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

• Project/Area Number: 25350978
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Chemical biology
• Research Institution: Kagawa 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: ¥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.

Research Products

• [Journal Article] Expression analyses of splice variants of zebrafish cyclin-dependent kinase-like 5 and its substrate, amphiphysin 1 (2016)
  • 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
  • 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
  • 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
  • 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
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Cell-based inhibitor screening identifies multiple protein kinases important for circadian clock oscillations (2015)
  • 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
  • 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-PAGE (2015)
  • 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
  • Peer Reviewed
• [Journal Article] Effects of calmodulin on expressionof lignin-modifyingenzymes in Pleurotus ostreatus (2014)
  • 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
  • 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
  • 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
  • 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
  • Peer Reviewed / Open Access
• [Journal Article] Application of two-dimensional electrophoresis using liquid-phase isoelectric focusing (2014)
  • 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
  • Peer Reviewed
• [Journal Article] In-gel kinase assay revisited (2014)
  • 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
  • Peer Reviewed
• [Journal Article] Nuclear translocation of doublecortin-like protein kinase and phosphorylation of a transcription factor JDP2 (2014)
  • 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
  • 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
  • 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
  • Peer Reviewed
• [Journal Article] Expression and gene knockdown of zebrafish Ca/calmodulin-dependent protein kinase I-LL (2013)
  • Author(s): Y. Senga, K .Yoshioka, I. Kameshita, N. Sueyoshi
  • Journal Title: Arch. Biochem. Biophys. Volume: 540 Pages: 41-52
  • 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
  • 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
• [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
• [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
• [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
• [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
• [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
• [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
• [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
• [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
• [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
• [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
• [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
• [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
• [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
• [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
• [Presentation] Exploration of an endogenous substrate for DCKL5, a protein kinase associated with X-linked neurodevelopmental disorders (2014)
  • 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
• [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
• [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
• [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
• [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
• [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
• [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
• [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
• [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
• [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
• [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
• [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
  • Invited
• [Remarks] 動物機能生化学研究室
  • URL: http://www.ag.kagawa-u.ac.jp/sueyoshi/index.html