Characterization of a mammalian homolog of the GCN2 eukaryoticinitiation factor 2a kinase

Juan J. Berlanga, Javier Santoyo and Ce sar de Haro

Centro de BiologõÂa Molecular `Severo Ochoa', Consejo Superior de Investigaciones CientõÂficas, Universidad AutoÂnoma de Madrid, Spain

In eukaryotic cells, protein synthesis is regulated in response to various environmental stresses by phos-

phorylating the a subunit of the eukaryotic initiation factor 2 (eIF2a). Three different eIF2a kinases have been

identified in mammalian cells, the heme-regulated inhibitor (HRI), the interferon-inducible RNA-dependent

kinase (PKR) and the endoplasmic reticulum-resident kinase (PERK). A fourth eIF2a kinase, termed GCN2, was

previously characterized from Saccharomyces cerevisiae, Drosophila melanogaster and Neurospora crassa. Here

we describe the cloning of a mouse GCN2 cDNA (MGCN2), which represents the first mammalian GCN2

homolog. MGCN2 has a conserved motif, N-terminal to the kinase subdomain V, and a large insert of 139 amino

acids located between subdomains IV and V that are characteristic of the known eIF2a kinases. Furthermore,

MGCN2 contains a class II aminoacyl-tRNA synthetase domain and a degenerate kinase segment, downstream

and upstream of the eIF2a kinase domain, respectively, and both are singular features of GCN2 protein kinases.

MGCN2 mRNA is expressed as a single message of < 5.5 kb in a wide range of different tissues, with the

highest levels in the liver and the brain. Specific polyclonal anti-(MGCN2) immunoprecipitated an eIF2a kinase

activity and recognized a 190 kDa phosphoprotein in Western blots from either mouse liver or MGCN2-

transfected 293 cell extracts. Interestingly, serum starvation increased eIF2a phosphorylation in MGCN2-

transfected human 293T cells. This finding provides evidence that GCN2 is the unique eIF2a kinase present in all

eukaryotes from yeast to mammals and underscores the role of MGCN2 kinase in translational control and its

potential physiological significance.

Keywords: cDNA cloning; mouse liver; protein kinase; serum starvation; translation initiation.

Phosphorylation of the a subunit of eukaryotic translationinitiation factor 2 (eIF2a) is one of the best characterizedmechanisms for down-regulating protein synthesis in mammal-ian cells in response to a variety of different cellular stressesincluding nutrient starvation, iron deficiency, heat shock andviral infection [1±4].

eIF2a kinases regulate the initiation of protein synthesis byphosphorylation of the a subunit of eIF2 on Ser51. GTP-boundeIF2 is necessary for delivering charged initiation tRNAMet tothe 40S ribosomal subunits, and after initiation of translation itis released as eIF2-GDP. The phosphorylated form of eIF2sequesters its own recycling factor, eIF2B, necessary for

exchange of GDP by GTP [5]. As only the GTP-bound formof eIF2 is able to initiate translation, sequestering of eIF2Bleads to a general reduction of protein synthesis.

All distinct eIF2a kinases share extensive identity in thekinase catalytic domain. Apart from the 12 conserved subdomainsfound in most protein kinases, they have additional character-istic features, including an insert between subdomains IV and Vwhich distinguishes them from other serine/threonine kinases[2,6]. In contrast to the catalytic domains, there is little simi-larity in their regulatory regions, which facilitates the differentstress signals controlling each eIF2a kinase. These kinases areactivated by distinct stimuli as follows: the heme-regulatedinhibitor (HRI) in rabbit reticulocytes by heme deficiency [7],the double-stranded RNA-dependent kinase (PKR) in human,mouse and rat cells by the occurrence of double-stranded RNAs(dsRNAs) [8] and the GCN2 of Saccharomyces cerevisiae byamino acid deprivation [4]. A third mammalian eIF2a kinase,termed PERK or PEK, has recently been identified as anendoplasmic reticulum (ER) kinase that is activated by ERstress [9]. PERK is expressed at high levels in the rat pancreas[10] and colocalizes with somatostatin in islet delta cells [11].

In contrast to PKR and HRI, which inhibit global proteinsynthesis in response to stress signals, the activation of GCN2in yeast leads to increased translation of one mRNA species, theGCN4 mRNA. This gene-specific regulation is mediated byfour short upstream ORFs in the 5 0-leader of GCN4 mRNA [4].Previously, GCN2 kinase has also been cloned and character-ized from Drosophila melanogaster (DGCN2) [12] and fromNeurospora crassa (CPC3) [13]. Expression of DGCN2 isregulated developmentally and at later stages becomesrestricted to a few cells of the central nervous system [12].

