The role of heme-binding, phosphorylation, and degradation in heme-regulated eukaryotic initiation factor 2α kinase (HRI)

• Project/Area Number: 21770130
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Single-year Grants
• Research Field: Functional biochemistry
• Research Institution: Tohoku University
• Principal Investigator: IGARASHI Jotaro Tohoku University, 多元物質科学研究所, 助教 (80375162)
• Project Period (FY): 2009 – 2010
• Project Status: Completed (Fiscal Year 2010)
• Budget Amount: ¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000); Fiscal Year 2010: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2009: ¥3,120,000 (Direct Cost: ¥2,400,000、Indirect Cost: ¥720,000)
• Keywords: ヘム / 自己リン酸化 / プロテアソーム / 結晶化 / ヘム調節インヒビター / 翻訳開始因子キナーゼ / 脱リン酸化 / ヘム制御モチーフ / 質量分析

Research Abstract

Heme-regulated eukaryotic initiation factor 2α (eIF2α) kinase (HRI), functions in response to heme concentration. Under normal conditions, heme binds to HRI and blocks its function. However, during heme shortage, heme dissociates from the protein, and autophosphorylation subsequently occurs. The molecular mechanism of heme-binding, autophosphorylation, and degradation in HRI remained to be established. In the present study, 1) we demonstrate that His119/His120 and Cys411 are the axial ligands for heme in human HRI, as well as mouse HRI. 2) With the aid of mass spectroscopy we identified 33 phosphorylated sites in mouse HRI overexpressed in Escherichia coli. We further generated 30 enzymes with mutations at the phosphorylated residues and examined the catalytic activities. The activities of Y193F, T485A, and T490A mutants were significantly lower than that of wild-type protein. Accordingly, we suggest that Tyr193, Thr485, and Thr490 are essential residues in the catalysis. 3) Due to degradation of HRI in cultured cell, we show that HRI degradation is induced by 20S proteasome in vitro.

