Regulation of protein synthesis by heme-regulated eIF-2α kinase

THE ~~~~N of the initiation of tr~slati~n by phosphorylation of the a-subunit of eukaryotic initiation factor 2 (elF-Za) has been &served under a variety of physiological ~~nditi~ns~ including viral infection, nutrient starvation and heat shock (reviewed in Ref. I>. ~~l~ti~n of protein synthesis by elF-Za phosphoryiation was first ob served in rabbit reticulocyte lysates during heme deficiency and the addition of low concentrations of doubiestranded (ds) RNA [reviewed in Ref. 2). During initiation of protein synthesis, elF-2 binds GTP and Met-tRN~ as a ternary complex and then forms the 43 S pr~nitiation complex with the 40 S ribosomd subunit. Upon binding of mRNA and gaining of the 60 S ribosomal subunit, elF-2-GTP is hydrolysed to elF-2-GDP.The recycling of eIF-2requires the exchange of bound GDP for GTP, which is catalysed by initiation factor elF-2B.When eiF-2a is phosphoryiated, the binding affinity of elF-2(oP)-CDP to elF-2B is much higher than that of eIF-2-GDPto efF-2B.Since eIF-2Bis limiting and present at a fraction of the amount of elF-2, elF-2B activity is effectively inhibited once the amount of phosphorylated eIF-2 exceeds the amount of elF-2B. The unavailability of elF-2B results in the shut-off of protein synthe&s.

Jane-JaneChenand Irving M. London Protein synthesis is regulated by the phosphorylticM of the or;jubunit of eukaryotic initiation faGtor 2 (elF-2a) in a variety of cells. At present, there are two distinct mammalian efF-2a kinases that have beg cioned, the double-Strande~RN~e~ndent elF-2a kinase (PKR) and the henwegu lated elF-2a kinase (HRI). IiRt is activated under conditions of heme W ficiency in immature erythroid cells, and its activity is inhibited by heme. The high levels of HRI in reticulocytes indicate that its major physiological role is the regulation of protein synthesis, pafticularfy of obin, according to the coneentratio18 of heme in these cells.

starvation; the subsequent phosphorylation of elF-2a is required for the increased translation of GCN4, which is a tr~sc~ption~ activator of genes responsible for amino acid biosynthesis. Each elF-2a kinase contains a unique sequence that may be responsible for its regulation (Fig. 1). For example, the amino-terminaf 160 amino acids of PKR contain two copies of a dsl?NA-bisding motif rich in basic amino acids, while the carboxyl terminus of GCN2 contains ofuledRa an essential 530-residue motif with sip Three elF-2a kinases have been nifkant homology to histidyl-tRNA syncloned to date (reviewed in Ref. 3 and thetase; this simiiarity has prompted references therein). They are rabbit and the suggestion that GCN2 senses amino rat HRJ, human and mouse PKR and acid starvation by binding uncharged yeast CCNO.These three eIF-2a kinasm tRNA The extreme carboxy-termlnaf share extensive homology in the Wnase 124 amino acids of GCN2 are required catalytic domains (ng. l), and all phos- for its interactions with the 60 S rlbophorylate elF-2a at Sertil. Both HRI somal subunit and are also required for and PKR can functioually substitute for CCN2 action in oiuo. MU is fouud GCN2 in translational control of GCN4 mostly in the postribosomal superin yeast? However, the regulr!ory natant and lacks the ribosomal associmechanisms and the molecular sizes of ation sequence of GCNZ. The heme these elF-2a kfnases are very different. binding domain of HRf has yet to be HRI (626 amino acids) is activated determined. There is a portion of the sequence under hemedeficient conditions and its kinase activity is Inhibited by heme. between two halves of the conserved PKR @O amino acids) Is induced by Mnase domalns, known as the kfnase interferon, is activated by low concen- insertion sequence, that is unique to trations of dsRNA (ng ml-*) and is in- each kinase. In HRI and CCN2 this is hibited by high concentrations of large (about 120 amino acids), while in dsRNA (pgml-3. Yeast GCW2 (1590 PKR it is only about 40 amino acids amino acids) is actlvated by amino acid long. The last 20 amino acids of the kinase insertion sequence of all three efF-2a kinases share significant hornJ-1.Chm and 1.M. iandon ar3 at the ology, suggesting a functional role. MassachusattsInstitute of Technology, Deletion of SIXamino acids (361 to 366) Harvard-MlTDivisionof HealthSciincesand from this region resuits in the loss of Technology, 77 MassachusettsAvenue, both autokfnase and eIF-2a kinase E25545, Cafnbrkfge,MA02139, USA. (8 1995,Elsevkv Science Ltd O!H%-0004/95/~.!50