Eur. J. Biochem. 265, 754±762 (1999) q FEBS 1999

Correspondence to C. de Haro, Centro de BiologõÂa Molecular `Severo

Ochoa', CSIC-UAM, Facultad de Ciencias, Canto Blanco, 28049 Madrid,

Spain. Fax: + 34 91 397 4799, Tel.: + 34 91 397 8432,

E-mail: cdeharo@cbm.uam.es

Abbreviations: DGCN2, Drosophila GCN2-like eIF2a protein kinase;

DMEM, Dulbecco's modified Eagle's medium; ECL, enhanced

chemiluminescence; eIF2a, the a subunit (38 kDa) of eukaryotic

polypeptide chain initiation factor 2; ER, endoplasmic reticulum; EST,

expressed sequence tag; GCN2, yeast general amino acid control eIF2a

protein kinase; HGCN2, human GCN2-like eIF2a protein kinase; HisRS,

histidyl-tRNA synthetase; HRI, heme-regulated inhibitor; IMAGE,

integrated molecular analysis of gene expression; MGCN2, mouse

GCN2-like eIF2a protein kinase; PKR, double-stranded RNA-dependent

kinase; TEMED, N,N,N 0,N 00-tetramethylethylenediamine.

Note: the novel nucleotide sequence data published here have been

submitted to the EMBL sequence data bank and are available under the

accession numbers AJ243533 and AJ243428.

(Received 24 May 1999, revised 19 July 1999, accepted 5 August 1999)

q FEBS 1999 Cloning of mouse GCN2 protein kinase (Eur. J. Biochem. 265) 755

It is noteworthy that the overall rate of protein synthesisdecreases within seconds when mammalian cells are deprivedof serum or of even a single essential amino acid [14]. Previousstudies provide evidence that eIF2a becomes more highlyphosphorylated when cells are deprived of amino acids orcontain a defective aminoacyl-tRNA synthetase [15,16]. Theprotein kinase(s) responsible for these translational effects hasnot been identified. In contrast, it is well known that bothGCN1-encoded and GCN20-encoded proteins are additionallyrequired for activation of yeast GCN2 in starved cells [17]. Ahuman homolog of yeast GCN1 has recently been identified[18]. All of this raises the question of whether a GCN2homolog in mammalian cells is responsible for the regulation ofpolypeptide chain initiation under these stress conditions.

In this report, we identify and characterize the firstmammalian member of the GCN2 protein kinase family andprovide evidence that mouse GCN2-like eIF2a protein kinase(MGCN2) mRNA is widely distributed in several tissues. Inaddition, our data indicate that MGCN2 activity is regulated byserum. The presence of GCN2 homologs in mouse and humancells indicates that GCN2, in contrast to other members ofeIF2a kinase family, is conserved in all eukaryotic cells. Thiswould involve GCN2 in some common mechanisms thatmodulate eIF2 function in eukaryotic translation initiation.

E X P E R I M E N T A L P R O C E D U R E S

Materials and preparations

All reagents were from Sigma except protein A±Sepharose,ammonium persulfate and [g-32P]ATP from Amersham Phar-macia Biotech and acrylamide, N,N 0-methylenebisacrylamide,N,N,N 0,N 0-tetramethylethylenediamine (TEMED) and SDSfrom Bio-Rad. Specific DNA primers were obtained fromIsogen Bioscience. Highly purified rabbit reticulocyte eIF2awas obtained as described previously [19]. Mouse liver extractswere also prepared as described previously [20]. A polyclonalantibody against eIF2a-P [21] and a monoclonal antibodyagainst eIF2a [15] that recognizes both the phosphorylated andthe unphosphorylated forms were provided by Dr NahumSonenberg (McGill University, Montreal, Canada). Recombi-nant human wild-type eIF2a and the eIF2a-S51A mutant wereobtained as described by Berlanga et al. [20].

Cloning and sequence analysis

blast searches of the GenBank sequence database were madeusing sequence profiles of DGCN2 [12]. The matchingsequence (GenBank accession number AA016507) corre-sponded to the integrated molecular analysis of gene expression(IMAGE) consortium clone number 440053 derived from amouse embryo library. The 5 0-end of the cDNA was clonedusing 5 0-RACE. Poly(A)+ RNA (1 mg) from mouse liver(Clontech) was used to obtain a mouse liver cDNA libraryusing the Marathon cDNA amplification kit (Clontech).Amplifications were performed by PCR using the antisenseprimer 5 0-AAGAAGTTGCCCTTCAGGATGTCACTGTCG-3 0,complementary to nucleotides 3231±3260 of MGCN2 cDNA,in combination with the adaptor primer AP1 (Clontech). The5 0-RACE products were subcloned into a pCR2.1 vector(Invitrogen) and sequenced across both strands. The sequencesof three independent clones were compared to detect errors thatcould have occurred during reverse transcription or PCRamplification. Clone 440053 and RACE products were subjectedto automated sequencing (Applied Biosystem). Sequence

analyses were performed using blast [22], fasta [23], gap(Wisconsin Package, Genetics Computer Group, University ofWisconsin, Madison) and clustal w [24].

Human GCN2 cDNA sequence generated from the expressedsequence tag (EST) database

During a blast search of sequences related to the mouseGCN2 sequence in the EST database, we found a number ofhuman ESTs showing a good match at different regions. Byseeking multiple independent occurrence of overlapping ESTsequences we found 15 ESTs. Their accession numbers areAA316106, H19145, AA423963, AA376437, AA128979,AA306702, AA370461, AA159576, AA044653, AA610503,H97872, AA422123, AA523306, AA043344 and AI129185.Thus, we generated a partial human cDNA sequence from allthese ESTs, which together make a continuous 2162 bp cDNAsequence (deposited in EMBL Nucleotide Sequence Databaseas accession number AJ243428). The ORF (nucleotides3±1646) encodes a 548 amino acid peptide almost identical tothe C-terminal region (amino acids 1101±1648) of mouseGCN2. This peptide is a portion of the putative human GCN2homolog.

