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Development of a new two-dimensional in-gel phosphatase assay and its application to protein phosphatase research

Research Project

Project/Area Number 25350978
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeMulti-year Fund
Section一般
Research Field Chemical biology
Research InstitutionKagawa University

Principal Investigator

Kameshita Isamu  香川大学, 農学部, 教授 (60127941)

Co-Investigator(Kenkyū-buntansha) SUEYOSHI NORIYUKI  香川大学, 農学部, 准教授 (90346635)
Project Period (FY) 2013-04-01 – 2016-03-31
Project Status Completed (Fiscal Year 2015)
Budget Amount *help
¥5,070,000 (Direct Cost: ¥3,900,000、Indirect Cost: ¥1,170,000)
Fiscal Year 2015: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000)
Fiscal Year 2014: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000)
Fiscal Year 2013: ¥2,730,000 (Direct Cost: ¥2,100,000、Indirect Cost: ¥630,000)
Keywords細胞内情報伝達 / タンパク質リン酸化 / プロテインホスファターゼ / カルモジュリンキナーゼ / 翻訳後修飾 / プロテインキナーゼカスケード / タンパク質リン酸化反応 / 活性制御機構 / ツーハイブリッドスクリーニング / 活性制御因子 / 二次元電気泳動 / インゲルアッセイ / 酸化還元 / 蛍光発生基質
Outline of Final Research Achievements

Two major objectives of this research project are as follows. (1) Development of a new two-dimensional in-gel phosphatase assay. (2) Elucidation of the regulatory mechanisms of PPM family phosphatases including CaM-kinase phosphatase (CaMKP) and nuclear CaMKP (CaMKP-N). In this project, we have successfully developed a unique two-dimensional in-gel phosphatase assay using fluorogenic substrate, 4-methylumbelliferyl phosphate (MUP). Futhermore, we clarified the regulatory mechanisms of cellular localization of CaMKP-N through proteolytic processing and found a new anchoring protein, protocadherin gamma subfamily C5, that regulate CaMKP activity in vivo.

Report

(4 results)
  • 2015 Annual Research Report   Final Research Report ( PDF )
  • 2014 Research-status Report
  • 2013 Research-status Report
  • Research Products

    (45 results)

All 2016 2015 2014 2013 Other

All Journal Article (17 results) (of which Int'l Joint Research: 1 results,  Peer Reviewed: 17 results,  Open Access: 5 results,  Acknowledgement Compliant: 4 results) Presentation (27 results) (of which Invited: 1 results) Remarks (1 results)

  • [Journal Article] Expression analyses of splice variants of zebrafish cyclin-dependent kinase-like 5 and its substrate, amphiphysin 12016

    • Author(s)
      Syouichi Katayama, Yukako Senga, Ami Oi, Yosuke Miki, Yasunori Sugiyama, Noriyuki Sueyoshi, Isamu Kameshita
    • Journal Title

      Gene

      Volume: 583 Issue: 1 Pages: 15-23

    • DOI

      10.1016/j.gene.2016.02.036

    • Related Report
      2015 Annual Research Report
    • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
  • [Journal Article] Critical Determinants of Substrate Recognition by Cyclin-Dependent Kinase-like 5 (CDKL5)2015

    • Author(s)
      Syouichi Katayama, Noriyuki Sueyoshi and Isamu Kameshita
    • Journal Title

      Biochemistry

      Volume: in press Issue: 19 Pages: 2975-2987

    • DOI

      10.1021/bi501308k

    • Related Report
      2015 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] The phosphatase-resistant isoform of CaMKI, Ca2+/calmodulin-dependent protein kinase Iδ (CaMKIδ), remains in its 'Primed' form without Ca2+-stimulation.2015

    • Author(s)
      Senga Y, Ishida A, Shigeri Y, Kameshita I, Sueyoshi N.
    • Journal Title

      Biochemistry

      Volume: 54 Issue: 23 Pages: 3617-3630

    • DOI

      10.1021/bi5012139

    • Related Report
      2015 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Regulation of Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP/PPM1F) by protocadherin-gamma C5 (Pcdh-gamma C5)2015

    • Author(s)
      Onouchi T, Kishino-Kaneko Y, Kameshita I, Ishida A, Sueyoshi N.
    • Journal Title

      Arch Biochem Biophys

      Volume: 585 Pages: 109-120

    • DOI

      10.1016/j.abb.2015.09.014

    • Related Report
      2015 Annual Research Report
    • Peer Reviewed / Acknowledgement Compliant
  • [Journal Article] Cell-based inhibitor screening identifies multiple protein kinases important for circadian clock oscillations2015

