Autophosphorylation of heme-regulated eukaryotic initiation factor 2a kinase and the role of the modification in catalysis Jotaro Igarashi 1, *, Takehiko Sasaki 1, *, Noriko Kobayashi 2 , Shinji Yoshioka 2 , Miyuki Matsushita 2 and Toru Shimizu 1 1 Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Japan 2 Bio-Medical Center, R&D Division, Nanotechnology Product Business Group, Hitachi High-Technologies Corporation, Hitachinaka, Ibaraki, Japan Introduction Inhibition of protein synthesis or translation is impor- tant to ensure cell survival during stress or emergency states [1,2]. Four eukaryotic initiation factor 2a (eIF2a) 3 kinases, heme-regulated eIF2a kinase (HRI), PKR, PERK and GCN2, inhibit translation upon recognition of stress or emergency states, such as heme shortage, virus infection, accumulation of denatured proteins or amino acid shortage, respectively. After Keywords autophosphorylation; eIF2a kinase; heme; mass spectrometry; mutation Correspondence J. Igarashi, Institute of Multidisciplinary Research for Advanced Materials, Tohoku University 2-1-1 Katahira, Aoba-ku, Sendai 980-8577, Japan Fax: +81 22 217 5605 Tel: +81 22 217 5605 E-mail: jotaro@tagen.tohoku.ac.jp *These authors contributed equally to this work (Received 2 September 2010, revised 23 November 2010, accepted 7 January 2011) doi:10.1111/j.1742-4658.2011.08007.x Heme-regulated eukaryotic initiation factor 2a (eIF2a) kinase (HRI), functions in response to heme shortage in reticulocytes and aids in the maintenance of a heme:globin ratio of 1:1. Under normal conditions, heme binds to HRI and blocks its function. However, during heme shortage, heme dissociates from the protein and autophosphorylation subsequently occurs. Autophosphorylation comprises a preliminary critical step before the execution of the intrinsic function of HRI; specifically, phosphorylation of Ser-51 of eIF2a to inhibit translation of the globin protein. The present study indicates that dephosphorylated mouse HRI exhibits strong intramo- lecular interactions (between the N-terminal and C-terminal domains) compared to phosphorylated HRI. It is therefore suggested that autophos- phorylation reduces the intramolecular interaction, which induces irrevers- ible catalytic flow to the intrinsic eIF2a kinase activity after heme dissociates from the protein. With the aid of MS, we identified 33 phos- phorylated sites in mouse HRI overexpressed in Escherichia coli. Phosphor- ylated sites at Ser, Thr and Tyr were predominantly localized within the kinase insertion region (16 sites) and kinase domain (12 sites), whereas the N-terminal domain contained five sites. We further generated 30 enzymes with mutations at the phosphorylated residues and examined their catalytic activities. The activities of Y193F, T485A and T490A mutants were signifi- cantly lower than that of wild-type protein, whereas the other mutant pro- teins displayed essentially similar activity. Accordingly, we suggest that Tyr193, Thr485 and Thr490 are essential residues in the catalysis. Abbreviations CID, collision-induced dissociation; ECD, electron-capture dissociation; eIF2a, eukaryotic initiation factor 2a; Ga-IMAC, gallium immobilized metal ion affinity chromatography; GCN2, general control nonderepressible 2; HRI, heme-regulated eIF2a kinase or heme-regulated inhibitor; KD, C-terminal kinase domain containing amino acids 145-619; KI, kinase insert with amino acids 244-371; NTD, N-terminal domain containing amino acids 1-144; P-eIF2a, phosphorylated-eIF2a ; PERK, PKR-like endoplasmic reticulum-related kinase; PKR, double strand RNA-dependent protein kinase. 918 FEBS Journal 278 (2011) 918–928 ª 2011 The Authors Journal compilation ª 2011 FEBS sensing stress, these eIF2a kinases phosphorylate a common substrate, eIF2a at Ser51, leading to the termination of protein synthesis [3]. HRI regulates the translation of hemoglobin proteins in reticulocytes in response to heme availability [4–10]. The protein remains inactive in the presence of sufficient heme, thus inhibiting catalysis under normal conditions, although it becomes active when heme dissociates from the protein during heme shortage. HRI is composed of an N-terminal domain (NTD) (amino acids 1–144) (Fig. 1, red area) and a C-termi- nal kinase domain (KD) (amino acids 145–619) (Fig. 1, green area) including a kinase insert (KI) region (amino acids 244–371) (Fig. 1, yellow area). Note that the amino acid numbering is based on the mouse HRI sequence from the present study (Fig. 1). Heme binding ⁄ recognition sites are located both within the NTD (His119 ⁄ His120) and the KD (Cys409), sug- gesting that significant global structural changes (and not simply heme binding to a protein