Research Products

• [Journal Article] A hydrogen-bonding network formed by the B10-E7-E11 residues of a truncated hemoglobin from Tetrahymena pyriformis is critical for stability of bound oxygen and nitric oxide detoxification. (2011)
  • Author(s): Igarashi, J., Kobayashi, K., Matsuoka, A.
  • Journal Title: J.Biol.Inorg.Chem. 16 Pages: 599-609
  • Peer Reviewed
• [Journal Article] Autophosphorylation of heme-regulated eukaryotic initiation factor 2α kinase, HRI, and the role of the modification in catalysis. (2011)
  • Author(s): Igarashi, J., Sasaki, T., Kobayashi, N., Yoshioka, S., Matsushita, M., Shimizu, T.
  • Journal Title: FEBS J. 278 Pages: 918-928
  • Peer Reviewed
• [Journal Article] Heme binding characteristics of the isolated PAS-B domain of mouse Per2, a transcriptional regulatory factor associated with circadian rhythms. (2011)
  • Author(s): Hayasaka, K., Kitanishi, K., Igarashi, J., Shimizu, T.
  • Journal Title: Biochim.Biophys.Acta 1814 Pages: 326-333
  • Peer Reviewed
• [Journal Article] A hydrogen-bonding network formed by the B10-E7-E11 residues of a truncated hemoglobin from Tetrahymena pyriformis is critical for stability of bound oxygen and nitric oxide detoxification (2011)
  • Author(s): Jotaro Igarashi
  • Journal Title: Journal of Biological Inorganic Chemistry Volume: 16 Pages: 599-609
  • Peer Reviewed
• [Journal Article] Autophosphorylation of heme-regulated eukaryotic initiation factor 2α kinase, HRI, and the role of the modification in catalysis (2011)
  • Author(s): Jotaro Igarashi
  • Journal Title: FEBS Journal Volume: 278 Pages: 918-928
  • Peer Reviewed
• [Journal Article] Catalysis and oxygen binding of Ec DOS, a heme-based oxygen-sensor enzyme from Escherichia coli. (2010)
  • Author(s): Kobayashi, K., Tanaka, A., Takahashi, H., Igarashi, J., Ishitsuka, Y., Yokota, N., Shimizu, T.
  • Journal Title: J.Biochem. 148 Pages: 693-703
  • Peer Reviewed
• [Journal Article] Important roles of Tyr43 at the putative heme distal side in oxygen recognition and stability of the Fe(II)-O 2 complex of YddV, a globin-coupled heme-based oxygen sensor diguanylate cyclase. (2010)
  • Author(s): Kitanishi, K., Kobayashi, K., Kawamura, Y., Ishigami, I., Ogura, T., Nakajima, K., Igarashi, J., Tanaka, A., Shimizu, T.
  • Journal Title: Biochemistry 49 Pages: 10381-10393
  • Peer Reviewed
• [Journal Article] Thiolate with α-lipoic acid remedies depressive behavior of mice induced by methyl-mercury. (2010)
  • Author(s): Kitanishi, K., Igarashi, J., Shimizu, T.
  • Journal Title: Curr.Top.Toxicol. 6 Pages: 23-29
• [Journal Article] Selective neuronal nitric oxide synthase inhibitors and the prevention of cerebral palsy. (2009)
  • Author(s): Ji, H., Tan, S., Igarashi, J., Li, H., Derrick, M., Martasek, P., Roman, L., Vasquez-Vivar, J., Poulos, T.L., Silverman, R.B.
  • Journal Title: Ann.Neurol. 65 Pages: 209-217
  • Peer Reviewed
• [Journal Article] The FG loop of a heme-based gas sensor enzyme, Ec DOS, functions in heme binding, autoxidation and catalysis. (2009)
  • Author(s): Ito, S., Igarashi, J., Shimizu, T.
  • Journal Title: J.Inorg.Biochem. 103 Pages: 1380-1385
  • Peer Reviewed
• [Journal Article] Role of Phe113 at the distal side of the heme domain of an oxygen-sensor (Ec DOS) in the characterization of the heme environment. (2009)
  • Author(s): Ito, S., Araki, Y., Tanaka, A., Igarashi, J., Wada, T., Shimizu, T.
  • Journal Title: J.Inorg.Biochem. 103 Pages: 989-996
  • Peer Reviewed
• [Journal Article] Crystal structures of constitutive nitric oxide synthases in complex with de novo designed inhibitors. (2009)
  • Author(s): Igarashi, J., Li, H., Jamal, J., Ji, H., Fang, J., Lawton, G., Silverman, R.B., Poulos, T.L.
  • Journal Title: J.Med.Chem. 52 Pages: 2060-2066
  • Peer Reviewed
• [Presentation] Molecular mechanism of NO-induced catalytic activation of mouse heme-regulated eukaryotic initiation factor 2α kinase, HRI. (2010)
  • Author(s): Igarashi, J., Sasaki, T., Iwashita, J.,Shimizu, T.
  • Organizer: Pacifichem 2010 Congress.Honolulu
  • Place of Presentation: HI, USA.
  • Year and Date: 2010-12-16
• [Presentation] ヘム調節インヒビター(HRI)のヘム・リン酸化による活性調節機構 (2010)
  • Author(s): 五十嵐城太郎, 佐々木健彦, 清水透
  • Organizer: 第33回日本分子生物学会年会・第82回日本生化学会大会合同大会(BMB2010)
