Crude proinhibitor was prepared by the procedure of Gross and Rabinovitz (2). The lysate used for this purpose and for translation assays was prepared as described by Hunt et al. (13) from 2- to 3-kg white New Zealand rabbits made anemic by daily injection of 25 mg of acetylphenylhydrazine for 4 days (1). Freshly prepared lysates were kept frozen in liquid nitrogen in small aliquots until used. Postribosomal supernatant fraction freshly prepared from the lysate (2) was chromatographed at 40 on carboxymethyl-Sephadex C-50 (Pharmacia) as described (ref. 2, Fig. 6). The peak fractions containing proinhibitor (about 1.3 mg of protein per ml; A280/A260 -1), if not used right away, were stored in liquid nitrogen. The proinhibitor content of these fractions was determined by treatment of an aliquot with N-ethylmaleimide, which converts proinhibitor to inhibitor (2), and the latter was assayed with the protein synthesis or the eIF-2 ternary complex formation assay. The protein synthesis assay measures the incorporation of [14C]lysine into acid-insoluble material under specified conditions. It was done essentially as described by Hunt et al. (13) with 25 ,l of lysate, with or without 34 ,M hemin, and other additions as indicated in the tables and figures. The samples (50 pi) were incubated for 60 min at 34°. The ternary complex assay is based on the observation that, in the presence of ATP, the inhibitor renders eIF-2 incapable of forming a ternary complex with Met-tRNAi and GTP. This is true when partially, but not highly, purified eIF-2 is used. Partially purified eIF-2 is incubated briefly with ATP and inhibitor, and the mixture is then supplemented with [35S]MettRNAi and GTP for ternary complex formation. A control without inhibitor is run simultaneously. Samples (40 Al) containing N-hydroxyethylpiperazine-N'-2 ethanesulfonic acid (Hepes) buffer, pH 7.6,25 mM; KCl, 50 mM; Mg(OAc)2, 0.75 mM; dithiothreitol, 1.25 mM; ATP, 0.5 mM; Artemis salina DE-180 _eIF-2, 27.6 ,g of protein; and inhibitor or other additions, were incubated for 1 min at 250 or 300, or for various times as stated. They were then supplemented with KCI (final concentration, 100 mM), 1.3 pmol of A. salina [a5S]Met-tRNAi,

1464

Biochemistry:

Datta et al.

and GTP (final concentration, 0.14 mM), brought to a volume of 50 ,Al, and incubated for a further 5 min at 300. The formation of ternary complex was measured as described (14). eIF-2 DE-180 was a fraction from ribosomal salt wash of developing A. salina embryos, eluted with 180 mM KCI from DEAE-cellulose as described (14). A. salina [`5S]MetAtRNAi (10,00015,000 cpm/pmol) was prepared by acylation of crude tRNA with A. salina aminoacyl tRNA synthetases as described (14). Preparation and Assay of cAMP-Dependent Protein Kinases and Catalytic Subunit. A commercial preparation (Sigma) of bovine heart protein kinase (BHK) was used unless otherwise specified. This was also true of the catalytic subunit prepared from Sigma BHK by the procedure described by Rubin et al. (15). Homogeneous BHK and catalytic subunit from homogeneous enzyme were used in some experiments. They were the kind gift of Drs. R. Rangel-Aldao and 0. M. Rosen, Albert Einstein College of Medicine. Protein kinase fractions (DEAE-cellulose peaks I, II, and III) were isolated from the postribosomal supernatant of rabbit reticulocyte lysate (Gibco) as described by Traugh et al. (16). In this procedure, the kinases are precipitated with ammonium sulfate at 50% saturation and fractionated by DEAE-cellulose chromatography. In one experiment, peak II was used without further purification (pkIIa) but the bulk of this fraction was further purified by chromatography on phosphocellulose (which does not retain the kinase but removes other proteins) and Sephadex G-200 filtration. The latter fraction is referred to as pkIIb. Protein kinase activity was assayed by measuring 32p incorporation from ['y-32P]ATP into histone. The reaction mixture (50 Al) contained Hepes buffer, pH 7.2, 25 mM; Mg(OAc)2, 4 mM; [,y-32P[ATP (130-150 cpm/pmol), 0.4 mM; bovine serum albumin, 30,g; histone (Sigma, type IIA), 50,gg; cAMP, 10,uM; and enzyme. After incubation for 8 min at 30°, the reaction was terminated by the addition of 1 ml of 10% trichloroacetic acid, containing 5 mM Na2HPO4, and the precipitated radioactivity was determined as in the protein synthesis assay. The assay was carried out under conditions in which the reaction velocity was proportional to enzyme concentration. One unit of kinase activity was defined as the amount of enzyme catalyzing the transfer of 1 pmol of 32p from [ky-32P]ATP to histone per min at 30'; the specific activity of the enzyme is expressed as units/,ug of protein. Protein was determined by the method of Lowry et al. (17), with bovine serum albumin as the standard. The specific activities of the enzymes used were: Sigma BHK, 11; homogeneous BHK, 85; pkIIa, 9; pklIb, 26. BHK (molecular weight 174,000) consists (18) of one cAMP-binding dimer (molecular weight 98,000) and two catalytic subunits (molecular weight 76,000). Thus, we estimated the specific activity of pure catalytic subunit as 85 X 174/76 = 195. The amounts of BHK and catalytic subunit used are given throughout as lAg of the pure protein. The amounts of pkIIa and pklIb are those actually used. RESULTS