activities of human PKR,while mutation of the conserved Met719 to v&e in this region of GCNL results in increased eIF-2a kinase activity. Kinase domains IX and X, which are highly conserved among these three eIF-2a kinases, are likefy to be involved in eff-2 binding. The HRI synthetic pep tide ~74, which is based on a sequence in domain IX, inhibits the elF-2a kinase activities of HRf and PKR? The crystal structure of the co-crystal of CAMPdependent protein kinase and its sub strate analog, the inhibitory peptide, demonstrates that three amino acids in domain IX are in contact with the inhibitory peptlde? In hemede#kient reticulocyte lysate, activation of HRI is accompanied by Its phosphoryiatlon and the phosphorylation of eiF-2a (reviewed in Refs 2, 7 and references therein). The observation that the mutation of Lys199 of HRI (the invariant Lys residue in kinase domain U) to Arg results in the loss of both HRf phosphorylation and elF-2a phosphorylatlon further supports the hypothesis that HRI is activated by autophosphorylation! Hemin has been shown to bind to HRI, and this binding results in the inhibition of both the autophosphoryfation and eMa pho+ phoryfation by HRI. In addition, hemin promotes intersubunit disulfide-bond formation between HRI molecules. In the absence of hemln, HRl exists as a dimer, held by noncovalent interactions, and is active. In the presence of hemln, m becomes an irmctbe, dfsMde-Unked lo6

and CSF-1RTKs. It is interesting that HRI and these ~?NQ RTKs ail possess Mnase insertion sequences that separate the conserve,-1 base domains. The kinase iltsertion sequerices

