Analysis of calmodulin-kinase cascade by using functional proteomics.

使用功能蛋白质组学分析钙调蛋白激酶级联。

基本信息

  • 批准号:
    17570115
  • 负责人:
  • 金额:
    $ 2.24万
  • 依托单位:
  • 依托单位国家:
    日本
  • 项目类别:
    Grant-in-Aid for Scientific Research (C)
  • 财政年份:
    2005
  • 资助国家:
    日本
  • 起止时间:
    2005 至 2006
  • 项目状态:
    已结题

项目摘要

To search for the substrates of Ca^<2+>/calmodulin-dependent protein kinase I (CaM-KI), we performed affinity chromatography purification using either the unphosphorylated or phosphorylated (at Thr^<177>) GST-fused CaM-KI catalytic domain (residues 1-293, Lys^<49>Glu) as the affinity ligand. Proteomic analysis was then carried out to identify the interacting proteins. In addition to the detection of two known CaM-KI substrates (CREB and synapsin I), we identified two Numb family proteins (Numb and Numbl) from rat tissues. These proteins were unphosphorylated and were bound only to the Thr^<177>-phosphorylated CaM-KI catalytic domain. This finding is consistent with the results demonstrating that Numb and Numbl were efficiently and stoichiometrically phosphorylated in vitro at equivalent Ser residues (Ser^<264> in Numb and Ser^<304> in Numbl) by activated CaM-KI and also by two other CaM-Ks (CaM-KII and CaM-KIV). Using anti-phosphoNumb/Numbl antibody, we observed the phosphorylation of … More Numb family proteins in various rat tissue extracts and we also detected the ionomycin-induced phosphorylation of endogenous Numb at Ser^<264> in COS-7 cells. The present results revealed that the Numb family proteins are phosphorylated in vivo as well as in vitro. Furthermore, we found that the recruitment of 14-3-3 proteins was the functional consequence of the phosphorylation of the Numb family proteins. Interaction of 14-3-3 protein with phosphorylated Numbl blocked dephosphorylation of Ser^<304>. Numb is thought to participate in clathrin-dependent endocytosis by directly interacting with the clathrin-associated adaptor complex AP-2, although the underlying mechanisms are unknown. Pull-down experiments showed that the phosphorylation of Numb impaired its binding to the AP-2 complex and simultaneously recruited 14-3-3 proteins in vitro. Based on experiments using Numb mutants, both the initial phosphorylation of Ser^<264> and the subsequent phosphorylation of Ser^<283> are sufficient to abolish the binding of Numb to AP-2 and to promote the interaction with 14-3-3 protein. These findings suggest a novel mechanism for the regulation of Numb-mediated endocytosis, namely through direct phosphorylation. Less
为了寻找Ca^2+/钙调蛋白依赖性蛋白激酶I(CaM-KI)的底物,我们使用未磷酸化或磷酸化(在Thr^<177>)GST融合的CaM-KI催化结构域(残基1-293,Lys^<49>Glu)作为亲和配体进行亲和色谱纯化。然后进行蛋白质组学分析以鉴定相互作用的蛋白。除了检测两种已知的CaM-KI底物(CREB和突触蛋白I),我们还从大鼠组织中鉴定了两种Numb家族蛋白(Numb和Numbl)。这些蛋白质未被磷酸化,仅与Thr^<177>-磷酸化的CaM-KI催化结构域结合。这一发现与以下结果一致,该结果表明Numb和Numbl在体外通过活化的CaM-KI以及另外两种CaM-Ks(CaM-KII和CaM-KIV)在等效的Ser残基(Numb中的Ser^和Numbl中的Ser^)处被有效且化学计量地磷酸化<264><304>。使用抗磷酸化Numb/Numbl抗体,我们观察到磷酸化的 ...更多信息 在各种大鼠组织提取物中的Numb家族蛋白,我们还检测了离子霉素诱导的COS-7细胞中内源性Numb在Ser^处的磷酸化<264>。本研究结果表明,Numb家族蛋白在体内以及在体外磷酸化。此外,我们发现14-3-3蛋白的募集是Numb家族蛋白磷酸化的功能结果。14-3-3蛋白与磷酸化的Numbl的相互作用阻断了Ser^的去磷酸化<304>。Numb被认为通过直接与网格蛋白相关的适配器复合物AP-2相互作用参与网格蛋白依赖性内吞作用,尽管其潜在机制尚不清楚。下拉实验表明,Numb的磷酸化损害了其与AP-2复合物的结合,同时在体外招募14-3-3蛋白。基于使用Numb突变体的实验,Ser^的初始磷酸化<264>和随后的Ser^的磷酸化<283>都足以消除Numb与AP-2的结合并促进与14-3-3蛋白的相互作用。这些发现提示了一种新的调节Numb介导的内吞作用的机制,即通过直接磷酸化。少

