Báo cáo khoa học: Autophosphorylation of heme-regulated eukaryotic initiation factor 2a kinase and the role of the modification in catalysis doc

Four eukaryotic initiation factor 2a eIF2a3 kinases, heme-regulated eIF2a kinase HRI, PKR, PERK and GCN2, inhibit translation upon recognition of stress or emergency states, such as heme

initiation factor 2a kinase and the role of the

modification in catalysis

Jotaro Igarashi1,*, Takehiko Sasaki1,*, Noriko Kobayashi2, Shinji Yoshioka2, Miyuki Matsushita2 and Toru Shimizu1

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.

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 addiautophosphoryla-tion 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 32P-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 autophosphorylaclarifica-tion 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.

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 dephosphoryphosphory-lated 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)1Æmg)1 HRI, which is significantly lower than that of phosphorylated HRI (18 nmol P-eIF2aÆmin)1Æmg)1 HRI) [17], implying an important role for multiple phosphorylations in HRI catalysis The half-inhibition constant (IC50s ± SD) of enzyme activity upon heme binding to dephosphorylated HRI

1.0

Phosphatase

Heme

*

0.8 0.6 0.4 0.2 0.0

Fig 2 Pulldown assay to detect interactions between His-tagged NTD (His6-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 His6 -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 His6-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 phosphodephospho-rylated HRI (lane 2).

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 IC50 values of heme for the

dephosphorylated H119A⁄ H120A and C409S mutant

proteins (Fig 3) The IC50 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

IC50value 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 IC50 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 IC50 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 (IC50= 0.39 lm) of dephosphorylated H119A⁄ H120A HRI is similar to that of wild-type KD (IC50= 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 IC50 value of the dephosphorylated wild-type HRI was increased to 2.4 lm On the other hand, the IC50value (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

–1 HRI)

[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

phorylated HRI versus log [heme] The kinase activities of

dephos-phorylated HRI were 50% of those of phosdephos-phorylated HRI Data

were analyzed using the equation: Y = bottom + (top ) bottom) ⁄

(1 + 10 X ) logIC 50 ) The IC50values 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].

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 32P-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.

Ion score

Ser6

Tyr145

Ser161

Ser257

Ser275 Ser276 Thr283

Tyr305 Thr306

Ser319 Ser320 Thr329 Ser332

Ser495 Ser502 b Tyr504 b

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.

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

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.

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

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.

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 4C 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.

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 vitrokinase 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

15C for 5 min, and the reaction initiated by adding 50 lm

ATP at 15C At the indicated times, the reaction was

termi-nated by adding sample buffer, and heat-denatured at 95C

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 abspres-ence 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 mm 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 IC50 value was conducted using

prism5 (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 lL of

0.1 m NH4HCO3 (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 37C 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 condiCorpora-tions 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 (17101002), and Special Education and Research Expenses from the Ministry of Education, Culture, Sports, Science, and Technology of Japan

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Supporting information

The following supplementary material is available: Fig S1 SDS⁄ PAGE to demonstrate homogeneity of the purified protein and dephosphorylation with k phosphatase

Fig S2 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