Construction of plasmids

The coding sequence corresponding to the mouse GCN2(MGCN2) was amplified by PCR using the full-length cDNAcloned into plasmid pT3T7D (Amersham Pharmacia Biotech)as a template. For the expression of MGCN2 in mammaliancells under the control of a cytomegalovirus promoter, the PCRoligonucleotides generated SmaI and XbaI sites, and theMGCN2 coding region was subcloned into a pcDNA3.1/Myc-His vector (Invitrogen) in frame with a C-terminal tag encodingthe myc epitope and a polyhistidine metal-binding peptide(pcMGCN2-wt). The deletion mutant MGCN2-D1±194 wasgenerated by amplification of a cDNA fragment that allowstranslation initiation from an ATG codon located 675 nucleo-tides downstream of the 5 0-end (methionine 195) and cloned inthe same vector (pcMGCN2-D1±194).

Antibodies against a synthetic MGCN2 peptide

Based on the MGCN2 cDNA coding sequence, a syntheticpeptide (C-GRAEPQESYSQRQ) was produced, correspondingto amino acids 11±23, with an additional residue (C) in itsN-terminal end. The peptide was synthesized as described bySantoyo et al. [12], coupled by the terminal cysteine residue tokeyhole limpet hemocyanin (Calbiochem) and rabbits wereimmunized as described by MeÂndez and de Haro [25]. Serumwas purified by affinity-chromatography as described pre-viously [12]. Plasmids pcMGCN2-wt and pcMGCN2-D1±194,encoding wild-type MGCN2 and the MGCN2 deletion mutant,respectively, were transcribed and translated in the TnT-coupledreticulocyte lysate system (Promega), in the presence of[35S]-labeled methionine (Amersham Pharmacia Biotech). Asexpected, our affinity-purified antibodies specifically immuno-precipitated the wild-type MGCN2 product (<190 kDa), butnot the mutant protein (<164 kDa) in which the first 194 aminoacids had been deleted (data not shown). For simplicity, theseaffinity-purified anti-(MGCN2-peptide) will be referred to asanti-(MGCN2).

MGCN2 mRNA expression

Mouse multiple tissue Northern blot (Clontech) was probedwith the full-length MGCN2 cDNA radiolabeled with

756 J. J. Berlanga et al. (Eur. J. Biochem. 265) q FEBS 1999

[a-32P]dCTP (Amersham Pharmacia Biotech) and analyzed byautoradiography. The cDNA probe was then removed to allowreprobing with mouse actin cDNA (Clontech) as a quantitycontrol probe.

Cell culture and transfection

293 and 293T cells were maintained in Dulbecco's modifiedEagle's medium (DMEM) containing 10% (v/v) fetal bovineserum. Cells were grown and transfected with 5 mg plasmidDNA per 100-mm or 60-mm dish as described previously [20].The plasmids used were pcMGCN2-wt and the control plasmidpcDNA 3.1/Myc/His/lacZ (Invitrogen).

Immunoprecipitation, affinity purification and eIF2a kinaseassay

At 48 h post-transfection, cells were washed once with NaCl/Pi

containing 90 mm sodium fluoride, 17.5 mm sodium molybdateand 17.5 mm b-glycerophosphate, and lysed in lysis buffer[20 mm Tris/HCl pH 7.8, 137 mm NaCl, 1 mm EGTA, 10%(v/v) glycerol, 1% (v/v) Triton X-100, 1 mm PhCH2SO2F,1 mm sodium orthovanadate, 10 mm sodium fluoride, 0.1 mmb-glycerophosphate, 1 mm tetrasodium diphosphate and a prote-ase inhibitor cocktail (Completew, Boehringer Mannheim)].Cell debris was removed by centrifugation and the proteinconcentration in the supernatant was measured using the Bio-Radprotein assay. The supernatants were either subjected toimmunoprecipitation with anti-(MGCN2) (5 mg of IgG) andprotein A±Sepharose as described previously [20], or incubatedwith 20 mL of TALONTM metal affinity resin (Clontech). After30 min at 4 8C, metal affinity resin was washed three timeswith lysis buffer containing 10 mm imidazole and twice with abuffer [20 mm Hepes/KOH pH 7.6, 50 mm KCl, 1 mm MgCl2,5% (v/v) glycerol] containing 10 mm imidazole, before elutionwith the same buffer containing 100 mm imidazole. Afterelution, dithiothreitol was added to adjust its concentration to1 mm in the sample solution. In the case of the mouse liverextracts, the supernatants were precleared with proteinA±Sepharose and then subjected to immunoprecipitation withanti-(MGCN2) as described previously [12]. The immunopre-cipitates, as well as the eluted proteins from the metal affinityresin, were assayed for their ability to phosphorylate eIF2a asreported previously [12,25] with modifications as described. Ina total volume of 30 mL, samples containing 10 mL of enzymefractions, as specified in the figure legends, were pre-incubatedfor 10 min at 30 8C in the presence of buffer containing 20 mmTris/HCl, pH 7.5, 0.2 mg´mL21 BSA, 50 mm ATP and 5 mmMg(OAc)2. All samples were subsequently incubated for20 min at 30 8C in the presence of purified rabbit reticulocyte