surface patch) are required for heme sensing by HRI [11]. The cata- lytic activation process of HRI comprises four steps. Specifically, in response to heme deficiency or low heme concentration: (a) heme dissociates from the HRI protein; (b) HRI is autophosphorylated at key residues; (c) HRI is autophosphorylated at multiple sites; and (d) subsequent phosphorylation at Ser51 of eIF2a is triggered. Therefore, it is important to explore the role of autophosphorylation of the HRI protein in the intramolecular protein–protein interac- tion (between the NTD and KD) and catalysis upon heme binding. Identification of autophosphorylated sites should further facilitate our understanding of the molecular mechanism underlying HRI function. As with other protein kinases, the activities of eIF2a kinases are regulated by the initial autophosphoryla- tion step. In addition to Ser ⁄ Thr residues, Tyr residues are phosphorylated in the eIF2a kinases, PKR and PERK [12,13]. To date, only a few autophosphoryla- tion sites have been identified for eIF2a kinases. In mouse HRI, Thr483 and Thr485 phosphorylation has been detected using 32 P-labeling and peptide sequenc- ing analysis, although only phosphorylated Thr485 was implicated in protein function [14]. In addition, phosphoamino acid analysis revealed phosphorylation at Tyr residues in mouse HRI [15]. Because eIF2a kinases are activated via multiple autophosphorylation steps, it is important to identify other phosphorylation sites in HRI and to establish the contribution of the autophosphorylation process to heme sensing and catalysis. The results obtained should aid the clarifica- tion of the general role of autophosphorylation in catalysis by eIF2a kinases as well as the molecular mechanism of stress sensing. In the present study, we overexpressed mouse HRI protein in Escherichia coli and examined the effects of phosphorylation on intramolecular protein–protein interactions, catalysis and heme sensing using both autophosphorylated and dephosphorylated HRI proteins. We aimed to identify autophosphorylation sites of HRI by MS. Based on the 33 phosphorylated sites identified using LC-MS ⁄ MS analysis, we gener- ated 30 mutant proteins deficient in phosphorylation and assessed their catalytic activities aiming to determine the role of phosphorylation in catalysis. Notably, phosphorylation at Tyr193, Thr485 and Thr490 appeared to be critical for intrinsic eIF2a kinase activity. Fig. 1. Amino acid sequence and domain architecture of HRI. HRI is composed of an NTD (amino acids 1–144) (red area) and a KD (amino acids 145–619) (green area) including a KI region (amino acids 244–371) (yellow area). The peptide fragments used to determine phosphorylated sites are underlined, and the phosphorylated sites identified after trypsin digestion, Ga-IMAC purification and LC-MS ⁄ MS detection are indicated in red. Previously identified phosphorylated sites are shown in bold. J. Igarashi et al. Autophosphorylation of an HRI FEBS Journal 278 (2011) 918–928 ª 2011 The Authors Journal compilation ª 2011 FEBS 919 Results and Discussion Dephosphorylation of HRI with k phosphatase and autophosphorylation in the presence of ATP Because purified mouse HRI overexpressed in E. coli is already phosphorylated, it is possible that the auto- phosphorylation of HRI observed in the present study was induced by expression in a heterologous host but did not occur in eukaryotic cells. To address this issue, we attempted to generate dephosphorylated HRI pro- tein by treatment with k phosphatase [16]. Initially, HRI was co-expressed with k phosphatase in E. coli. However, the yield of dephosphorylated HRI was only 10% of that of phosphorylated HRI as a result of the formation of inclusion bodies. Subsequently, we suc- cessfully obtained dephosphorylated HRI protein with k phosphatase and examined its autophosphorylation activity. Dephosphorylation of HRI was evident from differences in protein mobility on SDS ⁄ PAGE when purified without k phosphatase and when k phospha- tase-treated HRI proteins were compared (Fig. S1). No marked differences in oligomerization status were observed between autophosphorylated and k phospha- tase-treated HRI. As shown in Fig. S2 (upper panel), dephosphorylated HRI protein per se autophosphory- lates within 1–2 min after mixing with ATP in the absence of heme. Intrinsic HRI catalytic activity, in terms of the phosphorylation of eIF2a, was observed after autophosphorylation in the absence of heme (Fig. S2, lower panel). We conclude that dephospho- rylated HRI protein has properties similar to the native counterpart in terms of autophosphorylation and phosphorylation of eIF2a. Effects of dephosphorylation on