    • Author(s)
      Naohiro Kon, Yasunori Sugiyama, Hikari Yoshitane, Isamu Kameshita, Yoshitaka Fukada
    • Journal Title

      Communicative & Integrative Biology

      Volume: 8 Issue: 4 Pages: 1-4

    • DOI

      10.4161/19420889.2014.982405

    • Related Report
      2015 Annual Research Report
    • Peer Reviewed / Open Access / Acknowledgement Compliant
  • [Journal Article] Expression and phosphorylation state analysis of intracellular protein kinases using Multi-PK antibody and Phos-tag SDS-PAGE2015

    • Author(s)
      Sugiyama, Y., Katayama, S., Kameshita, I., Morisawa, K. Higuchi, T., Todaka, J., Kinoshita, E., Kinoshita-Kikuta, E., Koike, T., Taniguchi, T., Sakamoto, S.
    • Journal Title

      MethodsX

      Volume: 2 Pages: 469-474

    • Related Report
      2015 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Effects of calmodulin on expressionof lignin-modifyingenzymes in Pleurotus ostreatus2014

    • Author(s)
      Suetomi, T., Sakamoto, T., Tokunaga, Y., Kameyama, T.,Honda, Y., Kamitsuji, H., Kameshita, I., Izumitsu, K., Suzuki, K., Irie, T.
    • Journal Title

      Curr. Genet.

      Volume: 14 Issue: 2 Pages: 1-14

    • DOI

      10.1007/s00294-014-0460-z

    • Related Report
      2015 Annual Research Report 2014 Research-status Report
    • Peer Reviewed
  • [Journal Article] TandeMBP: Generation of a unique protein substrate for protein kinase assays.2014

    • Author(s)
      (1)Kameshita, I., Yamashita, S., Katayama, S., Senga, Y., Sueyoshi, N.
    • Journal Title

      J. Biochem.

      Volume: 156 Issue: 3 Pages: 147-154

    • DOI

      10.1093/jb/mvu025

    • Related Report
      2014 Research-status Report
    • Peer Reviewed / Open Access / Acknowledgement Compliant
  • [Journal Article] Cellular localization of CoPK12, a Ca2+/calmodulin-dependent protein kinase in mushroom Coprinopsis cinerea, is regulated by N-myristoylation and limited proteolysis.2014

    • Author(s)
      Kaneko K, Tabuchi M, Sueyoshi N, Ishida A, Utsumi T, Kameshita I
    • Journal Title

      Journal of Biochemistry

      Volume: in press Issue: 1 Pages: 51-61

    • DOI

      10.1093/jb/mvu018

    • Related Report
      2014 Research-status Report
    • Peer Reviewed
  • [Journal Article] CaMKII is essential for the cellular clock and coupling between morning and evening behavioral rhythms.2014

    • Author(s)
      Kon N, Yoshikawa T, Honma S, Yamagata Y, Yoshitane H, Shimizu K, Sugiyama Y, Hara C, Kameshita I, Honma K, Fukada Y.
    • Journal Title

      Genes Dev

      Volume: 28 Issue: 10 Pages: 1101-1110

    • DOI

      10.1101/gad.237511.114

    • Related Report
      2014 Research-status Report
    • Peer Reviewed / Open Access
  • [Journal Article] Application of two-dimensional electrophoresis using liquid-phase isoelectric focusing2014

    • Author(s)
      杉山康憲、片山将一、馬場裕美、末吉紀行、亀下勇
    • Journal Title

      SEIBUTSU BUTSURI KAGAKU

      Volume: 58 Issue: 2 Pages: 56-58

    • DOI

      10.2198/sbk.58.56

    • NAID

      130004704687

    • ISSN
      0031-9082, 1349-9785
    • Related Report
      2014 Research-status Report
    • Peer Reviewed
  • [Journal Article] In-gel kinase assay revisited2014

    • Author(s)
      石田敦彦、亀下勇
    • Journal Title

      SEIBUTSU BUTSURI KAGAKU

      Volume: 58 Issue: 2 Pages: 62-64

    • DOI

      10.2198/sbk.58.62

    • NAID

      130004704689

    • ISSN
      0031-9082, 1349-9785
    • Related Report
      2014 Research-status Report
    • Peer Reviewed
  • [Journal Article] Nuclear translocation of doublecortin-like protein kinase and phosphorylation of a transcription factor JDP22014

    • Author(s)
      T. Nagamine, S. Nomada, T. Onouchi, I. Kameshita, N. Sueyoshi
    • Journal Title

      Biochem. Biophys. Res. Commun.