may be involved

in

dime&&ion. A conserved hem+regulatory motif has been identified recently in erythroid 5 aminoievuiinate synthase precursor, an enzyme of the heme-biosynthetic pathway14. His RS This motif is also found in other heme-binding proteins, 1590 such as HAP1 (a yeast transcription factor for cytochrome genes), hemopexin m-1 and cataiase. The motif conThe @uctwe of the eIF2a kinases. The three kinases are aligned by their conserved kinase catalytic Kl, kinase insertion sequence. Numbers refer to tains an invariant Cys-Pro ing region in PKR and the ribosorne-binding region sequence preceded preferuences unique to each kinase. His RS, region ably by a basic amino acid ase; ~74, region corresponding to HRI synthetic residue. In both rabbit and rat HRf, there are two such motifs that do not appear in GCN2 Purified HRI binds ATP; however, if and PKR: CysfWPro407 (in the kinase the HRI has previousiy been treated insertion sequence) and Cys548Pro549 with hemin, the AW binding is signifi- (in the extreme carboxyi terminus). The cantly redu~ed~~. This result suggests importance of the invariant Cys residue that the inhibition of both auto- in the heme-regulatory motif is consistphosphorylation and the phosphoryl- ent with the regulation of HRf by direct ation of elF-2orby heme is the result of binding of hemin and the promotion of inhibition of AW binding to HFU.Since intersubunit disulfide-bond formation sulfhydryi groups important for the hemin also promotes disulfide-bond by hemin in HRl. Recently, it has been formation of the lntersubunit diauifide formation, it could be inferred that free shown that heme promotes HAP1 dibond in reticulocyte lysaks? In ad- sulfhydryl groups are irn~rt~n~ for the rner~z~t~~nin yeast cells? In addition, it dition, these studies also indicate that binding of ATP to HRf. This is consistent has been reported that only the dimeric with an earlier finding that fhe presence form of nitric oxide synthase is active of ATP prevents the inactivation of and binds heme? iysate HRl by high concentrations of NE&l’! Hemin may inhibit the binding of AW to HRl by inducing a conforHRl has aiso been shown to be actimational change in HRi that brings the vated in hemin-suppiemented iysaks essenntialsulfhydryi groups into close by heat shock or sulfhydryl reagents proximity and consequenUy prom&es (reviewed in Ref. 7). The 90 Mla heatdisulfid&ond formation. shock protein (Hsp90) has been Formation of a disulfiddinked dimer reported to co-purify with HRI, and to is not unique to HRl, and has been inhibit protein synthesis in heminreported for two receptor tyrosine supplemented reM&cyte lysaks17+ ldnases (RTKs), the colony-stimulating However, the effect of Hsp90 on the factor-l @SF-I) receptor and the eff-2a kinase activity of purified HRI plateletderived growth factor (PDGF) is unclear. Szyszka et al. reported that receptor. ActivaUon of all RTKs appears the phosphorylated form of HspW to invoive receptor dimerization. The enhanced the eIF-2a kinase activity of bindingofCSF4 @!?f* 12)andPDGF’3to HRl@,while Mendez et al. reported that their respective receptors stimulates the eMa kinase activity of HRI was both noncovaIent dimerhtion and independent of HspW (Ref. 20). Re dm dlmerizatlon of the re- centiy, Mendez and de Haro reported Ceptonr. The disulftde-llnked dimeriz- that casein kinase 11may be involved in ation of the CSF-1receptor is thought to the activation of HlU to a state that is be involved in limuced receptor no longer responsive to heme (hemeintern;allzauon’*, but the exact role irreversible l-W)% This effect of casein remains to be determined in both PDCF kinase II on HRI reqWes the presence