intramolecular protein–protein interactions We employed a pulldown assay to examine the effects of dephosphorylation on protein–protein inter- actions between the isolated NTD and KD using His-tagged NTD (His6-NTD) and both phosphory- lated and dephosphorylated KD (Fig. 2). Interactions between NTD and KD were stronger in the presence than the absence of heme (compare lanes 1 and 2 versus lanes 3 and 4; Fig. 2), which is consistent with previous studies [17]. However, the interactions between His6-NTD and dephosphorylated KD were stronger than those between His6-NTD and the phosphorylated domains (Fig. 2B). Analogous results were obtained with a D440N mutant of KD (the cat- alytic residue is mutated, thus abrogating phosphory- lation). This finding indicates that the interactions between NTD and KD are not attributable to the effects of k phosphatase but rather to phosphoryla- tion per se. In particular, heme-induced enhancement of protein–protein interactions involving dephospho- rylated KD was significantly higher than that observed with phosphorylated KD. These results sug- gest that multiple phosphorylations inhibit protein– protein interactions between isolated NTD and KD domains, leading to the suppression of intramolecu- lar interactions (between N-terminal and C-terminal domains) within the HRI protein. Moreover, the heme-sensing ability of HRI is markedly inhibited by multiple phosphorylations. Effects of dephosphorylation on heme sensitivity Next, we examined the catalytic activities of dephos- phorylated HRI, and compared the catalytic and heme-sensing properties of phosphorylated and dephosphorylated HRIs. The effects of heme on the kinase activities of dephosphorylated HRI proteins are shown in Fig. 3. Specific activity of the eIF2a kinase reaction of dephosphorylated HRI in the absence of heme was 8.0 nmol phosphorylated-eIF2a (P-eIF2a)Æmin )1 Æmg )1 HRI, which is significantly lower than that of phosphorylated HRI (18 nmol P-eIF2aÆmin )1 Æmg )1 HRI) [17], implying an important role for multiple phosphorylations in HRI catalysis. The half-inhibition constant (IC 50 s ± SD) of enzyme activity upon heme binding to dephosphorylated HRI 1.0 Phosphatase Heme * 0.8 0.6 0.4 0.2 0.0 KD recovery 1234 AB Fig. 2. Pulldown assay to detect interactions between His-tagged NTD (His 6 -NTD) and KD. (A) SDS ⁄ PAGE band patterns reveal His 6 - NTD and KD interactions. Lane M is the marker band. Only in the presence of heme (lanes 2 and 4) were interactions between His 6 - NTD and KD detected. In the absence of heme (lanes 1 and 3), interactions between His 6 -NTD and KD were not observed. (B) Densitometric analysis demonstrates that interactions between His 6 -NTD and KD are influenced by phosphorylation of KD because this binding was significantly (*; P < 0.05) stronger for dephospho- rylated HRI (lane 4) than phosphorylated HRI (lane 2). Autophosphorylation of an HRI J. Igarashi et al. 920 FEBS Journal 278 (2011) 918–928 ª 2011 The Authors Journal compilation ª 2011 FEBS was 2.4 ± 1.4 lm, which is similar to that of phos- phorylated HRI (2.1 lm) [17]. Because His119⁄ His120 and Cys409 are the heme-binding sites of mouse HRI, we measured the IC 50 values of heme for the dephosphorylated H119A ⁄ H120A and C409S mutant proteins (Fig. 3). The IC 50 value obtained for the dephosphorylated H119A ⁄ H120A mutant protein (0.39 ± 1.3 lm) was 10-fold lower than that of the phosphorylated enzyme (3.7 lm) [17]. By contrast, the IC 50 value of the dephosphorylated C409S mutant was 5.5 ± 1.3 lm, which is comparable to that (5.1 lm)of the phosphorylated mutant protein. Notably, the opti- cal absorption spectra of heme-bound dephosphoryl- ated HRI proteins were essentially similar to those of heme-bound phosphorylated HRI (Fig. S3). In HRI activation, the sensing of heme concentration is critical for function. We did not detect differences in heme sensitivity between proteins with mutations at the heme-binding sites (His119 ⁄ His120 and Cys409) and wild-type full-length enzyme [17]. However, when KD (an N-terminal deleted mutant protein) was used to examine heme sensitivity, marked differences in heme-binding ability were evident between wild-type and the C409S mutant protein. Specifically, the IC 50 values of wild-type and C409S KD proteins for heme were 0.25 lm and > 10 lm, respectively [17]. In the present study, we observed significant differences in heme sensitivity between dephosphorylated wild-type and mutant proteins (Fig. 3), and the IC 50 values for dephosphorylated wild-type, H119A ⁄ H120A and C409S HRI proteins were 2.4, 0.39 and 5.5 lm, respec- tively. The high heme sensitivity (IC 50 = 0.39 lm)of dephosphorylated H119A ⁄ H120A HRI is similar to