      Volume: 446 Issue: 1 Pages: 73-78

    • DOI

      10.1016/j.bbrc.2014.02.075

    • Related Report
      2013 Research-status Report
    • Peer Reviewed
  • [Journal Article] Identification of amphiphysin 1 as an endogenous substrate for CDKL5, a protein kinase associated with X-linked neurodevelopmental disorder.2013

    • Author(s)
      Sekiguchi M, Katayama S, Hatano N, Shigeri Y, Sueyoshi N, Kameshita I.
    • Journal Title

      Archives of Biochemistry and Biophysics

      Volume: 535(2) Issue: 2 Pages: 257-267

    • DOI

      10.1016/j.abb.2013.04.012

    • Related Report
      2013 Research-status Report
    • Peer Reviewed
  • [Journal Article] Ink-native electrophoresis: An alternative to blue-native electrophoresis more suitable for in-gel detection of enzymatic activity.2013

    • Author(s)
      Kaneko K, Sueyoshi N, Kameshita I, Ishida A,
    • Journal Title

      Anal. Biochem.

      Volume: 440 Issue: 2 Pages: 142-144

    • DOI

      10.1016/j.ab.2013.05.026

    • Related Report
      2013 Research-status Report
    • Peer Reviewed
  • [Journal Article] Expression and gene knockdown of zebrafish Ca/calmodulin-dependent protein kinase I-LL2013

    • Author(s)
      Y. Senga, K .Yoshioka, I. Kameshita, N. Sueyoshi
    • Journal Title

      Arch. Biochem. Biophys.

      Volume: 540 Pages: 41-52

    • Related Report
      2013 Research-status Report
    • Peer Reviewed
  • [Journal Article] An active C-terminally truncated form of Ca2+/calmodulin-dependent protein kinase phosphatase-N (CaMKP-N/PPM1E).2013

    • Author(s)
      Ishida A, Tsumura K, Oue M, Takenaka Y, Shigeri Y, Goshima N, Ishihara Y, Hirano T, Baba H, Sueyoshi N, Kameshita I, Yamazaki T.
    • Journal Title

      BioMed Res. Int.

      Volume: 2013 Pages: 134813-134813

    • DOI

      10.1155/2013/134813

    • NAID

      120006557539

    • Related Report
      2013 Research-status Report
    • Peer Reviewed
  • [Presentation] 抗がん剤を処理したヒト白血病細胞HL-60の細胞内キナーゼの発現およびリン酸化動態の解析2015

    • Author(s)
      杉山康憲、片山将一、亀下勇、森澤啓子、樋口琢磨、戸高寛、木下英司、木下恵美子、小池透、坂本修士
    • Organizer
      第88回日本生化学会・第38回日本分子生物学会合同大会
    • Place of Presentation
      神戸
    • Year and Date
      2015-12-04
    • Related Report
      2015 Annual Research Report
  • [Presentation] 担子菌Coprinopsis cinereaの成長菌糸に存在するCaMキナーゼ(CoPK02)の性質2015

    • Author(s)
      山下雅史、片山将一、千賀由佳子、杉山康憲、末吉紀行、亀下勇
    • Organizer
      第88回日本生化学会・第38回日本分子生物学会合同大会
    • Place of Presentation
      神戸
    • Year and Date
      2015-12-03
    • Related Report
      2015 Annual Research Report
  • [Presentation] Amphiphysin 1のCLAP領域はCyclin-dependent kinase-like 5によってリン酸化されるために重要である2015

    • Author(s)
      片山将一、大井愛海、三木洋祐、末吉紀行、亀下勇
    • Organizer
      第88回日本生化学会・第38回日本分子生物学会合同大会
    • Place of Presentation
      神戸
    • Year and Date
      2015-12-03
    • Related Report
      2015 Annual Research Report
  • [Presentation] Protocadherin gamma subfamily C5 (Pcdh-C5)によるCaMK phosphatase (CaMKP-N/PPM1E) の分解抑制2015

    • Author(s)
      小野内貴士、石田敦彦、亀下勇、末吉紀行
    • Organizer
      第88回日本生化学会・第38回日本分子生物学会合同大会
    • Place of Presentation
      神戸
    • Year and Date
      2015-12-03
    • Related Report
      2015 Annual Research Report
  • [Presentation] CaMキナーゼホスファターゼはニューロフィラメントLと結合して重合を阻害する2015