Translation in reticulocyte lysates Protein synthesis in hemin-containing reticulocyte lysates is inhibited not only by inhibitor, i.e., by cAMP-independent eIF-2 kinase (3-5), but by cAMP-dependent protein kinases or their catalytic subunits. Fig. 1A compares the kinetics of inhibition by BHK and inhibitor. Fig. 1B shows the kinetics of inhibition by BHK catalytic subunit at two different concentrations. It is evident that both BHK and its catalytic subunit are strong inhibitors of translation in hemin-containing lysates. It

Proc. Natl. Acad. Sci. USA 74 (1977) 10Ko

z

0

C

0 0

S

60 -

o>

2.0 - s

l

40 z-.

z 2c

0

-o 0=

2c

z

0

_

0 cc

|

0.65 1.30 BHK (pg)

1.95

zE0=

0 v

10)o

%z .p

c c

f-"

D

6

6-

2 0 TIME (minutes)

0.15 0.30 OA5 CATALYTIC SUBUNIT (pg)

FIG. 1. Inhibition of protein synthesis in reticulocyte lysates by cAMP-dependent BHK and its catalytic subunit. Protein synthesis assay as described in Materials and Methods. (A) Effect of kinase. All samples contained 34 gM hemin. (0) No further additions; (0) BHK, 1.3 ,ug, cAMP, 10 ,uM; (-) inhibitor, 10 ,g. (B) Effect of catalytic subunit. (0) Hemin, 34 ALM; (-) no hemin; (o) hemin, 34MuM, catalytic subunit, 0.15 ,ug; (U) hemin, 34MM, catalytic subunit, 0.3 rug. (C and D) Inhibition of protein synthesis as a function of the concentration of protein kinase and catalytic subunit, respectively. The different symbols in panel C correspond to different experiments. 10096 values of hemin-stimulated activity are 45,419 cpm (average) for C and 46,984 cpm for D.

can be estimated from Fig. iC that 0.85 jig of BHK (containing 0.37 ,g of catalytic subunit) in a 50-Al reaction mixture caused 50% inhibition of translation relative to sample without hemin taken as 100% inhibited. With catalytic subunit 50% inhibition was produced by 0.17 ,ug/50 ,ul, and 0.5 ,tg caused better than 95% inhibition (Fig. 1D). Thus, the catalytic subunit is about 5-fold more active than the whole kinase. Stimulation by cAMP of the inhibitory effect of bovine heart