eIF-2 (0.5 mg) as substrate and 5 mCi of [g-32P]ATP(3000 Ci´mmol21). Incubations were terminated by the addi-tion of SDS sample buffer, and samples were analyzed byelectrophoresis on 10% SDS/PAGE as described previously[12,20]. After electrophoresis, in some experiments the proteinswere transferred to an Immobilon-P membrane (MilliporeCorp.) and immunodetected using either anti-(MGCN2) oranti-(Myc) (Invitrogen) as described previously [12,20].

In vivo phosphorylation of eIF2a

At 36 h post-transfection, cells were maintained in DMEMsupplemented with 10% fetal bovine serum as control ortransferred to DMEM supplemented with either 2% or 0% fetalbovine serum (0% = serum deprivation conditions). After 12 h,cells were washed and lysed as described above. Proteins weresubjected to SDS/PAGE, blotted onto 0.25 mm nitrocellulosemembranes and sequentially probed with mouse anti-(Myc),rabbit anti-(eIF2a-P) [21] and mouse anti-(eIF2a) [15]. Afterextensive washing, the immunoreactive bands were detected byenhanced chemiluminiscence (ECL, Amersham PharmaciaBiotech). The relative intensities of bands were quantified byusing a Biolmage Densitometer (Milligen/Biosearch). The datashown are representative of three different experiments withsimilar results.

R E S U LT S

Cloning of MGCN2 cDNA

A partial cDNA clone (nucleotides 2961±5212) from mouseembryos (EST 440053/GenBank accession no. AA016507)shows an ORF (nucleotides 2961±5036) encoding a polypep-tide of 692 amino acids with similarity to the C-terminal region[last kinase subdomain and the entire histidyl-tRNA synthetase(HisRS) domain] of Drosophila and yeast GCN2 [12,26]. Whenthis cDNA was used as a probe in Northern blot analysis ofpoly(A)+ RNA from mouse liver, a single 5.5-kb transcriptwas detected (data not shown). To obtain the full-length cDNA,5 0-RACE experiments were performed using mouse liverpoly(A)+ RNA. Such experiments produced an overlappingpartial cDNA fragment (R5-2B clone, 1±3259 nucleotides).The full-length cDNA (5212 nucleotides) was obtained fromthese two overlapping clones (EMBL nucleotide sequencedatabase, accession number AJ243533). The first ATG in thissequence, which is located 93 nucleotides from the 5 0-end ofthe cDNA, begins an ORF of 4944 bp (nucleotides 93±5036).This ATG constitutes a bona fide initiation codon, because it ispreceded by an in-frame stop codon and is surrounded by thesequence GCCATGG, which matches the consensus sequence

Fig. 1. MGCN2 encodes the mouse GCN2 kinase homolog. (A) Alignment of the peptide sequence of mouse GCN2 and their D. melanogaster,

S. cerevisiae and N. crassa homologs. The sequences were aligned using the clustal w alignment program, with some manual adjustments made in regions

of limited similarity. MGCN2, mouse GCN2 eIF2a kinase; DGCN2, Drosophila GCN2 eIF2a kinase; GCN2, S. cerevisiae GCN2 eIF2a kinase; and CPC3,

N. crassa GCN2 eIF2a kinase. Gaps introduced to optimize the alignment are represented by dots. Kinase subdomains are identified by roman numerals

directly above the appropriate regions. The conserved motifs, m1, m2 and m3, of the HisRS-related region are also indicated. Amino acid numbering is shown

to the left. The partial peptide sequence, corresponding to amino acids 11±23, used to obtain anti-(MGCN2) is mark by a line above the sequence. Identical

residues in at least three of the four sequences are indicated by a black background, while similar amino acids in at least three of the four sequences are

indicated by a gray background. (B) Diagram of MGCN2 domain structure. The 1648 amino acid MGCN2 sequence is illustrated by a larger box. The figure

is drawn to scale. Highlighted domains include the N-terminal (cross-hatched box); the `degenerate kinase' (vertical dashed box) that is related to subdomains

I±XI of eukaryotic protein kinases; the conserved two lobes of the eIF2a kinase domain (black), separated by a large insert (white box); and the HisRS-like

domain (stippled boxes) that includes the three motifs (m1, m2 and m3) conserved among the class II aminoacyl-tRNA synthetases. The numbers refer to the

amino acid residues.

q FEBS 1999 Cloning of mouse GCN2 protein kinase (Eur. J. Biochem. 265) 757

758 J. J. Berlanga et al. (Eur. J. Biochem. 265) q FEBS 1999

for strong mammalian initiator codons [27]. The 3 0-UTR is 176nucleotides in length and contains a putative polyadenylationsignal (AATAAA, nucleotides 5194±5199). The full-lengthMGCN2 cDNA encodes for a protein of 1648 amino acids(Fig. 1A), with a predicted molecular mass of 186.4 kDa. Ahigh degree of similarity was found to all eIF2a kinases andspecifically with all GCN2 kinases reported previously (Fig.1A). MGCN2 contains all 12 conserved catalytic subdomainsof eukaryotic Ser/Thr protein kinases (Fig. 1A) with the con-served sequence of eIF2a kinases N-terminal of subdomain V.In addition, MGCN2 bears a large insert of < 139 amino acidsbetween subdomains IV and V, typical of eIF2a kinases(Fig. 1B).

