Identification and functional analysis of substrates of disease-associated protein kinases using multiple phosphoproteomic technologies

• Project/Area Number: 26440101
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Cell biology
• Research Institution: The University of Tokushima
• Principal Investigator: KOSAKO Hidetaka 徳島大学, 先端酵素学研究所(オープンイノベ), 教授 (10291171)
• Co-Investigator(Renkei-kenkyūsha): MATSUDA Noriyuki 東京都医学総合研究所, 生体分子先端研究分野, プロジェクトリーダー (10332272) ; NAKAYA Michio 九州大学, 薬学研究院, 准教授 (80464387) ; ISHIZAKI Toshimasa 大分大学, 医学部, 教授 (70293876)
• Project Period (FY): 2014-04-01 – 2017-03-31
• Project Status: Completed (Fiscal Year 2016)
• Budget Amount: ¥5,070,000 (Direct Cost: ¥3,900,000、Indirect Cost: ¥1,170,000); Fiscal Year 2016: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2015: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000); Fiscal Year 2014: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
• Keywords: シグナル伝達 / プロテオーム / リン酸化 / キナーゼ / PINK1 / ERK / PKD / ユビキチン / T細胞 / 核膜孔複合体 / ROCK

Outline of Final Research Achievements

Many diseases are associated with mutations in protein kinases. To fully and therapeutically understand the complex signaling networks, it is essential to develop analytical strategies for the global identification and functional characterization of in vivo substrates of individual protein kinases. In this study, we have identified novel substrates of three disease-associated protein kinases including PINK1, ERK and PKD by using the IMAC/2D-DIGE method, Phos-tag Western blotting and mass spectrometry. Furthermore, we uncovered molecular mechanisms by which each kinase regulates cellular functions through these substrates.

Research Products

• [Journal Article] Global Identification of ERK Substrates by Phosphoproteomics Based on IMAC and 2D-DIGE. (2017)
  • Author(s): H. Kosako, K. Motani.
  • Journal Title: Methods Mol Biol. Volume: 1487 Pages: 137-149
  • DOI: 10.1007/978-1-4939-6424-6_10
  • ISBN: 9781493964222, 9781493964246
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Protein kinase D regulates positive selection of CD4+ thymocytes through phosphorylation of SHP-1 (2016)
  • Author(s): Ishikawa E, Kosako H, Yasuda T, Ohmuraya M, Araki K, Kurosaki T, Saito T, Yamasaki S
  • Journal Title: Nat Commun Volume: 7 Issue: 1 Pages: 12756-12756
  • DOI: 10.1038/ncomms12756
  • NAID: 120006535062
  • Peer Reviewed / Open Access
• [Journal Article] Analog-to-digital Conversion in the Cellular Signaling System (2016)
  • Author(s): 新土優樹，小迫英尊，佐甲靖志，高橋恒一
  • Journal Title: Seibutsu Butsuri Volume: 56 Issue: 6 Pages: 334-336
  • DOI: 10.2142/biophys.56.334
  • NAID: 130005170660
  • ISSN: 0582-4052, 1347-4219
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Conversion of graded phosphorylation into switch-like nuclear translocation via autoregulatory mechanisms in ERK signalling. (2016)
  • Author(s): Y. Shindo, K. Iwamoto, K. Mouri, K. Hibino, M. Tomita, H. Kosako, Y. Sako and K. Takahashi.
  • Journal Title: Nature Communications Volume: 7 Issue: 1 Pages: 10485-10485
  • DOI: 10.1038/ncomms10485
  • NAID: 120006535049
  • Peer Reviewed / Open Access
• [Journal Article] PKA regulates PINK1 stability and Parkin recruitment to damaged mitochondria through phosphorylation of MIC60. (2016)
  • Author(s): S. Akabane, M. Uno, N. Tani, S. Shimazaki, N. Ebara, H. Kato, H. Kosako, and T. Oka.
  • Journal Title: Molecular Cell Volume: 62
  • Peer Reviewed
• [Journal Article] Phosphorylated ubiquitin chain is the genuine Parkin receptor. (2015)
  • Author(s): K. Okatsu, F. Koyano, M. Kimura, H. Kosako, Y. Saeki, K. Tanaka and N. Matsuda
  • Journal Title: The Journal of Cell Biology Volume: 209 Issue: 1 Pages: 111-128
  • DOI: 10.1083/jcb.201410050
  • Peer Reviewed / Open Access
• [Journal Article] Ubiquitin is phosphorylated by PINK1 to activate parkin. (2014)
  • Author(s): Koyano, F., Okatsu, K., Kosako, H., Tamura, Y., Go, E., Kimura, M., Kimura, Y., Tsuchiya, H., Yoshihara, H., Hirokawa, T., Endo, T., Fon. E. A., Trempe, J. F., Saeki, Y., Tanaka, K., and Matsuda, N.
  • Journal Title: Nature Volume: 510(in press) Issue: 7503 Pages: 162-166
  • DOI: 10.1038/nature13392
  • Peer Reviewed
• [Journal Article] Epithelial protein lost in neoplasm modulates platelet-derived growth factor-mediated adhesion and motility of mesangial cells (2014)
  • Author(s): Haruko Tsurumi, et. al
  • Journal Title: Kidney International Volume: Epub Issue: 3 Pages: 548-557
  • DOI: 10.1038/ki.2014.85
  • Peer Reviewed / Open Access
• [Presentation] Dissection of protein kinase D signaling during thymocyte development using various phosphoproteomic strategies. (2016)
  • Author(s): H. Kosako, E. Ishikawa, S. Yamasaki
  • Organizer: HUPO 2016
  • Place of Presentation: Taipei, Taiwan
  • Year and Date: 2016-09-19
  • Int'l Joint Research
• [Presentation] Phos-tagなどのリン酸化プロテオミクス技術の結集によるキナーゼ基質の同定と機能解析 (2015)
  • Author(s): 小迫 英尊
  • Organizer: BMB2015
  • Place of Presentation: 神戸ポートアイランド（兵庫県神戸市）
  • Year and Date: 2015-12-02
  • Invited
• [Presentation] 先端プロテオミクス技術によるタンパク質キナーゼ基質の同定と機能解析 (2015)
  • Author(s): 小迫 英尊
  • Organizer: 第15回日本蛋白質科学会年会
  • Place of Presentation: あわぎんホール（徳島県徳島市）
  • Year and Date: 2015-06-26
  • Invited
• [Presentation] Identification and Functional Analysis of Protein Kinase Substrates using Various Proteomic Technologies, (2015)
  • Author(s): 小迫英尊
  • Organizer: Keystone Symposia
  • Place of Presentation: Colorado, USA
  • Year and Date: 2015-01-13
• [Presentation] リン酸化プロテオミクスによるキナーゼ基質の同定と機能解析 (2014)
  • Author(s): 小迫英尊
  • Organizer: 日本プロテオーム学会2014年会
  • Place of Presentation: つくば国際会議場（茨城県つくば市）
  • Year and Date: 2014-07-17
  • Invited
• [Remarks] 細胞情報学分野：：藤井節郎記念医科学センター
  • URL: http://www.fujii.tokushima-u.ac.jp/cellsignaling/
• [Remarks] 細胞情報学分野 :: 藤井節郎記念医科学センター
  • URL: http://www.fujii.tokushima-u.ac.jp/cellsignaling/