    • Author(s)
      尾崎華、加藤剛志、中川綾子、石原康宏、末吉紀行、亀下勇、谷口隆信、山崎岳、石田敦彦
    • Organizer
      第88回日本生化学会・第38回日本分子生物学会合同大会
    • Place of Presentation
      神戸
    • Year and Date
      2015-12-02
    • Related Report
      2015 Annual Research Report
  • [Presentation] Dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A)によるCyclin-dependent kinase-like 5 (CDKL5) のリン酸化とその意義2015

    • Author(s)
      大井愛海、片山将一、三木洋祐、波多野直哉、杉山康憲、末吉紀行、亀下勇
    • Organizer
      第88回日本生化学会・第38回日本分子生物学会合同大会
    • Place of Presentation
      神戸
    • Year and Date
      2015-12-01
    • Related Report
      2015 Annual Research Report
  • [Presentation] Ca2+/CaM-dependent protein kinase I(CaMKI)によるDistal-less homeobox 1 (Dlx1)のリン酸化とオステオカルシンの転写制御2015

    • Author(s)
      秋月一駿、千賀由佳子、杉山康憲、亀下勇、末吉紀行
    • Organizer
      第88回日本生化学会・第38回日本分子生物学会合同大会
    • Place of Presentation
      神戸
    • Year and Date
      2015-12-01
    • Related Report
      2015 Annual Research Report
  • [Presentation] CaMKIとCaMKIのホスファターゼ抵抗性に注目した活性調節機構の比較解析2015

    • Author(s)
      千賀由佳子、石田敦彦、茂里康、亀下勇、末吉紀行
    • Organizer
      第88回日本生化学会・第38回日本分子生物学会合同大会
    • Place of Presentation
      神戸
    • Year and Date
      2015-12-01
    • Related Report
      2015 Annual Research Report
  • [Presentation] マルチPK抗体とPhos-tagを利用した細胞内リン酸化動態の新規解析法2015

    • Author(s)
      杉山康憲、亀下勇、坂本修士
    • Organizer
      第66回日本電気泳動学会総会
    • Place of Presentation
      東京
    • Year and Date
      2015-10-19
    • Related Report
      2015 Annual Research Report
  • [Presentation] カルモジュリン依存性プロテインキナーゼIアイソフォーム(CaMKIαとCaMKIδ)の自己リン酸化に着目した比較解析2015

    • Author(s)
      秋月一駿、千賀由佳子、杉山康憲、亀下勇、末吉紀行
    • Organizer
      第56回日本生化学会 中国・四国支部例会
    • Place of Presentation
      松江市
    • Year and Date
      2015-05-30
    • Related Report
      2015 Annual Research Report
  • [Presentation] Rett症候群の発症に関わるCyclin-dependent kinase-like 5(CDKL5)のアミノ酸変異と基質リン酸化活性2015

    • Author(s)
      大井愛海、片山将一、杉山康憲、末吉紀行、亀下勇
    • Organizer
      第56回日本生化学会 中国・四国支部例会
    • Place of Presentation
      松江市
    • Year and Date
      2015-05-30
    • Related Report
      2015 Annual Research Report
  • [Presentation] CaMKIdeltaによるDistal-less homeobox 1 を介した骨形成メカニズム2014

    • Author(s)
      千賀由佳子、杉山康憲、亀下勇、末吉紀行
    • Organizer
      日本分子生物学会年回
    • Place of Presentation
      横浜
    • Year and Date
      2014-11-25 – 2014-11-28
    • Related Report
      2014 Research-status Report
  • [Presentation] ゼブラフィッシュに存在する2種類のCDKL5のスプライスバリアントの比較解析2014

    • Author(s)
      片山将一、千賀由佳子、大井愛海、三木洋祐、杉山康憲、末吉紀行、亀下勇
    • Organizer
      日本分子生物学会年会
    • Place of Presentation
      横浜
    • Year and Date
      2014-11-25 – 2014-11-28
    • Related Report
      2014 Research-status Report
  • [Presentation] プロトカドヘリンγC5を介したCaMKホスファターゼの制御メカニズム2014

    • Author(s)
      小野内貴士、亀下勇、末吉紀行
    • Organizer
      日本分子生物学会年会
    • Place of Presentation
      横浜
    • Year and Date
      2014-11-25 – 2014-11-28
    • Related Report
      2014 Research-status Report
  • [Presentation] PPMホスファターゼは金属配位部位に隣接するCys残基の酸化還元によって活性制御を受ける2014

    • Author(s)
      馬場裕美、末吉紀行、亀下勇
    • Organizer
      日本分子生物学会年会
    • Place of Presentation
      横浜
    • Year and Date
      2014-11-25 – 2014-11-28
    • Related Report
      2014 Research-status Report
  • [Presentation] Exploration of an endogenous substrate for DCKL5, a protein kinase associated with X-linked neurodevelopmental disorders2014