The deduced amino acid sequence of MGCN2 showedhighest overall similarity to DGCN2 of Drosophila [12], GCN2of yeast [26] and CPC3 of Neurospora crassa [13], with 33, 26and 27% identity, and 44, 37 and 36% similarity, respectively.Furthermore, MGCN2 has four regions with characteristicfeatures of GCN2-like kinases: the N-terminal region, thedegenerate kinase domain, the eIF2a kinase domain and theHisRS-like domain (Fig. 1B).

Notably, a block of conserved sequence at the N-terminalwas found between MGCN2 of mouse (amino acids 1±200) andDGCN2 of Drosophila (amino acids 1±193) with 37% identityand 50% similarity. This N-terminal sequence of MGCN2 alsoshowed significant similarity to the rest of the GCN2 proteins(Fig. 1A) but to no other polypeptides. These data suggest thatthe N-terminal region may represent a novel domain for GCN2protein kinases with unknown function.

The highest sequence conservation was observed in thekinase domain (amino acids 587±1001; Fig. 1). The MGCN2kinase domain is more closely related to the DrosophilaDGCN2 (46% identity and 55% similarity), although it shareshigh homology with all of the known eIF2a kinases. Thisdegree of similarity is higher if we consider that the greatestvariation is concentrated in the unique insertion sequencelocated between catalytic subdomains IV and V (Fig. 1A). Inaddition, 11 residue positions dispersed among the catalyticdomains of the eIF2a kinases were shown to be conservedamong the family members, but absent in the majority of otherprotein kinases [28]. MGCN2 contains the same residues at allof these positions. These findings suggested that MGCN2encodes a functional eIF2a kinase.

Downstream of the eIF2a kinase domain, MGCN2 containsan amino acid sequence (amino acids 1021±1492) related to

HisRS (Fig. 1B), a unique feature of the GCN2 protein kinasefamily. All HisRS-like domains are characterized by threemotifs (m1, m2 and m3) conserved among class II aminoacyl-tRNA synthetases, plus sequences unique to HisRS proteins.Thus, like yeast GCN2 and N. crassa CPC3, MGCN2 possessesthe sequences AAGGRYD (called `histitine B'), locatedimmediately N±terminal of m3, and well conserved. However,the HisRS-like domain of MGCN2 is somewhat more similar toDrosophila DGCN2 than to the other GCN2 proteins (31%identity and 46% similarity).

In addition, the N-terminal region directly preceding thecatalytic domain of MGCN2 encodes a degenerate kinasedomain (amino acids 280±537) also found in all known GCN2proteins. MGCN2 contains residues related to subdomains I±XIand lacks the nearly invariant amino acids Phe and Gly insubdomain VII, which participates in ATP binding (Fig. 1A).Taken together, these data indicate that MGCN2 is indeed themammalian homolog of GCN2 protein kinase.

In addition, a search of the nucleotide database (GenBank)identified multiple human ESTs that closely matched portionsof mouse MGCN2. By using several of these human ESTs withoverlapping sequences we constructed a sequence of 2162 bp(EMBL nucleotide sequence database accession numberAJ243428) encoding a polypeptide 548 residues long and94% identical to the C-terminal region (amino acids 1101±1648) of mouse MGCN2 (data not shown). We conclude thatMGCN2 represents a member of the GCN2 kinase family inmammalian cells.

Expression and characterization of MGCN2 cDNA

MGCN2 mRNA expression was examined by Northern blottingusing the full-length MGCN2 cDNA as a probe (Fig. 2). Asingle 5.5 kb mRNA was detected at varying levels in most ofthe tissues examined and corresponded to the full-length cDNAthat we cloned. There appear to be some differences in levels ofexpression in different tissues, thus, the MGCN2 gene washighly expressed in liver and brain. Lower levels of MGCN2mRNA were detected in skeletal muscle and spleen (Fig. 2). Itis interesting to note that the mRNA levels of both mHRI andPEK eIF2a kinases were also very low in skeletal muscle cells;however, elevated significantly in spleen cells [10,20]. As withMGCN2, ESTs corresponding to human GCN2 (HGCN2)mRNA were found in essentially all tissues (Table 1).

Table 1. Expression of HGCN2 mRNAs in different human tissues.