  • Place of Presentation: 神戸
  • Year and Date: 2010-12-10
• [Presentation] ヘム調節インヒビター(HRI)のヘム・リン酸化による活性調節機構 (2010)
  • Author(s): 五十嵐城太郎
  • Organizer: 第33回日本分子生物学会年会・第82回日本生化学会年会合同大会(BMB2010)
  • Place of Presentation: 神戸
  • Year and Date: 2010-12-10
• [Presentation] NO-induced activation of a heme-sensor, eIF2α kinase, in association with binding to cysteine and heme. (2010)
  • Author(s): Igarashi, J., Sasaki, T., Iwashita, J.,Shimizu, T.
  • Organizer: 6th International Conference, Biology, Chemistry, and Therapeutic Applications of Nitric Oxide.
  • Place of Presentation: Kyoto,Japan.
  • Year and Date: 2010-06-15
• [Presentation] ヘム調節インヒビター(HRI)のヘム・リン酸化による反応制御機構 (2010)
  • Author(s): 五十嵐城太郎, 佐々木健彦, 清水透
  • Organizer: 日本生化学会東北支部第76回例会・シンポジウムム
  • Place of Presentation: 福島
  • Year and Date: 2010-05-08
• [Presentation] ヘム調節インヒビター(HRI)のヘム・リン酸化による反応制御機構 (2010)
  • Author(s): 五十嵐城太郎
  • Organizer: 日本生化学会東北支部第76回例会・シンポジウム
  • Place of Presentation: 福島
  • Year and Date: 2010-05-08
• [Presentation] Heme-regulated eukaryotic initiation factor 2α kinase associated with protein translation. (2010)
  • Author(s): Igarashi, J.,Shimizu, T.
  • Organizer: Gordon Research Conference on Protein Cofactors, Radicals and Quinones.
  • Place of Presentation: Ventura, CA, USA.
• [Presentation] Heme-regulated eukaryotic initiation factor 2α kinase associated with protein translation (2010)
  • Author(s): Jotaro Igarashi
  • Organizer: Gordon Research Conference on Protein Cofactors, Radicals and Quinones
  • Place of Presentation: Ventura, CA, USA
• [Presentation] ヘム調節インヒビター(HRI)の活性調節機構:ヘム結合及びリン酸化について (2009)
  • Author(s): 五十嵐城太郎, 佐々木健彦, 岩下隼, 清水透
  • Organizer: 第82回日本生化学会大会会
  • Place of Presentation: 神戸
  • Year and Date: 2009-10-22
• [Presentation] Molecular mechanism of HRI : Heme binding and autophosphorylation (2009)
  • Author(s): 五十嵐城太郎
  • Organizer: 第82回日本生化学会大会
  • Place of Presentation: 神戸
  • Year and Date: 2009-10-22
• [Presentation] Oxygen stability and crystal structure of Tetrahymena truncated hemoglobin. (2009)
  • Author(s): Igarashi, J., Matsuoka, A.
  • Organizer: 14th International Conference on Biological Inorganic Chemistry (ICBIC14).
  • Place of Presentation: Nagoya, Japan.
  • Year and Date: 2009-07-29
• [Presentation] Oxygen stability and crystal structure of Tetrahymena truncated hemoglobin (2009)
  • Author(s): 五十嵐城太郎
  • Organizer: 14th International Conference of Biological Inorganic Chemistry
  • Place of Presentation: 名古屋
  • Year and Date: 2009-07-29
• [Presentation] The heme-binding site of the heme-regulated inhibitor (HRI), and the role of heme regulatory motif in heme sensing. (2009)
  • Author(s): Igarashi, J.,Murase, M., Shimizu, T.
  • Organizer: 16th International Conference on Cytochrome P450, Biochemisty, Biphysics, Functional Genomics.
  • Place of Presentation: Nago, Japan.
  • Year and Date: 2009-06-23
• [Presentation] The heme-binding site of the heme-regulated inhibitor(HRI), and the role of the heme regulatory motif in heme sensing (2009)
  • Author(s): 五十嵐城太郎
  • Organizer: 16th International Conference on Cytochrome P450
  • Place of Presentation: 名護
  • Year and Date: 2009-06-23
• [Presentation] ヘム調節インヒビター(HRI)の-酸化窒素による活性調節機構:S-ニトロシル化の検討 (2009)
  • Author(s): 五十嵐城太郎, 岩下隼, 佐々木健彦, 清水透
  • Organizer: 第9回日本NO学会学術集会会
  • Place of Presentation: 静岡
  • Year and Date: 2009-05-08
• [Book] Emerging Role of Heme as a Signal and the Gas-Sensing Site : Heme-Sensing and Gas-Sensing Proteins.Handbook of Porphyrin Science (Kadish, K.M., Smith, K.M., Guilard, R.Eds.) (2011)
  • Author(s): Igarashi, J., Kitanishi, K., Shimizu, T.
  • Publisher: World Scientific (Hackensack, NJ, USA)
• [Book] Handbook of Porphyrin Science Vol 15 (2011)
  • Author(s): Jotaro Igarashi(分担執筆)
  • Publisher: World Scientific
• [Book] 酵素利用技術体系(小宮山眞編)(シトクロムP450.) (2010)
  • Author(s): 五十嵐城太郎, 清水透
  • Publisher: エヌ・ティー・エス(東京)
• [Book] 酵素利用技術体系 (2010)
  • Author(s): 五十嵐城太郎(分担執筆)
  • Publisher: エヌ・ティー・エス
• [Book] の分子生物学 第2版(大村恒雄, 石村巽, 藤井義明編)(のヘムーチオレート蛋白質の構造と機能.P450) (2009)
  • Author(s): 五十嵐城太郎, 清水透, 他
  • Publisher: 講談社(東京)
• [Book] P450の分子生物学(大村恒雄、石村巽、藤井義明編) (2009)
  • Author(s): 五十嵐城太郎
  • Total Pages: 303
  • Publisher: 講談社
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