and rabbit reticulocyte protein kinases is shown in Table 1. The table also gives experiments with homogeneous BHK and its catalytic subunit. Ternary complex formation The inhibition of ternary complex formation upon phosphorylation of the small subunit of eIF-2 by ATP is a convenient, sensitive method of assay for the reticulocyte inhibitor. The reaction is fast (Fig. 2A), and, with short incubation times, proportional to the inhibitor concentration within a limited concentration range (Fig. 2B). The inactivation of eIF-2 is clearly a consequence of the phosphorylation of the factor for, as shown in Fig. 2A, there was no inhibition when ATP was omitted. As further shown in Fig. 2A, cAMP-dependent protein kinases, whether from bovine heart or rabbit reticulocytes, or the catalytic subunit, were inactive in this assay. The small inhibition caused by Sigma BHK may have been due to contamination with proinhibitor. It is clear from these results that inhibition of translation in reticulocyte lysates by cAMP-dependent kinases is indirect. Conversion of proinhibitor to inhibitor The most likely explanation for the inhibition of translation in lysates by cAMP-dependent protein kinase is that it acts as a kinase kinase to catalyze the conversion of proinhibitor to inhibitor by transfer of phosphate from ATP. This would be in strict analogy to the activation of phosphorylase kinase by

Biochemistry:

Datta et al.

Proc. Natl. Acad. Sci. USA 74 (1977)

Table 1. Inhibition of protein synthesis in lysates by cAMP-dependent protein kinase and its catalytic subunit

Additions Exp. 1

cAMP (10MM) BWK(0.22 ug) BHK (0.22 mg), cAMP (10 A M) BHK (0.55 Ag) BHK (0.55 jg), cAMP (10 MM) Exp. 2

BHK* (4,Mg) BHK* (6,4g) Exp. 3

Total 22,166 20,867

pkIIb (2mg) pkIIb (2 Mg), cAMP (12.5 MM) pkIIb (4 Mg) pkIIb (4 Mg), cAMP (12.5 MM)

60

z

0

Inhibition, %

!

40

40

20

20

z

*1

,

10,376t

0

11,790

22,689

10,491 12,313

11 0

19,866 18,808

9,490 8,432

20 29

16,292 27,760t 74,392 45,572 26,020

5,916

50

45,632 17,812 0

62 100

13,858t 26,785

Homogeneous C (0.5 Mg) Exp. 4

Net due to hemin

80so-BBI

-A p 60 -

[14C]Lys incorporated, cpm

1465

12,927

12,660 22,294t 58,994 57,979

0

100

36,700 35,685

3

51,186 52,702

28,892 30,408

21 17

49,701

27,407

25

Assays as described in Materials and Methods. Catalytic subunit (C) contained 2 mg of bovine serum albumin per ml as a stabilizer. At the concentrations present with C in Exp. 3, bovine serum albumin by itself had no effect on translation by the lysate. Concentration of hemin (when present) was 34 MM. * Homogeneous BHK. t No hemin.

cAMP-dependent protein kinase (9, 10). To test this hypothesis, crude proinhibitor was prepared and incubated with or without catalytic subunit or cAMP-dependent protein kinases and ATP and the reaction mixtures were assayed for inhibitor formation with the ternary complex or protein synthesis assay. As seen in Fig. 3, there was little or no inhibition of ternary complex formation with proinhibitor alone (bars 2 and 4) but significant inhibition was observed when proinhibitor was incubated with catalytic subunit and ATP (bars 3 and 5). The poor proportionality of the assay (inhibition in sample 5 should have been 26% rather than 18%) may have been caused by the presence of protein phosphatase in the crude proinhibitor preparations. The experiment of Fig. 4 shows that conversion of proinhibitor to inhibitor required catalytic subunit and ATP. Under these conditions the conversion was complete because as much inhibitor was formed by incubation with catalytic subunit and ATP as by N-ethylmaleimide treatment. The smaller conversion in samples to which no catalytic subunit had been added may be due to endogenous cAMP-dependent protein kinase (and traces of cAMP) or self activation (see ref. 9) or both. The experiment of Table 2 illustrates the effect of cAMPdependent bovine heart and rabbit reticulocyte protein kinases, as well as catalytic subunit, on the proinhibitor-inhibitor conversion assayed by protein synthesis in the reticulocyte lysate.