项目成果

期刊论文数量(9)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
Phosphorylation of Numb regulates its interaction with the clathrin‐associated adaptor AP‐2
  • DOI:
    10.1016/j.febslet.2006.09.043
  • 发表时间:
    2006-10
  • 期刊:
  • 影响因子:
    3.5
  • 作者:
    H. Tokumitsu;N. Hatano;Shigeyuki Yokokura;Yuka Sueyoshi;N. Nozaki;R. Kobayashi
  • 通讯作者:
    H. Tokumitsu;N. Hatano;Shigeyuki Yokokura;Yuka Sueyoshi;N. Nozaki;R. Kobayashi
Phosphorylation of Numb family proteins :Possible involvement of Ca^<2+>/calmodulin-dependent protein kinases.
Numb家族蛋白的磷酸化:可能涉及Ca^2/钙调蛋白依赖性蛋白激酶。
  • DOI:
  • 发表时间:
    2005
  • 期刊:
  • 影响因子:
    0
  • 作者:
    K.Matsuoka;H.Tabunoki;T.Kawai;S.Ishikawa;M.Yamamoto;R.Sato;T.Ando;Satoshi Watanabe;Hiroshi Tokumitsu et al.
  • 通讯作者:
    Hiroshi Tokumitsu et al.
Knockdown of nuclear Ca2+/calmodulin-dependent protein kinase phosphatase causes developmental abnormalities in zebrafish
  • DOI:
    10.1016/j.abb.2006.09.034
  • 发表时间:
    2007-01-15
  • 期刊:
  • 影响因子:
    3.9
  • 作者:
    Nimura, Takaki;Sueyoshi, Noriyuki;Kameshita, Isamu
  • 通讯作者:
    Kameshita, Isamu
Phosphorylation of Numb family proteins. : Possible involvement of Ca^<2+>/calmodulin-dependent protein kinases.
Numb 家族蛋白的磷酸化。
  • DOI:
  • 发表时间:
    2005
  • 期刊:
  • 影响因子:
    0
  • 作者:
    T.Fujii;M.G.Suzuki;T.Kawai;K.Tsuneizumi;A.Ohnishi;M.Kurihara;S.Matsumoto;T.Ando;Hiroshi Tokumitsu et al.
  • 通讯作者:
    Hiroshi Tokumitsu et al.
Spatiotemporal expression of four isoforms of Ca2+/calmodulin-dependent protein kinase I in brain and its possible roles in hippocampal dendritic growth
  • DOI:
    10.1016/j.neures.2006.09.013
  • 发表时间:
    2007-01-01
  • 期刊:
  • 影响因子:
    2.9
  • 作者:
    Kamata, Akifumi;Sakagami, Hiroyuki;Kondo, Hisatake
  • 通讯作者:
    Kondo, Hisatake
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TOKUMITSU Hiroshi其他文献