    • Author(s)
      katayama, S., Sueyoshi, N., Kameshita, I.
    • Organizer
      第15回IUBMB
    • Place of Presentation
      台北
    • Year and Date
      2014-10-22 – 2014-10-24
    • Related Report
      2014 Research-status Report
  • [Presentation] 大腸菌発現系を用いた高活性型CaMKP-Nの取得とその利用2014

    • Author(s)
      朝波彰、小野内貴士、馬場裕美、亀下勇、末吉紀行
    • Organizer
      日本生化学会本会
    • Place of Presentation
      京都
    • Year and Date
      2014-10-15 – 2014-10-18
    • Related Report
      2014 Research-status Report
  • [Presentation] カルシウムがなくても潜在的活性化状態にあるホスファターゼ抵抗性CaMKIアイソフォーム2014

    • Author(s)
      千賀由佳子、石田敦彦、茂里康、亀下勇、末吉紀行
    • Organizer
      日本生化学会本会
    • Place of Presentation
      京都
    • Year and Date
      2014-10-15 – 2014-10-18
    • Related Report
      2014 Research-status Report
  • [Presentation] 金属依存性プロテインホスファターゼPPM1Bの機能解析2014

    • Author(s)
      樫村明理、渡辺真以、馬場裕美、小野内貴士、亀下勇、末吉紀行
    • Organizer
      日本生化学会中四国支部例会
    • Place of Presentation
      愛媛
    • Year and Date
      2014-06-05 – 2014-06-06
    • Related Report
      2014 Research-status Report
  • [Presentation] Ca2+/カルモジュリン依存性プロテインキナーゼホスファターゼの機能解析2014

    • Author(s)
      小野内貴士、馬場裕美、亀下勇、末吉紀行
    • Organizer
      日本生化学会中四国支部例会
    • Place of Presentation
      愛媛
    • Year and Date
      2014-06-05 – 2014-06-06
    • Related Report
      2014 Research-status Report
  • [Presentation] TandeMBP: MBPを超えるプロテインキナーゼ用人工基質の創製

    • Author(s)
      山下翔、片山将一、末吉紀行、亀下勇
    • Organizer
      日本生化学会
    • Place of Presentation
      横浜
    • Related Report
      2013 Research-status Report
  • [Presentation] CDKL5によるAmphiphysin1のリン酸化とCDKL5の基質特異性の解析

    • Author(s)
      片山将一、関口茉里、三木洋祐、末吉紀行、亀下勇
    • Organizer
      日本生化学会
    • Place of Presentation
      横浜
    • Related Report
      2013 Research-status Report
  • [Presentation] CaMKIδはCaMKIαとのN末端領域のアミノ酸の相違によりホスファターゼ抵抗性を有するようになる

    • Author(s)
      千賀由佳子、亀下勇、石田敦彦、末吉紀行
    • Organizer
      日本分子生物学会
    • Place of Presentation
      神戸
    • Related Report
      2013 Research-status Report
  • [Presentation] DCLKの核移行に伴うキナーゼ活性の制御メカニズム

    • Author(s)
      野間田祥悟、永峰賢、亀下勇、末吉紀行
    • Organizer
      日本分子生物学会
    • Place of Presentation
      神戸
    • Related Report
      2013 Research-status Report
  • [Presentation] Pcdhgc5を介したCaMKP-Nの活性化メカニズム

    • Author(s)
      小野内貴士、亀下勇、末吉紀行
    • Organizer
      日本分子生物学会
    • Place of Presentation
      神戸
    • Related Report
      2013 Research-status Report
  • [Presentation] メタル依存性プロテインホスファターゼ(PPM)の酸化的修飾による活性制御

    • Author(s)
      馬場裕美、末吉紀行、渡辺真以、樫村明理、茂里康、五島直樹、亀下勇
    • Organizer
      日本分子生物学会
    • Place of Presentation
      神戸
    • Related Report
      2013 Research-status Report
  • [Presentation] CDKL5とCaMキナーゼIの新規基質タンパク質

    • Author(s)
      亀下勇
    • Organizer
      蛋白研セミナー
    • Place of Presentation
      大阪大学
    • Related Report
      2013 Research-status Report
    • Invited
  • [Remarks] 動物機能生化学研究室

    • URL

      http://www.ag.kagawa-u.ac.jp/sueyoshi/index.html

    • Related Report
      2015 Annual Research Report

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Published: 2014-07-25   Modified: 2019-07-29  

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