2 4 6 TIME (minutes)

8

0

0.3

0.6

INHIBITOR

0.9

1.2

(pg protein)

FIG. 2. Assay of inhibitor through its effect on ternary complex formation. (A) Kinetics of inhibition. Partially purified eIF-2 was incubated for various times at 300, with the indicated supplements, before GTP and [35S]Met-tRNAi were added. The samples were then incubated for a further 5 min and assayed for ternary complex formation as described in Materials and Methods. (0) Inhibitor, 1.5 Mg of protein, ATP, 0.4 mM; (0) inhibitor, 0.75 Mg of protein, ATP, 0.4 mM; (v) BHK, 0.38,ug, cAMP, 5,MM, ATP, 0.4 mM; (o) inhibitor, 1.5,ug of protein (no ATP); (&) either BHK catalytic subunit, 0.3 ,g, ATP, 0.4 mM, or cAMP-dependent rabbit reticulocyte protein kinase (pkIlb), 2, g of protein, cAMP, 5 MM, ATP, 0.4 mM. The retention of 35S radioactivity by ternary complex formation at zero time was 8400 cpm. (B) Inhibition of ternary complex formation as a function of the inhibitor concentration. eIF-2 was incubated with ATP, 0.4 mM, and without or with the indicated amounts of inhibitor for 1 min at 250 before assay. The retention of 35S radioactivity by ternary complex formation in the absence of inhibitor was 7926 cpm.

No conversion was generally detected in the absence of added kinase (Exps. 1 and 2). However, in some cases (not shown), addition of ATP-Mg2+ alone caused some conversion, as already noted in ternary complex assay experiments (Fig. 4). The conversion observed in the presence of cAMP-dependent reticulocyte kinase was enhanced by cAMP and by ATP (Exp. 2). cAMP-dependent protein kinase from rabbit reticulocytes and catalytic subunit appeared to be equally effective in promoting proinhibitor-inhibitor conversion (Exp. 3), for the same number of enzyme units in each case (about 20) caused the same conversion (about 65%). 20

15-

o5 0

1 2345

FIG. 3. Conversion of proinhibitor to inhibitor by BHK catalytic subunit. Samples (25 Ml) containing Hepes buffer, pH 7.6, 20 mM; KCl, 50 mM; Mg(OAc)2, 2 mM; ATP, 0.4 mM; without or with proinhibitor and/or BHK catalytic subunit as indicated were incubated for 8 min at 30°. Aliquots were then assayed for inhibitor by the ternary complex formation assay. (1) No proinhibitor, no catalytic subunit. (2) Proinhibitor, 0.107 Mg of protein, no catalytic subunit. (3) Proinhibitor, 0.107 Mg of protein, catalytic subunit 0.15,Mg. (4) Proinhibitor, 0.214 Mg of protein, no catalytic subunit. (5) Proinhibitor, 0.214 Mg of protein, catalytic subunit 0.15 Mg. The retention of 3IS radioactivity by ternary complex formation in a control sample without proinhibitor was 6531 cpm.

Biochemistry:

1466

Datta et al.

Proc. Natl. Acad. Sci. USA 74 (1977)

R 30

Table 2. Conversion of proinhibitor to inhibitor by cAMP-dependent protein kinase and its catalytic subunit

0 0

or e

[14C ]Lys,

20 H-

uj

Additions

0

OCL

z

zI.. 10

k

0

0

0.075

I 0.30 0.45 0.15 PROINHIBITOR (lg protein)