that of wild-type KD (IC 50 = 0.25 lm), suggesting that the C-terminal domain itself has sensitivity in the sub- micromolar range. It appears that Cys409 is necessary for heme binding and heme sensitivity in sub-micromo- lar range. His119 or His120 would modulate the sensi- tivity in the low micromolar range because the IC 50 value of the dephosphorylated wild-type HRI was increased to 2.4 lm. On the other hand, the IC 50 value (5.5 lm) of the dephosphorylated C409S HRI was distinct from that (> 10 lm) of the C409S KD. These results suggest that His119 ⁄ His120 may partially compensate for Cys409 in the dephosphorylated C409S HRI. In addition, His119 or His120 alone is possibly sufficient, in the dephosphorylated form, for heme bind- ing and modulation of heme sensitivity at 5 lm, even in the absence of Cys409. Identification of the autophosphorylation sites of HRI overexpressed in E. coli To further clarify the details of autophosphorylation, we aimed to identify phosphorylation sites in HRI using MS. The HRI protein was initially digested with trypsin and phosphorylated peptides recovered with gallium immobilized metal ion affinity chromatography (Ga-IMAC). Phosphorylated peptides were subjected to LC linked to MS. The sequence coverage of MS analysis was 35.8% (222 residues ⁄ 619 amino acids). A mascot search (http://www.matrixscience.com) was conducted to identify peptides (Fig. S4). The phosphor- ylated sites of mouse HRI, together with the peptide sequences employed to identify these sites and the LC- MS ⁄ MS data, are summarized in Fig. 1 and Table 1. In total, 33 phosphorylated sites, including 23 Ser, seven Thr and three Tyr residues, were identified. The number of phosphorylated sites was almost consistent with the quantification of phosphates using BIOMOL Specific activity (nmol P-elF2α·min –1 ·mg –1 HRI) Heme (µM) [Heme] (µ M) A B Fig. 3. (A) Phosphorylation of eIF2a (black triangle) by wild-type, H119A ⁄ H120A and C409S dephosphorylated HRI proteins detected via Coomassie staining of a SDS-acrylamide gel containing Phos-tag acrylamide and manganese. The reaction was terminated at 4 min, and phosphorylated eIF2a proteins were compared with various heme concentrations. (B) The dose-response curve of eIF2a kinase activities of wild-type, H119A ⁄ H120A and C409S dephos- phorylated HRI versus log [heme]. The kinase activities of dephos- phorylated HRI were 50% of those of phosphorylated HRI. Data were analyzed using the equation: Y = bottom + (top ) bottom) ⁄ (1 + 10 X ) logIC 50 ). The IC 50 values of wild-type, H119A ⁄ H120A and C409S mutant proteins were 2.4, 0.39 and 5.5 l M, respectively. The IC 50 values of wild-type, H119A ⁄ H120A and C409S mutants of phosphorylated HRI were reported as 2.1, 3.7 and 5.1 l M, respectively [17]. J. Igarashi et al. Autophosphorylation of an HRI FEBS Journal 278 (2011) 918–928 ª 2011 The Authors Journal compilation ª 2011 FEBS 921 Green (Enzo Life Sciences International, Inc., Plym- outh Meeting, PA, USA) in k phosphatase-treated HRI protein (28 sites). In general, phosphorylated sites were located within three domains or regions; specifically, NTD, KD and KI. NTD contained only five sites at Ser5, Ser6, Ser34, Ser41 and Ser125. We identified 12 sites in KD at Ser144, Tyr145, Thr160, Ser161, Tyr163, Ser495, Thr532, Thr535, Ser545, Ser547, Ser609 and Ser612. KI had 16 phosphorylation sites; specifically, Ser252, Ser257, Ser273, Ser274, Ser275, Ser276, Thr283, Ser293, Ser304, Tyr305, Thr306, Ser317, Ser319, Ser320, Thr329 and Ser332. Interestingly, many of the phosphorylated sites are located in KI, whereas only five sites are phosphorylated in NTD. However, we failed to identify Thr483 and Thr485 as phosphory- lation sites, which is inconsistent with previous data obtained from 32 P-labeling experiments [14]. We sus- pect that the cysteine residues were not adequately pro- tected in our examination of peptides containing Thr483 and Thr485. Multiple phosphorylations of HRI are essential for generation of the active form. However, Thr485 is the Table 1. Phosphorylation sites of mouse HRI identified by fragmentation analysis using LC-MS ⁄ MS. Position Peptide sequence P-site m ⁄ zzM r (experimental) M r (calculated) Ion score 1–11 MLGG SSVDGER Ser5 634.2251 2 1266.4356 1266.4353 67 Ser6 12–37 DTDDDAAGAVAAPPAIDFPAEV SDPK Ser34 879.0528 3 2634.1366 2634.1374 52 38–51 YDE SDVPAELQVLK Ser41 843.3845 2 1684.7544 1684.7597 70 93–100 a LLCQTFIK Ser97 b 551.7808 2 1101.5470 1101.5294 16 123–129 AI THLMR Thr125 461.2209 2 920.4272 920.4303 31 137–148 a QDPCQDNSYMQK Ser144 845.2712 2 1688.5278 1688.5249 12 Tyr145 153–162 EIAFEAQ TSR Thr160 656.2490 2 1310.4834 1310.4945 25 Ser161 163–175 YLNEFEELAILGK Tyr163 