of Hsp90. Although heme-irreversibie ~ntersubun~~ disufffde bonds (Fig* 2). c~nc~ntrati~, The ~s~~ati~ of th HRI can be produced in U&D, its physio Although the mechanism by which proteins with HRl is ir: and is logical relevance in ~ticui~~~ is Hsp70 and dithiothre~tol prevent ac- influ~c~ by hemin, GTPand unclear. Purified HRl that contains no tivation of HRl in heme deficiency is redox state. Hsp90 is an active elF-2a Mnase, is currently unknown, it is possible that Using a purified preparation of HRf heme responsive and is a potent Hsp70, a chaperone, could change the that was enriched in Hsp90 and coninhibitor of protein synthesisiu; these conformation of HRf and allow dithio- tains casein kinase If, Mendez et ai. findings indicate that Hsp9O is not threitoi to reduce the critical disulfide have presented a model for essential for the kinase actfvitfes of to free sulfhydryfs, which can then be iation of HRf In which an active HRf or for its ref@ation by heme oxidized by hemin. In support of this HRWisp90 heterodii is in Am. However, in reticutocyte iysate hypothests, it has been observed that and in whkh NE34 promotes the preparations with different leveis of the kinase activity of HRf is more sensi- dissociation of Hsp90 from this heteroHsp90, the restoration of protein tive to hemin in the presence of dithio- dimer? This proposed model is incomsynthesis by the delayed addition of threitol (Ref. 25; J-J. Chen and P .f. patible with several studtes that show hemin is greater in lysates with higher Chefalo, unpublished). Alternatively, it that there is no sign&ant change in the levels of HSP90 (Ref. 22). This observa- is also possible that sulfhydryl groups molecufar size of HIU upon actition suggests that lisp90 may be of HRf are requfred for the binding of vation by NEM, and HRf remains as a involved in the heme-mediated in- Hsp70, since the association of Hsp70 dimer 9c11z. activation of active HW in reticulocyte with HRf also requires dithiothreitoi? lysates. Oxidation of these sulfhydryls of HRf in A dynamic interaction of HRl with iysates may lead to its activation during fnhibitors of protein synthesis inheat-shock proteins (Hsp90, Hsp70 and oxidative stressz*28.Thus, these studies itiation with properties similar to those ~59) has been demonstrated by co suggest that HRl may contain different of HRi have been partiafly purified from immunoadsorption of HRl with these sets of sulfhydryls, which may be regu- human and mouse immature erythroid proteins from reticuloeyte lysates? lated differently by hemin and Hsp70. cells. Recently, efF-ti kinases from both The association of HRf with Hsp90 and As described above, HRI is a cysteine- uninduced and induced murfne erythropS9, but not Hsp70, is enhanced by rich protein with 11 cysteines per sub- leukemia (MEL) cells have been purified hemin. The level of Hsp70 in iysates unit, and thus provides a large number and shown to be very simifar to rabbit appears to be inversely related to the of possible sets of sulfhydryls in the reticufocyte HRf? HRf-fike activity has degree of translational inhibition in HRI homodimer. also been reported in some nonerythHRf purified to near homogeneity, roid celt types such as Ehrlich ascites hemin-suppiemented lysates under conditions of heat and oxidative stress? free of Hsp70 and HspW, is active and is cells and HeLa cells. Most recently, These results suggest that Hsp70 may responsive to hemin in terms of both Olmsted et ~1.~ purified two elF-2a be required to maintain HRl in an kinase activity and intersubunit disul- kinases, which appear to be distinct inactive form. Indeed, the addition of fide formation (J-J. Chen, t? J. Chefalo from HRl and PKR, from Rhrlich ascites denatured protein, which sequesters and ELRan, unpubifshed). These results ceils. Studies using a monoclonaf antibody Hsp70, activates HRi in hemin- demonstrate that the HRf homodimer is supplemented lysates24. Most recently, active and heme responsive, and that raised against rabb?t retkuiocyte HRf Hsp70 has been shown to prevent the heat-shock proteins are not essential suggested that HRl may be erythroid activation of HRf in heme deficiency by for the regulation of HRl by heme in specific; HRI was detected only in the reducing the optimal concentration of uitro.However, the regulation of HRI by peripheral blood and bone marrow hemin required to suppress HRf acti- heme in.retkulocyte lysates is probably of anemk rabbits3r. Further studfes vation? This effect of Hsp70 requires more complex and may involve heat- indfcate that thb is the case. HRf dfthiothreitol (a dfsufffde-reducing agent) shock proteins for fine tuning and rapid mRNA, polypeptide and protefn Mnase and, to a lesser extent, GTI? These adjustment to changes of heme activity are detected in the bone observations fit well with the earlfer report that heavy metal ions activate HRf by inhfbitfng the capacity of the hemfn-supplemented reticulocytes to reduce disulffde bonds%, and wfth the require Hemin ment of a disulfidereducing system to mafntafn the maxtmal rate of initiation of pro tein synthesis27. Most significantly, all these studies point to the importance of suffhydryl groups in the ref@tlation of HRI actfvfty, in agreeinactive dimer Active chimer ment with our proposed model of the regulation of hemin via sulfhydryl oxim-2 Regulation of HRI by hemin viaintersubunit disulfide-bond formation of HRI homodimer~. dation and the formation of