0.45

FIG. 4. Conversion of proinhibitor to inhibitor by BHK catalytic subunit: requirement for catalytic subunit and ATP. The experiment was carried out in two stages, the first toform inhibitor by phosphorylation -of the proinhibitor, the second to assay for inhibition of ternary complex formation. In the first stage, three samples, each (250 Ml) containing Hepes buffer, pH 7.6, 20 mM; KCl, 50 mM; and proinhibitor, 160 Mig of protein, freshly prepared by carboxymethylcellulose-Sephadex chromatography, were set up with additional supplements as follows: (a) catalytic subunit, 3.6 Mg; (b) Mg(OAc)2,' 2 mM, ATP, 0.4 mM; (c) catalytic subunit 3.6 Mg, Mg(OAc)2,2 mM, ATP, 0.4 mM. After incubation for 8 min at 300, the samples were chilled and each was separately chromatographed on a small carboxymethylcellulose-Sephadex column to remove the catalytic subunit. This also removes better than 99% of the ATP. In the second stage, inhibitor formation was assayed in aliquots of the appropriate column fractions with the ternary complex formation assay as in Fig. 3. The preincubation with ATP-Mg2+ and eIF-2 was for 1 min at 300 and the eIF-2 contained 32.2 Mg of protein. For determination of the total proinhibitor present, samples of proinhibitor were incubated with N-ethylmaleimide, 5 mM, for 8 min at 340, and the excess Nethylmaleimide was neutralized with dithiothreitol. The retention of 35S radioactivity by ternary complex formation in a control sample without proinhibitor was 7288 cpm. (U) Catalytic subunit; (ca) ATP-Mg2+; (0) catalytic subunit, ATP-Mg2+; (M) N-ethylmaleimide.

DISCUSSION The results presented in this paper are consistent with the model of Fig. 5, in which cAMP-dependent protein kinase plays the same role in activating cAMP-independent eIF-2 kinase as it does in activating cAMP-independent phosphorylase kinase (9, 10). Much work remains to be done to show that enzymatic activation and inactivation of eIF-2 kinase involves phosphorylation and dephosphorylation, beginning with purification of the proinhibitor which is now available only in crude form. The main fact arising from this study is that protein synthesis can be, and probably is, regulated by cAMP. Our present results are restricted to reticulocytes, but there is little doubt that they will apply also to other cells.* Pryor and Berthet showed in l6o that cAMP inhibits protein synthesis in rabbit liver slices (19). The inverse relationship between cell growth and cAMP levels disclosed by the work of Pastan and others (20) can now be explained in view of the identical relationship between protein synthesis and cAMP levels suggested by our work. Tomkins and collaborators (21) showed that cAMP and prostaglandin E1 (which raises the intracellular level of cAMP) inhibited the * An ATP-dependent inhibitor of ternary complex formation is present in A. salina ribosomal salt wash and has been partially pUrified (C. de Haro and S. Ochoa, unpublished data). Moreover, BHK catalytic subunit strongly inhibits polypeptide chain initiation in wheat germ and A. salina systems (J. M. Sierra and A. Datta, unpublished data).

Exp. 1 N-Ethylmaleimide (5 mM) None ATP, cAMP (12.5 AM) pkIIa (11 Mg), ATP, cAMP (12.5 MM) BHKt (2,4g), ATP, cAMP (12.5 MM) Ct (0.2 ig), ATP Exp. 2 N-Ethylmaleimide (5 mM) pkIlb (2jfng) CAMP (6MM)

cAMP (6,MM)

pkIlb (2;1-g),

pkIIb (2 Mg), cAMP (61MM), ATP Exp. 3 N-Ethylmaleimide (5 mM) pkIlb (0.8 Mig), cAMP (12.5 MM), ATP C (0.09 Ag), ATP

incorporated, cpm

Inhibition of translation* cpm

%

10,540

100

31,618f 21,078

34,574

-

32,600

0 0

23,540

8,078

76

25,242 20,956

6,376 10,662

60 100

50,306 66,750 68,740

18,119 1,675

100 9 0

61,168

7,257

40

57,593

10,832

60

48,380

18,056

100

55,3i4 54,664

11,122 11,772

62 65

68,245t

66,436t

Proinhibitor, 15 Ml (about 20,Mg of protein), prepared as described in Materials and Methods, was incubated for 8 min at 300, with the indicated additions, in a final volume of 20,Ml. Samples contained proinhibitor except as noted: ATP, when present, was added as 0.5 mM ATP-Mg2+. Aliquots, 6 Ml, were withdrawn for assay of inhibitor formation in the reticulocyte lysate translation system in the presence of hemin. The amount of potential inhibitor was determined for each experiment by incubation of a sample of proinhibitor with N-ethylmaleimide for 8 min at 300 and the excess N-ethylmaleimide was neutralized with dithiothreitol. The ['4C]lysine incorporation values in assay samples in the absence of added hemin were as follows: Exp. 1,17240 cpm; Exp. 2, 24166; Exp. 3, 22779. * Extent to which proinhibitor is converted to inhibitor during incubation. Conversion by N-ethylmaleimide is taken as 100%. t Homogeneous protein. C, catalytic subunit. No proinhibitor.