809.8862 2 1617.7578 1617.7691 89 188–196 LDGQH YAIK Tyr193 b 562.7601 2 1123.5056 1123.5063 20 246–263 VPIQLP SLEVLSEQEGDR Ser252 1084.9967 2 2167.9788 2167.9803 83 Ser257 270–286 DNE SSSSIVFAELTPEK Ser273 1086.8777 2 2171.7408 2171.7391 42 Ser274 1086.8788 2 2171.7430 2171.7391 42 Ser275 Ser276 Thr283 287–296 EKPFGE SEVK Ser293 615.2781 2 1228.5416 1228.5377 36 297–312 NENNNLV SYTANLVVR Ser304 1030.4192 2 2058.8238 2058.8214 54 Tyr305 Thr306 313–338 NSSE SESSIELQEDGLTDLSVRPVVR Ser317 1082.7552 3 3245.2438 3245.2257 72 Ser319 Ser320 Thr329 Ser332 488–507 a VGTCLYASPEQLEGSQYDA Thr490 b 845.9799 3 2534.9179 2534.8756 24 Ser495 Ser502 b Tyr504 b 531–538 ATVLTGVR Thr532 448.7290 2 895.4434 895.4528 35 Thr535 448.7307 2 895.4468 895.4528 38 542–548 IPE SLSK Ser545 427.2018 2 852.3890 852.3994 39 542–548 IPESL SKR Ser547 505.2566 2 1008.4986 1008.5005 27 607–615 QL SLLSQDR Ser609 1139.5358 1 1138.5285 1138.5383 32 Ser612 570.2753 2 1138.5360 1138.5383 36 a Detected only carbamidomethylated sample using iodoacetamide. b These residues are unreliable when considering the low ion score of phosphorylated peptides and few fragment ions adjacent to phosphorylated residues; thus, we could not clearly determine whether these residues were phosphorylated. Autophosphorylation of an HRI J. Igarashi et al. 922 FEBS Journal 278 (2011) 918–928 ª 2011 The Authors Journal compilation ª 2011 FEBS only phosphorylated residue identified to date that might be critical for HRI activation [14]. In the present study, we identify 33 phosphorylated sites in HRI. The reaction with autophosphorylated HRI protein and k phosphatase showed that 28 nmol of phosphates were released from 1 nmol of HRI for 1 h. This indi- cated that most of the phosphorylated sites in HRI were identified in the present study, although the sequence covereage was not high (36%). Interestingly, KI contained 16 phosphorylation sites, whereas NTD and KD had five and 12 phosphorylation sites, respec- tively. However, mutation of phosphorylated residues in KI resulted in proteins with catalytic activities essen- tially similar to those of wild-type enzyme (described below). Thus, we propose that KI is phosphorylated principally to increase the solubility or stability of par- ticular peptide regions in the HRI protein, although such phosphorylation is not associated with catalytic function. Cys409 is one of the heme binding ⁄ sensing sites located near KI. Thus, multiple phosphorylations of KI after heme dissociation possibly prevent protein interactions with heme and promote the intrinsic eIF2a kinase activity. Effects of mutations at phosphorylated Ser, Thr and Tyr on catalysis To determine the detailed significance of HRI auto- phosphorylation with respect to catalysis, we generated 30 mutations at phosphorylated Ser, Thr and Tyr resi- dues and examined the catalytic activities of the mutant proteins (Fig. 4). Interestingly, the activities of Y193F, T485A and T490A mutant proteins were sig- nificantly lower than that of wild-type protein (Fig. 4). Although the K196R (lacking ATP binding) and T490A mutants showed very low activity, the effects of the Y193F and T485A mutations were less substantial. This appeared to reflect differences in HRI autophos- phorylation status, in that some (but not all) Y193F and T485A mutant protein could autophosphorylate in a manner similar to that of wild-type protein (Fig. S5). In an effort to mimic phosphorylated Thr490, the cata- lytic activity of the T490D mutant protein was assayed. The activity was almost the same as that of T490A, and thus significantly lower than that of wild- type protein. This indicates that phosphorylation of Thr490 is an early key event in the autophosphoryla- tion of HRI. Tyr193 lies within catalytic subdomain II and is highly conserved among all eIF2a kinases (Tyr293 in PKR and Tyr-615 in PERK) (Fig. 5A) [18–20]. Given the function of highly conserved Tyr residues in cataly- sis, it is reasonable to assume that Tyr193 plays a criti- cal role in HRI activity. On the basis of a structural model, we speculate that Tyr193 is located at the dimer interface and is thus trans-phosphorylated by the kinase domains of other subunits of HRI (Fig. 5B). Unfortunately, because of the low ion score (i.e. a measure of how well the observed MS ⁄ MS spectrum matches that of the relevant peptide) of the phosphory- lated peptide, as well as the availability of only few fragment ions adjacent to Tyr193 (Fig. S4), Tyr193 phosphorylation status in mouse HRI remains unclear. Thr485 and Thr490 are critical for HRI activation. On the basis of amino acid alignments (Fig. 5A), we propose that Thr485 and Thr490 are located within the activation loop between