Tissue EST accession number

Aorta endothelial cells AA306702

Brain H19145, R19609, R19927

Colon AA838501

Epididymus AA335658

Fetal heart AA010941

Fetal lung AA376437, N75547

Kidney AA583690, AI015537

Lung AA316106, AA523306, AA632948

Melanocyte H97872

Neuroepithelial cells AA216651

Pancreas AA159551

Prostate AA230276, AA370461, AA652761, AA737237, AA767974

Thyroid AA610503

Uterus AA044653

q FEBS 1999 Cloning of mouse GCN2 protein kinase (Eur. J. Biochem. 265) 759

MGCN2 is a phosphoprotein and phosphorylates eIF2a onresidue serine 51

To verify that MGCN2 is an eIF2a kinase, we immunopreci-pitated the enzyme from mouse liver extracts by using anti-(MGCN2). The isolated immune complexes were incubatedwith [g-32P]ATP in the presence of highly purified rabbitreticulocyte lysate eIF2 as a substrate, and the radiolabeledproducts were analyzed by SDS/PAGE and Western blot asdescribed above. A labeled polypeptide with a molecular massof < 190 kDa was produced by MGCN2 immune complexes

(Fig. 3A, lane 3). This phosphopolypeptide was recognized bythe anti-(MGCN2) in a Western blot assay (Fig. 3A, lane 1).Moreover, MGCN2 immune complexes phosphorylated thea subunit of the eIF2 (Fig. 3A, lane 3). The immunoprecipita-tion was specific because it was prevented by addition of thepeptide immunogen in the immunoprecipitation assay (Fig. 3A,lanes 2 and 4).

In addition, we expressed the plasmids encoding MGCN2 orthe b-galactosidase (as negative control) in 293T (Fig. 3B) or293 (Fig. 4) cells as Myc±His-tagged fusion proteins. Recom-binant proteins were either purified by the TALONTM metalaffinity resin (Fig. 3B, lanes 1 and 2) or immunoprecipitated byanti-(MGCN2) (Fig. 3B, lanes 3 and 4), and the recombinantproteins were subjected to the same type of analysis. Bothaffinity-purified recombinant MGCN2 and isolated MGCN2immune complexes underwent phosphorylation and were fullyactive in phosphorylating eIF2a (Fig. 3B, lanes 1 and 3). Thesephosphorylated MGCN2 proteins were recognized by the anti-(Myc) in a Western blot assay (Fig. 3B, lanes 5 and 7). Asexpected, we detected recombinant b-galactosidase in theaffinity-purified preparation (Fig. 3B, lane 6) but not in theimmune complexes (Fig. 3B, lane 8). Moreover, recombinantMGCN2 specifically phosphorylated eIF2a at Ser51 (Fig. 4,lane 1, bottom), because phosphorylation was not observed inthe assay mixture containing the mutant substrate eIF2a-S51A(Fig. 4, lane 2, bottom). The phosphorylated MGCN2 (lanes 1and 2, middle) was also recognized by anti-(MGCN2) in animmunoblot (lanes 1 and 2, top).

The data presented in Fig. 3B indicate that the levels ofrecombinant MGCN2 (lanes 5 and 7) correlate with the levelsof MGCN2 phosphorylation (lanes 1 and 3). Furthermore,although the levels of recombinant MGCN2 detected in theimmune complexes were much higher than that in the affinity-purified preparation, the levels of eIF2a phosphorylation werevery similar. Thus, the eIF2a kinase activity of the MGCN2

Fig. 2. Tissue-specific expression of MGCN2 transcripts. Northern blot

of poly(A)+ RNA prepared from various mouse tissues (Clontech) was

hybridized with MGCN2 cDNA that encompasses the entire ORF (upper

panel). The size of the MGCN2 mRNA is 5.5 kb. The migration of RNA

size markers is indicated on the left. As a control of RNA levels, the same

blot was rehybridized with a mouse actin cDNA probe (lower panel).

Fig. 3. Autokinase and eIF2a kinase activities

of both partially purified and recombinant

MGCN2. (A) In vitro phosphorylation of

purified rabbit reticulocyte eIF2 by immune

complexes prepared from mouse liver extracts as

described under Experimental procedures using

anti-(MGCN2) in the absence (lanes 1 and 3) or

presence (lanes 2 and 4) of the competing

peptide. Proteins were separated by SDS/PAGE

and transferred to an Immobilon-P membrane

followed by autoradiography (right panel, lanes 3

and 4). The same membrane was probed with

anti-(MGCN2) (left panel, lanes 1 and 2) in a

Western blot analysis. (B) Recombinant MGCN2

is a phosphoprotein and phosphorylates the

a subunit of eIF2. In vitro kinase reactions

contained either metal affinity resin purified

proteins (lanes 1 and 2) or anti-(MGCN2)

immune complexes (lanes 3 and 4) prepared from

293T cells 48 h after transfection with either

MGCN2 (lanes 1 and 3) or b-galactosidase (lanes

2 and 4) expression plasmids. The reactions also

included purified rabbit reticulotycte eIF2. All

samples were assayed as described for (A) with

the exception that, after autoradiography (upper

panel, lanes 1±4), the same membrane was

probed with monoclonal anti-(Myc) (lower panel,

lanes 5±8).

760 J. J. Berlanga et al. (Eur. J. Biochem. 265) q FEBS 1999

immune complexes was lower than expected (Figs 3B and 4).The same was found with the MGCN2 immune complexesisolated from mouse liver extracts (Fig. 3A). These resultssupport the idea that anti-(MGCN2) might interfere with theeIF2a kinase activity of MGCN2. Nevertheless, we concludethat the isolated MGCN2 from mouse liver extracts andrecombinant MGCN2 are the same phosphoprotein, and thatboth specifically phosphorylate eIF2a on Ser51, the sameresidue that is targeted by all known eIF2a kinases.