celldar inhibitor of IQChen,J-J. et&. (1991) fbc. NetlAcad. Sci. usA68,31s-319 ~~~~~~~~nsho~ 11 Hunt, T. (1979) in Miami Winter to cause oncogenictransformation. sympsium: ~~~to~rein(~l. 16) Thus,eW-2~ldnasesmaybeconsidered (Russet, T. R., Brew, K., Schultz, J. and ads), pp. 321-345, as tumorsuppressors. AlthougheiF-2u tlaber, H., Press is the only knownphysiologicalsub 22 Li,Academic W. and Stanley, E. R. (1991) EA&?J. IO, &rateof HRfsofar,it hasbeenreported 277-286 that HRIand FXR can phosphorylate 13 Li, W. and Schiessinger. 1. (1991) 1. Cdl. the inhibitor of a ubiquitousnuclear 14 f3io/.ll,37!S6-3761 lathrop, J. T. and Timko, M. P. (1993) S&ice tramriptional factor9NF-ICB (Refs38, 39). Thus, HRI, which is presentin 15 o., 3)Pmc. N&i hemopoktic progenitorcells (J. S. Acad.sci~usA90,2851-28!% Crosby, 1. M. Londonand J-J.Chen, 16 Bask, K. J., Thiil, B. A., Lucas, S. and UnpubBshed), may have other sub Stuahr, 0. J. (1993) /. Bill. Cbm. 268, s&ate(s) in immature nucleated 21l2O-21129 hemopoMccelfs.It will be interesting 17 Rose, 0. W. et a/. (1967) 8iochemistly 26, 6563-6587 to seewhetherHRfplaysa role in the 18 Rose, D. W., Wekh, W. growthanddifferentiationof erythroid Hardasty, B, (1989) 1%

orofthe58

6239-6244

C&S.

Melloretalhavecloned

R., Kramer, 6. and Hardesty, B. (1989) The observationthat HRI is pro- 19 Szyszka, Biodremistry28,1435-1436 ducedat the highestlevelsin erythroid 20 Mendez, R., Moreno, A. and cellsfurthersupportsthephysiological de Hare, C. (1992) 1. f3iol. Chem. 267, role of HRI in the regulationof pro11!NO-11507 R. and de Hare, C. (1994) 1. Biol. tein synthesis,especiallyof hemo- 21 Mendez, Chem.269,6170-6176 globin,the principalproteinin these 22 Matts, R. L. and Hurst, R. (1992) 1. Biol. Chem. celize. The overexpression or knock-out 267,181~18174 of the geneencodingHRfshouldpro- 23 Mat& R. L., Xu, Z., Pal, 1. K. and Chen, J-J. (1992) i. Biol. Cttem. 267, videmorevaluableinformationon the 18160-18167 physbgkai role of HRiin erythroid- 24 Matts, R. L., Hurst, R. and Xu, Z. (1993) cell proliferationand differentiation, 6iochemisfrv32,7323-7328 and studieson the structure-function25 Gross, M., Olin, A., Hessefort, S. and Bender, S. (1994) J. Viol. Chem. 269, relationships of HRf should in22738-22748 creaseour understanding of the mol- 26 Matts, R. L., Schatz, 1. R., Hurst, R. and ecularmechanismof regulationof HfU Kagen, R. (1991) 1. Bid. Chem, 266, 12695-12702 by heme. 27 Jackson, R. J., Herbert, P., Campbell, E. A. and Hunt, T. (1983) Eur. J. Biwhem. 131,

313-324

Work from the authors’iaburatory 28 Jackson, R. J., Campbell, E. A.. Herbert, P. and Hunt, T. (1983) Eur. 1. Biochem. 131, wassupportedby grantsfromthe NIH 289-301 (D&16272)and the NationalScience 29 Mallor, H. et al. (1993) Eur. 1. Bochem. 211, Found&~ @MB440!5781). We thank 529-53B L., Hen&w, E. C. and J. Crosbyand I? Chefalofor prep- 30 Olmsted, E. A., 0’~. Panniers, R. (1993) 1. Biol. C&m. 268, arationof the figures.We also thank R Matts for critical readingof the 32 11552-12559 Pal, 1. K., Chen, J-J. and London, I. M. (1991) manuscript. B~istry30,2655-2562 32 Crosby, J. S., Lee, K, London, I. M. and Chen,J-J. (1994)MoL Cd. We 14,

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Academii

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