growth of cultured mouse lymphoma S49 cells but were much less effective on a variant that had low levels of cAMP-dependent protein kinase. Thus, the inhibition by cAMP of both protein synthesis and cell growth is mediated by cAMP-dependent protein kinase. Probably inhibition of cell growth is a consequence of protein synthesis inhibition. The conversion of proinhibitor to inhibitor is prevented by hemin (2), but its mode of action is unknown. Hemin probably blocks phosphorylation of the proinhibitor by physiological concentrations of cAMP-dependent protein kinase. We emphasize the word physiological because relatively large amounts of kinase, as added in our experiments, inhibited translation in the presence of hemin. We have some indirect evidence that hemin may interfere with the dissociation and release of the catalytic subunits of cAMP-dependent protein kinase by cAMP. In hemin-containing lysates the inhibitory effect of cAMPdependent reticulocyte kinase + cAMP per unit of enzyme

Biochemistry:

Datta et al.

Proc. Natl. Acad. Sci. USA 74 (1977)

3':5'-CYCLIC AMP ---- HEMIN? PROTEIN KINASE

INACTIVE .IF-2 KINASE

("PROINHIBITOR") +

ACTIVE

eIF-2

KINASE

("INHIBITOR")

(00~

ATP

AIF-2

eIF-2-P

ATP

FIG. 5. Regulation of polypeptide chain initiation by cAMP.

activity was very low when compared with that of the free catalytic subunit (data from Table 1, Exp. 4, and Fig. 1D), whereas kinase and subunit had the same effect on the proinhibitor-inhibitor conversion in the absence of hemin (Table 2, Exp. 3). A reported inhibition by hemin of cAMP-dependent protein kinases from rabbit reticulocytes (22) would be consistent with the view that bemin interferes with release'of the~ catalytic subunit. However, we have no direct proof. for this mode of action, and the subject requires further investigation. We thank Christa Melcharick for help with some of the preparations. A.D. is a Visiting Scientist from the Jawaharlal Nehru University, New Delhi, India. C. de H. is a postdoctoral fellow of the USA-Spain Cultural Cooperation Program. J.M.S. is a postdoctoral trainee of the Center of Molecular Biology, Madrid, Spain; Fulbright Travel Fellow Awardee.

1. Clemens, M. J., Henshaw, E. C., Rahaminoff, H. & London, I. M. (1974) "Met-tRNAe binding to 40S ribosomal subunits: A site for the regulation of initiation of protein synthesis by hemin," Proc. Natl. Acad. Sci. USA 71, 2946-2950. 2. Gross, M. & Rabinovitz, M. (1972) "Control of globin synthesis by hemin: Factors influencing formation of an inhibitor of globin chain initiation in reticulocyte lysates," Biochim. Biophys. Acta 287,340-352. 3. Farrell, P., Balkow, K., Hunt, T. & Jackson, R. (1976) "Evidence that protein synthesis in reticulocyte lysates is controlled by the reversible phosphorylation of the initiation factor IF-E2 (IFMP)," Abstracts Cambridge EMBO Workshop on Cytoplasmic Control of Eukaryotic Protein Synthesis. 4. Levin, D. H., Ranu, R. S., Ernst, V. & London, I. M. (1976) "Regulation of protein synthesis in reticulocyte lysates: Phosphorylation of methionyl-tRNAf binding factor by protein kinase activity of translational inhibitor isolated from heme-deficient lysates," Proc. Natl. Acad. Sci. USA 73,3112-3116. 5. Kramer, G., Cimadevilla, J. M. & Hardesty, B. (1976) "Specificity of the protein kinase activity associated with the hemin-controlled