MGCN2 is activated by serum starvation

It is well known that overall rates of protein synthesis inmammalian cells are inhibited during amino acid, glucose andserum starvation [14,29] by mechanisms involving eIF2a phos-phorylation, although the specific kinases and/or phosphatasesthat regulate phosphorylation of eIF2a under these conditionsare not known [14]. This regulatory system has been studiedmost intensively in yeast, where GCN2 is activated underconditions of amino acid deficiency [4]. We tested whetherMGCN2 could be activated by serum deficiency conditions.The data presented in Fig. 5 demonstrated that the recombinantMGCN2 is indeed activated by serum starvation. Thus, 293Tcells transfected with MGCN2 or b-galactosidase (as negativecontrol) expression plasmids accumulate large quantities of theMyc-tagged fusion proteins, and when levels of expressionreached a certain threshold, cells were maintained in mediumcontaining 0, 2 or 10% serum for 12 h before lysis as describedabove. Using immunoblot analysis with the appropriate anti-bodies we detected recombinant MGCN2 (Fig. 5A, lanes 1±3,top), eIF2a-P (middle) and total eIF2a (bottom) levels in thecell extracts. Whereas the levels of recombinant MGCN2, aswell as of the endogenous eIF2 (both phosphorylated and

unphosphorylated forms), were very similar under all threeexperimental conditions, the eIF2a phosphorylation observedin vivo was highly induced in serum-starved cells (Fig. 5A,middle). This increased level of eIF2a-P correlates with thedegree of serum deprivation. Quantitative evaluation of thisexperiment (Fig. 5B) reveals that the in vivo eIF2a-P levelsincreased more than twofold in MGCN2-transfected cells byserum starvation. It should be noted that, in good agreementwith previous results (Fig. 3), expression of MGCN2 in the293T cells maintained in serum-containing medium (10%serum control) yielded a basal eIF2a-P level (Fig. 5A, middle).

D I S C U S S I O N

Phosphorylation of eIF2a by GCN2 kinase mediates gene-specific translational control of GCN4 in S. cerevisiae. TheGCN4 gene encodes a transcriptional activator of amino acidbiosynthetic genes, and GCN2 activity is required for increasedtranslation of GCN4 mRNA under amino acid starvation. In thisreport, we describe the characterization of a new mammalian

Fig. 4. Recombinant MGCN2 specifically phosphorylates the a subunit

of eIF2 at Ser51. In vitro kinase reactions contained anti-(MGCN2)

immune complexes prepared from 293 cells 48 h after transfection with

either MGCN2 (lanes 1 and 2) or b-galactosidase (lanes 3 and 4) expression

plasmids. The reactions also included recombinant wild-type human eIF2a

(lanes 1 and 3) or eIF2a-S51A (lanes 2 and 4). All samples were assayed as

described in Fig. 3. The following results are shown: top, immunoblot

showing levels of MGCN2 protein; middle, autoradiogram of the in vitro

autokinase activity of MGCN2; bottom, autoradiogram of the eIF2a kinase

activity of MGCN2.Fig. 5. In vivo eIF2a phosphorylation by MGCN2 is induced by serum

starvation. 293T cells expressing MGCN2 or b-galactosidase by transient

transfection were maintained in medium containing 0, 2 or 10% serum for

12 h before lysis as described under Experimental procedures. (A) Equal

amounts of protein extracts (150 mg) were subjected to 10% SDS/PAGE

and immunoblot with monoclonal anti-(Myc) (upper), or polyclonal anti-

(eIF2a-P) (middle). The membrane was then stripped and immunoblotted

with monoclonal anti-(eIF2a) (lower). (B) Quantitative analysis of the

results in (A). The relative intensities of the bands were quantified and

expressed as the ratio between eIF2a-P and eIF2a signals. The unit was

defined as the value obtained for cells expressing b-galactosidase in

medium containing 10% serum. This experiment is representative of three

others which yielded similar results.

q FEBS 1999 Cloning of mouse GCN2 protein kinase (Eur. J. Biochem. 265) 761

eIF2a kinase, the mouse liver homolog of GCN2 (MGCN2).MGCN2 phosphorylates the a subunit of eIF2 at residue Ser51,as do all members of the eIF2a kinase family, and contains amodified HisRS-related domain, a unique feature of the GCN2kinase family.

Mouse MGCN2 is 46% identical to Drosophila DGCN2within the catalytic domain and contains all 12 conservedsubdomains and all residues known to be present among eIF2akinases [28]. This domain, as in all eIF2a kinases, possesses alarge (139 residues) insert between subdomains IV and V. TheeIF2a kinase insert region varies greatly in both length andsequence between PKR, HRI, PERK and GCN2 [7±9,30].Within this region, the main characteristic features of MGCN2,as in the other GCN2 kinases, are the stretches of serinescombined with highly acidic sequences. In addition, MGCN2contains a particular sequence N-terminal of this domain whichis very rich in prolines. The functional significance of thisregion is still unknown.