subdomains VII and VIII. Thr485 is well conserved in the eIF2a kinases and cor- responds to Thr446 of PKR, Thr981 of PERK and Thr899 of GCN2 [18–20]. By contrast to earlier studies Specific activity (nmol P-elF2α·min –1 ·mg –1 HRI) Fig. 4. In vitro kinase activities of wild-type and mutant proteins. SDS ⁄ PAGE of Phos-tag acrylamide and manganese used for the evaluation of phosphorylated eIF2a protein (Fig. S6A, B). The K196R mutation is within the ATP binding site (Fig. 5B), and thus the mutant protein dis- plays very low activity. K196R activity was used as a negative control. Experiments were repeated at least three times, and are shown as the mean ± SD. Note that the S502A mutant protein showed very low activity (Fig. S6C). However, the protein band on the SDS ⁄ PAGE was largely downshifted as a result of proteolysis (Fig. S6D). By contrast, the S502D mutant was expressed normally and displayed activity similar to the wild-type protein. J. Igarashi et al. Autophosphorylation of an HRI FEBS Journal 278 (2011) 918–928 ª 2011 The Authors Journal compilation ª 2011 FEBS 923 showing that Thr483 and Thr485 are phosphorylated [14], we did not detect phosphorylation at both sites. However, the T485A mutant protein showed signifi- cantly lower activity than the wild-type counterpart (Fig. 4), which is consistent with the previous proposal that Thr485 of HRI is critical for catalysis [14]. Thr490 of mouse HRI is also well conserved and cor- responds to Thr451 of PKR, Thr986 of PERK and Thr904 of GCN2 (Fig. 5A) [18–20]. The results obtained in the present study suggest that Thr490 is phosphorylated and essential in catalysis (Table 1 and Fig. 4). Regulatory mechanism of HRI Figure 6 shows a schematic representation of the regu- latory mechanism of HRI. On the basis of results obtained in a previous study, we propose that global protein rearrangements, including intramolecular protein–protein interactions, are required for heme sensing because the axial ligands of the heme, His119 ⁄ His120 in the N-terminal domain and Cys409 in the C-terminal domain, are heme-sensing sites [17]. This heme-sensing function is operative under normal con- ditions when heme concentrations are physiologically sufficient. However, under conditions of heme short- age, heme dissociates from the HRI protein, followed by protein autophosphorylation. It is currently unclear why autophosphorylation is required, although an increase in protein solubility and structural alterations induced by autophosphorylation may be important in catalysis. Data from the present study confirm a previ- ous suggestion that Thr485 is involved in catalysis. We show, for the first time, that Tyr193, Thr485 and Thr490 are key residues for multiple phosphorylations, and that dephosphorylation enhances intramolecular interactions between the N- and C-terminal domains of HRI critical for heme sensing, and between heme and protein. Therefore, multiple autophosphorylation steps are possibly important for preventing heme rebinding, inhibiting interactions between heme and protein after dissociation, and shifting the equilibrium of the biochemical reaction irreversibly towards phos- phorylation of eIF2a. Before intrinsic eIF2a kinase activity is acquired, heme must initially dissociate from the HRI binding site composed of His119 ⁄ His120 and Cys409 because auto- phosphorylation and eIF2a kinase activities are com- pletely inhibited by 10 lm heme (Fig. S2). Once heme is released from HRI, the intramolecular interactions A B Fig. 5. (A) Amino acid alignment of the kinase domains of human PKR, PERK, GCN2 and mouse HRI. Phosphorylated sites in the subdomain II and activation loop between subdomains VII and VIII are shown in bold. Phosphorylation sites determined in the present study are shown in red, and the residues important for catalysis identified in the present study are underlined. (B) Homology model of the kinase domain of HRI based on human PKR data (Protein data bank code: 2A19). HRI sequence missing in PKR, key autophosphorylated residues (Tyr193, Thr485 and Thr490), as well as Lys196 mediating ATP binding, are shown in magenta, cyan and violet, respectively. Autophosphorylation of an HRI J. Igarashi et al. 924 FEBS Journal 278 (2011) 918–928 ª 2011 The Authors Journal compilation ª 2011 FEBS between NTD and KD become weak (Fig. 2, lanes 1 and 3), after which autophosphorylation takes place as the second step. Intramolecular interactions are further decreased and the enzyme becomes active. Activated autophosphorylated HRI phosphorylates eIF2a at Ser51 in the fourth step (Fig. 6 and S2). Large-scale proteomics studies in HeLa cells indicate that human HRI has eight potential phosphorylated sites [21,22]. These residues, corresponding to those in the mouse sequence, are phosphorylated. A number of these residues contain the phosphorylation motif specific for casein kinase 1 (S-X-X-S ⁄ T), casein kinase 2 (S ⁄ T-X-X-E) and glycogen synthase kinase 3 (S-X-X-X-S). Phosphorylation of HRI by casein kinase 2 has been described previously [23]. These findings suggest that other kinases, including casein kinase 1, casein kinase 2 and glycogen synthase kinase 3, phosphorylate HRI, which functions upstream of these signaling cascades. Conclusions We demonstrate that autophosphorylation of mouse HRI occurring after heme dissociation is important for weakening intramolecular interactions between the N-terminal and C-terminal domains critical for heme sensing, thus confirming the importance of the heme binding site, His-119, or His-120 and Cys-409, for heme sensing. We identified 33 autophosphorylated sites using MS, and identified Tyr193, Thr485 and Thr490 as important residues in catalysis based on site-directed mutagenesis of the phosphorylation sites. Experimental procedures Materials Phos-tag acrylamide was purchased from Phos-tag Consor- tium Co. (Osaka, Japan). Oligonucleotides were obtained from Nihon Gene Research Laboratories (Sendai, Japan). k Protein phosphatase was purchased from New England Biolabs Japan (Tokyo, Japan). Other reagents were pur- chased from Wako Pure Chemical Industries (Osaka, Japan). Reagents were of the highest commercial grade available and used without further purification. Site-directed mutagenesis, protein expression and purification Mutagenesis was conducted using the QuikChange site- directed mutagenesis kit obtained from Stratagene (La Jolla, CA, USA). The oligonucleotide sequences are sum- marized in Table S1. Mutations were confirmed by DNA sequencing. Mouse HRI was overexpressed in the E. coli strain BL21(DE3) Codon Plus RIL (Stratagene). Protein expression was induced with 50 lm isopropyl-b-d-galactoside, as described previously [11,17,24]. The protein purification pro- cedures for full-length and N-terminal truncated KD mutant (amino acids 1–145 of full-length HRI were deleted) HRI and NTD proteins were similar to those described previously [11,17,24]. To generate dephosphorylated protein, HRI was treated with k protein phosphatase for 3 h at 4 ° C after cleavage of the His-tag. The term ‘phosphorylated protein (full-length or KD)’ refers to an enzyme prepared without the use of k protein phosphatase, whereas ‘dephosphorylated Fig. 6. Hypothetical regulatory mechanism of HRI. Heme association ⁄ dissociation at the heme-sensing site of HRI regulates the eIF2a kinase reaction. (1) Heme association with full-length HRI blocks catalysis, whereas heme dissociation exposes the active site and permits catalysis. (2) Next, HRI is autophosphorylated at key residues, including Tyr193, Thr485 and Thr490. (3) Thereafter, HRI autophosphorylates multiple residues. (4) Finally, HRI becomes an active eIF2a kinase, phosphorylating the substrate at Ser51. J. Igarashi et al. Autophosphorylation of an HRI FEBS Journal 278 (2011) 918–928 ª 2011 The Authors Journal compilation ª 2011 FEBS 925 protein (full-length or KD)’ refers to an enzyme treated with k protein phosphatase. His6-tagged eIF2a expression and purification is described elsewhere [11]. Enzyme assay and pulldown assay In vitro kinase assays were performed in according with pre- vious studies [17]. Briefly, the reaction mixture, consisting of 20 mm Tris ⁄ HCl (pH 7.7), 60 mm KCl, 2 mm magnesium acetate, 0.35 lm HRI and 10 lg of eIF2a, was incubated at 15 °C for 5 min, and the reaction initiated by adding 50 lm ATP at 15 °C. At the indicated times, the reaction was termi- nated by adding sample buffer, and heat-denatured at 95 °C for 5 min. Samples were loaded on a 7.5% SDS gel contain- ing 50 lm Phos-tag acrylamide and 100 lm manganese chlo- ride. The specific activities were calculated from a time course experiment. P-eIF2a interacted with the Phos-tag manganese complex so that mobility was slower than that of eIF2a. A pulldown assay using His6-tagged NTD and KD on Ni nitrilotriacetic acid-agarose column (Qiagen KK, Tokyo, Japan) has been described previously [17]. Briefly, His6-NTD (amino acids 1–138) and phoshorylated KD or k protein phosphatase-treated KD (amino acids 146–619) pro- teins were mixed with each other (100 pmol each) in the pres- ence or absence of hemin (100 pmol). The mixture was loaded on to the Ni-Sepharose 6 column (GE Healthcare, Little Chalfont, UK). Next, the column was washed with 20 mm Tris ⁄ HCl (pH 8.0), 150 mm NaCl and 100 mm imid- azole, and His6-tagged protein eluted with 20 mm Tris ⁄ HCl (pH 8.0), 150 mm NaCl and 200 m m imidazole. The