IL-34依存的に発生する新規単球系細胞の分化機構;濾胞樹状細胞によるIL-34特異的作用の解析
以IL-34依赖性方式发育的新型单核细胞的分化机制,分析IL-34对滤泡树突状细胞的特异性作用;
  • DOI:
  • 发表时间:
    2014
  • 期刊:
  • 影响因子:
    0
  • 作者:
    MAGARI Masaki;OGAWA Sayaka;TOYA Yuji;HIEDA Kentaro;YAMANE Fumihiro;MATSUI Kazue;NISHIKAWA Yumiko;KANAYAMA Naoki;TOKUMITSU Hiroshi;OHMORI Hitoshi;小川紗也香,山根文寛,松井一恵,鳥家雄二,西川裕美子,德光浩,金山直樹,大森斉,曲正樹
  • 通讯作者:
    小川紗也香,山根文寛,松井一恵,鳥家雄二,西川裕美子,德光浩,金山直樹,大森斉,曲正樹
濾胞樹状細胞依存的に発生する新規単球系細胞による胚中心B細胞の活性化
以滤泡树突细胞依赖性方式发育的新型单核细胞激活生发中心 B 细胞
  • DOI:
  • 发表时间:
    2014
  • 期刊:
  • 影响因子:
    0
  • 作者:
    MAGARI Masaki;OGAWA Sayaka;TOYA Yuji;HIEDA Kentaro;YAMANE Fumihiro;MATSUI Kazue;NISHIKAWA Yumiko;KANAYAMA Naoki;TOKUMITSU Hiroshi;OHMORI Hitoshi;小川紗也香,山根文寛,松井一恵,鳥家雄二,西川裕美子,德光浩,金山直樹,大森斉,曲正樹;鳥家雄二,長尾峻久,小川紗也香,岩崎映理子,松井一恵,西川裕美子,金山直樹,德光浩,大森斉,曲正樹
  • 通讯作者:
    鳥家雄二,長尾峻久,小川紗也香,岩崎映理子,松井一恵,西川裕美子,金山直樹,德光浩,大森斉,曲正樹
SRSF1-3 promotes nuclear localization of AID by inhibiting nuclear export of AID&#8232;
SRSF1-3通过抑制AID的核输出促进AID的核定位
  • DOI:
  • 发表时间:
    2015
  • 期刊:
  • 影响因子:
    0
  • 作者:
    152KAWAGUCHI Yuka;KAWAMOTO Naoko;MIYAZAKI Satoshi;NARIKI Hiroaki;YOKOYAMA Kazuki;TOKUMITSU Hiroshi;MAGARI Masaki;KANAYAMA Naoki
  • 通讯作者:
    KANAYAMA Naoki
IL-34/CSF-1R-dependent generation of a novel class of monocytic cells with unique B cell-stimulating activities
IL-34/CSF-1R 依赖性产生一类具有独特 B 细胞刺激活性的新型单核细胞
  • DOI:
  • 发表时间:
    2015
  • 期刊:
  • 影响因子:
    0
  • 作者:
    MAGARI Masaki;OGAWA Sayaka;TOYA Yuji;HIEDA Kentaro;YAMANE Fumihiro;MATSUI Kazue;NISHIKAWA Yumiko;KANAYAMA Naoki;TOKUMITSU Hiroshi;OHMORI Hitoshi
  • 通讯作者:
    OHMORI Hitoshi

TOKUMITSU Hiroshi的其他文献

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{{ truncateString('TOKUMITSU Hiroshi', 18)}}的其他基金

Comprehensive identification and signal transduction analysis of calmodulin-targets by functional proteomics
通过功能蛋白质组学对钙调蛋白靶标进行全面鉴定和信号转导分析
  • 批准号:
    21570143
  • 财政年份:
    2009
  • 资助金额:
    $ 2.24万
  • 项目类别:
    Grant-in-Aid for Scientific Research (C)
Identification and characterization of a novel target for CaM-kinase cascade
CaM-激酶级联新靶标的鉴定和表征
  • 批准号:
    19570134
  • 财政年份:
    2007
  • 资助金额:
    $ 2.24万
  • 项目类别:
    Grant-in-Aid for Scientific Research (C)
Physiological function of calcium/calmodulin-dependent protein kinase cascade
钙/钙调蛋白依赖性蛋白激酶级联的生理功能
  • 批准号:
    14580649
  • 财政年份:
    2002
  • 资助金额:
    $ 2.24万
  • 项目类别:
    Grant-in-Aid for Scientific Research (C)
Regulation of gene expression mediated by Ca^<2+>/calmodulin-dependent protein kinase cascade
Ca^2/钙调蛋白依赖性蛋白激酶级联介导的基因表达调节
  • 批准号:
    12680637
  • 财政年份:
    2000
  • 资助金额:
    $ 2.24万
  • 项目类别:
    Grant-in-Aid for Scientific Research (C)

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