Like all other GCN2 kinases, MGCN2 contains the threeconserved motifs found in class II aminoacyl tRNA synthetaseswithin its HisRS-related domain. However, certain residuesconserved in genuine HisRS proteins were absent in the GCN2-related kinases. Thus, the MGCN2 HisRS-like domain lacks theinvariant Arg in m3 as well as the amino acid stretchLVRGLDYY (called `histidine A') located N-terminal of m3,which contribute to ATP and histidine binding, respectively[31]. It was shown that the HisRS-related domain of yeastGCN2 binds uncharged tRNA and that the HisRS-likesequences are required for the activation of yeast GCN2 byamino acid starvation [32]. The presence of a HisRS-relateddomain in MGCN2 suggests that the mammalian enzyme mightfunction in response to amino acid starvation. It is especiallynoteworthy that the N-terminus of the GCN2-related kinasesdisplays a significant identity with unknown functions and noobvious similarity to any known protein in the currentdatabases. This region may represent an additional regulatorydomain capable of responding to some cellular stress conditionsother than amino acid deprivation.

Autophosphorylation is an important step for the activationof the eIF2a kinases [2,4,7,8]. It has recently been shown thatThr446 and Thr551 in the PKR activation loop are some of theautophosphorylation sites in vivo [33]. Interestingly, MGCN2contains two Thr residues (Thr898 and Thr903) at exactly thesame positions relative to subdomain VIII as those occupiedby the authophosphorylated residues in PKR. Accordingly,MGCN2 immune complexes prepared from either mouse liveror MGCN2-transfected 293 cell extracts immunoprecipitatedwith anti-(MGCN2), have shown autophosphorylating activityin vitro.

The tissue distribution of MGCN2 mRNA expression differsfrom HRI and PERK [10,20]. While Northern analysis suggeststhat MGCN2 is widely distributed among mouse tissues, it isexpressed in liver and brain at higher levels. This observationwas confirmed by Western blot assays that showed MGCN2 inliver extracts and by in situ hybridization assays on mouseembryos that revealed ubiquitous MGCN2 expression levelsduring development (unpublished results). We previouslyreported a highly specific expression pattern of DrosophilaDGCN2 at the central nervous system cells of the ventral cordin late embryogenesis [12]. All of these findings togethersuggest that GCN2 in higher eukaryotic cells may play either arole in development or a physiological role in a tissue that isvery sensitive to nutrient starvation.

In addition to viral infection and hemin deficiency, knownactivators of PKR and HRI, respectively, a variety of cellular

stresses have been observed to elicit the phosphorylation ofmammalian eIF2a including: starvation of amino acids, glucoseor serum; growth factor deprivation; and heat shock [14]. Manyof these stress conditions do not appear to be mediated by PKRand HRI, suggesting that additional mammalian eIF2a kinases,such as MGCN2, may mediate translational control in responseto many of these conditions. Here, we have shown thatrecombinant MGCN2 was activated by serum starvation inMGCN2-transfected 293T cells. Thus, the eIF2a phos-phorylation observed in vivo was greatly induced in serum-starved MGCN2-transfected cells.

We also wished to determine whether MGCN2 kinase, likeyeast GCN2, had the ability to be activated by amino acidstarvation. However, we were unable to detect any effect. Thus,in MGCN2-transfected 293T cells, amino acid depletion did notbring about a detectable change in eIF2a phosphorylation. Thereason for this result is not immediately clear. Although incertain other mammalian cell types, amino acid deficiency hasbeen shown to cause increased phosphorylation of eIF2a[15,34], more recent studies suggest that this does not seem tobe the case, at least in Chinese hamster ovary cells [35] and L6myoblasts [36]. Future research on MGCN2 should shed lighton the physiological control mechanism(s) of the mammalianGCN2. It is possible that, in contrast to yeast GCN2,mammalian GCN2s play important and specific roles inregulating translation in response to different stimuli otherthan amino acid starvation.

The data reported here indicate that we have succeeded incharacterizing the first mammalian homolog of yeast GCN2from mouse liver and provide strong evidence that MGCN2 isindeed a protein kinase responsible for phosphorylation ofeIF2a when mammalian cells are subjected to serum depriva-tion. Our data indicate that GCN2 is the founding and bestconserved member of the eIF2a kinase family.

It is not yet known whether mammalian cells induce theexpression of specific gene products at the translational levelunder conditions where eIF2a-phosphorylation is increased,and whether a GCN4-type mechanism can operate in highercells. The strong similarities in the control of eIF2a-phos-phorylation by nutrient starvation in yeast and higher organismssuggest that this is one hypothesis, and it is currently beinginvestigated.

A C K N O W L E D G E M E N T S

We thank Dr Rolando Rivera-Pomar for his critical reading of the

manuscript. We also thank Jose Alcalde for excellent technical assistance.

J. J. B. is the recipient of a postdoctoral fellowship from the Comunidad de

Madrid (Spain). This work was supported in part by Grant BIO4-CT95-

0045 from the European Commission Directorate General XII Biotechnol-

ogy Program and by an institutional grant from the FundacioÂn RamoÂn

Areces (to the Centro de BiologõÂa Molecular `Severo Ochoa').

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