1467

repressor Qf rabbit reticulocyte," Proc. Natl. Acad. Sci. USA 73, 3078-3082. 6. Weissbach, H. & Ochoa, S. (1976) "Soluble factors required for eukaryotic protein synthesis," Annu. Rev. Biochem. 45, 191216. 7. Safer, B., Anderson, W. F. & Merrick, W. C. (1975) "Purification and physical properties of homogeneous initiation factor MP from rabbit reticulocytes," J. Biol. Chem. 250, 9067-9075. 8. Staehelin, T., Trachsel, H., Erni, B., Boschetti, A. & Schreier, M. H. (1975) "The mechanism of initiation of mammalian protein synthesis," in Proceedings of the Tenth FEBS Meeting, eds. Chapeville, F. & Grunberg-Manago, M. (North Holland/ American Elsevier), Vol. 39, pp. 309-323. 9. Hayakawa, T., Perkins, J. P. & Krebs, E. G. (1973) "Studies on the subunit structure of rabbit skeletal muscle phosphorylase kinase," Biochemistry 12, 574-580. 10. Cohen, P. (1973) "The subunit structure of rabbit-skeletal-muscle phosphorylase kinase and the molecular basis of its activation reactions," Eur. J. Biochem. 34, 1-14. 11. Beuzard, I. & London, I. M. (1974) "The effect of hemin and double-stranded RNA on a and d globin synthesis in reticulocyte and Krebs II ascites cell-free systems and the relationship of these effects to an initiation factor preparation," Proc. Natl. Acad. Sci. USA 71, 2863-2866. 12. Gilbert, J. M. & Anderson, W. F. (1970) "Cell-free hemoglobin synthesis II. Characteristics of the transfer ribonucleic aciddependent assay system," J. Biol. Chem. 245, 2342-2349. 13.- Hunt, T., Vanderhoff, G. & London, I. M. (1972) "Control of globin synthesis: The role of heme," J. Mol. Biol. 66, 471-481. 14. Filipowicz, W., Sierra, J. M. & Ochoa, S. (1975) "Polypeptide chain initiation in eukaryotes: Initiation factor MP in Artemia salina embryos," Proc. Natl. Acad. Sci. USA 72, 3947-3951. 15. Rubin, C. E., Erlichman, J., & Rosen, 0. M. (1972) "Molecular forms and subunit composition of a cyclic adenosine 3',5'monophosphate-dependent protein kinase purified from bovine heart muscle," J. Biol. Chem. 247,36-44. 16. Traugh, J. A., Mumby, M. & Traut, R. R. (1973) "Phosphorylation of ribosomal proteins by substrate-specific protein kinases from rabbit reticulocytes," Proc. Natl. Acad. Sci. USA 70,373-376. 17. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) "Protein measurement with the Folin phenol reagent," J. Biol. Chem. 193,265-275. 18. Erlichman, J., Rubin, C. S. & Rosen, 0. M. (1973) "Physical properties of a purified cyclic adenosine 3':5'-monophosphatedependent protein kinase from bovine heart muscle," J. Biol. Chem. 248, 7607-7.609. 19. Pryor, J. & Berthet, J. (1960) "The action of adenosine 3',5'monophosphate on the incorporation of leucine into liver proteins," Biochim. Biophys. Acta 43, 556-557. 20. Pastan, I. H., Johnson, G. S. & Anderson, W. B. (1975) "Role of cyclic nucleotides in growth control," Annu. Rev. Biochem. 44, 491-522. 21. Daniel, V., Litwack, G. & Tomkins, G. M. (1973) "Induction of cytolysis of cultured lymphoma cells by adenosine 3':5'-cyclic monophosphate and the isolation of resistant variants," Proc. Natl. Acad. Sci. USA 70,76-79. 22. Hirsch, J. D. & Martelo, 0. J. (1976) "Inhibition of rabbit reticulocyte protein kinases by hemin," Biochem. Biophys. Res. Commun., 71,926-932.