input sample, washed flow-through and elution fractions were ana- lyzed by SDS ⁄ PAGE. Proteins were visualized with Phastgel Coomassie Brilliant Blue R350 (GE Healthcare) staining. Gel images were acquired using LAS-3000 (Fujifilm, Tokyo, Japan) and quantified with multi-gauge software (Fujifilm). To calculate specific activity and KD recovery values, the ratios of P-eIF2a over eIF2a, and KD over His6-NTD were obtained from densitometric analysis, respectively. Nonlin- ear fitting to obtain the IC 50 value was conducted using prism 5 (GraphPad Software, La Jolla, CA, USA). Trypsin digestion and phosphopeptide purification Full-length HRI protein was degraded using immobilized l-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin obtained from Pierce (Thermo Scientific, Rockford, IL, USA) in accordance with the manufacturer’s instruc- tions. Purified proteins (100 lg) were dissolved in 50 lLof 0.1 m NH 4 HCO 3 (pH 8.0). Immobilized l-1-tosylamido-2- phenylethyl chloromethyl ketone-treated trypsin (20 lL) was added to protein samples. The reaction mixture was incubated at 37 °C for 20 h. Trypsin beads were separated from the digestion mixture by centrifugation. Cys residues were protected using iodoacetamide before trypsin digestion. Phosphopeptide purification was conducted using a specific phosphopeptide isolation kit from Pierce (Thermo Scientific) with Ga-IMAC, in accordance with the manufac- turer’s instructions. LC We performed phosphopeptide separation and concentration with Nano-LC using a capillary column (diameter 75 mm, length 100 mm; column packing 3 lm) (Nikkyo Technos Co., Tokyo, Japan) with a flow rate of 200 nLÆmin )1 . For separation, solvent A (2% acetonitrile in 0.1% formic acid) plus 5% solvent B (98% acetonitrile in 0.1% formic acid) (v ⁄ v), followed by a linear gradient up to 40% solvent B for 60 min, was applied. Collision-induced dissociation (CID) and electron-capture dissociation (ECD) LIT q-TOF MS and MS ⁄ MS analyses MS was performed at Hitachi High Technologies Corpora- tion (Tokyo, Japan). Analytical conditions for the CID measurements were: MS mode, trap mode; ionization, ESI (positive ionization); nebulizer gas flow, 0.8 LÆmin )1 ; spray potential, 1500 V; detector potential, 2050 V; scan range, m ⁄ z 50–2000. Analytical conditions for the ECD measure- ments were: MS mode, trap mode; ionization, ESI (positive ionization); nebulizer gas flow, 0.6 LÆmin )1 ; spray potential, 1500 V; detector potential, 2150 V; scan range, m ⁄ z 50– 2000. MS ⁄ MS data were analyzed using mascot software. Details of the analyses are summarized in Fig. S4. Acknowledgements This work was supported in part by Grants-in-Aid for Scientific Research to J.I. (2177130 and 21117501) and T.S. 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Autophosphorylation of dephosphorylated HRI monitored using western blotting with anti-HRI serum and phosphorylation of eIF2a by HRI detected via Coomassie staining of a SDS-acrylamide gel con- taining Phos-tag acrylamide and manganese. J. Igarashi et al. Autophosphorylation of an HRI FEBS Journal 278 (2011) 918–928 ª 2011 The Authors Journal compilation ª 2011 FEBS 927 [...]... activities towards eIF2a of the mutant proteins Table S1 Oligonucleotides used for the site-directed mutagenesis experiments 928 This supplementary material can be found in the online version of this article Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors Such materials are peer-reviewed and may be re-organized for online delivery,.. .Autophosphorylation of an HRI J Igarashi et al Fig S3 Optical absorption spectra of heme-bound phosphorylated and dephosphorylated HRI proteins Fig S4 mascot analyses for LS-MS ⁄ MS data for autophosphorylated peptide fragments Fig S5 Phosphorylation status of HRI detected via Coomassie staining of an SDS-acrylamide gel containing Phos-tag acrylamide and manganese Fig S6 SDS ⁄ PAGE containing Phos-tag... materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be addressed to the authors FEBS Journal 278 (2011) 918–928 ª 2011 The Authors Journal compilation ª 2011 FEBS . heme-regulated eIF2a kinase or heme-regulated inhibitor; KD, C-terminal kinase domain containing amino acids 145-619; KI, kinase insert with amino acids 244-371; NTD, N-terminal domain containing. to explore the role of autophosphorylation of the HRI protein in the intramolecular protein–protein interac- tion (between the NTD and KD) and catalysis upon heme binding. Identification of autophosphorylated sites. synthase kinase 3 (S-X-X-X-S). Phosphorylation of HRI by casein kinase 2 has been described previously [23]. These findings suggest that other kinases, including casein kinase 1, casein kinase 2 and