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Development of Real-time Imaging Method of Protein Tyrosine Phosphorylation

蛋白质酪氨酸磷酸化实时成像方法的开发

基本信息

批准号：
09670115
负责人：
KAWANISHI Toru
金额：
$ 1.98万
依托单位：
National Institute of Health Sciences
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

项目摘要

We have tried to develop fluorescence probes for imaging of protein tyrosine phosphorylation. First, the genes coding SH2 domains in Grb2/Ash and PLCγ1 were fused with pEGFP-N and -C vectors (Clonetech) and the pEGFP-SH2 vectors were produced for the expression of EGFP-SH2. Four conjugated proteins, showing the almost same fluorescence spectra as EGFP, have been expressed in E.Coli. The proteins could be expressed in Hela cell, RBL-2H3 cell, HegG2 cell, and so on. However, no reproducible change in the localization of the fluorescence has been observed after the treatment with the stimulants of tyrosine phosphorylation. Second, the fluorescence probes, whose fluorescence wavelength is changed by FRET between ECFP and EYFP, have been developed. The plasmids for the expression of CFP-SH2 (Grb2/Ash) -YFP, YFP-SH2 (Grb2/Ash) -CFP, and YFP-SH2 (PLCγ1) -CFP were produced. The peptides, which are recognized by the SH2 domains in EGF receptor and PDGF receptor β, and those, whose tyrosine are phosphorylated, are both chemically synthesized. The fluorescence spectrum is different between YFP-SH2 (PLCγ1) -CFP incubated with the synthesized peptide and that incubated with the phosphorylated peptide. The plasmids for the expression of the fused proteins of the polypeptide (which are designed from N-terminal and C-terminal of EGF receptor and PDGF receptor), whose tyrosine are phosphorylated, and ECFP or EYFP have been produced. The fluorescence spectrum of one (CFP- (20amino acids residue around the tyrosine residue phosphorylated in PDFG receptor) -YFP) of the fused proteins has been changed after the incubation with the homogenate debris of HeLa cell, RBL-2H3 cell and so on, in the presence of ATP and EGF (PDGF). Unfortunately, the significant change of the fluorescence of those probes following the stimulation of tyrosine phosphorylation has not been detected in the cells, yet.

我们尝试开发用于蛋白质酪氨酸磷酸化成像的荧光探针。首先，将Grb2/Ash和PLCγ1中编码SH2结构域的基因与pEGFP-N和-C载体(Clonetech)融合，并产生pEGFP-SH2载体用于表达EGFP-SH2。四种缀合蛋白在大肠杆菌中表达，显示出与 EGFP 几乎相同的荧光光谱。该蛋白可在Hela细胞、RBL-2H3细胞、HegG2细胞等细胞中表达。然而，在用酪氨酸磷酸化刺激剂处理后，没有观察到荧光定位的可重复变化。其次，开发了通过ECFP和EYFP之间的FRET改变荧光波长的荧光探针。制作了用于表达CFP-SH2(Grb2/Ash)-YFP、YFP-SH2(Grb2/Ash)-CFP和YFP-SH2(PLCγ1)-CFP的质粒。 EGF受体和PDGF受体β的SH2结构域识别的肽以及酪氨酸被磷酸化的肽都是化学合成的。与合成肽一起孵育的YFP-SH2(PLCγ1)-CFP与与磷酸化肽一起孵育的YFP-SH2(PLCγ1)-CFP的荧光光谱不同。制备了酪氨酸磷酸化的多肽融合蛋白(从EGF受体和PDGF受体的N端和C端设计)和ECFP或EYFP的表达质粒。融合蛋白之一（CFP-（PDFG受体磷酸化酪氨酸残基周围的20个氨基酸残基）-YFP）与HeLa细胞、RBL-2H3细胞等匀浆碎片在ATP和EGF（PDGF）存在下孵育后，荧光光谱发生变化。不幸的是，尚未在细胞中检测到这些探针在刺激酪氨酸磷酸化后荧光的显着变化。

项目成果

期刊论文数量（0）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

川西徹: "高速共焦点顕微鏡"細胞. 31. 14-18 (1999)

Toru Kawanishi：“高速共聚焦显微镜”细胞。 31. 14-18 (1999)

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川西徹: "共焦点レーザー走査顕微鏡を用いた細胞内カルシウムイオンの高速高分解能画像化"日本薬理学雑誌. 112. 89-95 (1998)

Toru Kawanishi：“使用共焦激光扫描显微镜对细胞内钙离子进行高速、高分辨率成像”《日本药理学杂志》112. 89-95 (1998)。

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M.TAKAHASHI, Y.MISAWA, N.WATANABE, T.KAWANISHI, H.TANAKA, K.SHIGENOBU, and Y.KOBAYASHI: "Role of P-glycoprotein in human natural killer-like cell line-mediated cytotoxicity."Exp. Cell Res. 253. 396-402 (1999)

M.TAKAHASHI、Y.MISAWA、N.WATANABE、T.KAWANISHI、H.TANAKA、K.SHIGENOBU 和 Y.KOBAYASHI：“P-糖蛋白在人类自然杀伤样细胞系介导的细胞毒性中的作用。”Exp。

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Toru kawanishi,Hiroki Asoh,Takashi Kato,Chikako Uneyama,Kazuhiro Toyoda,Reiko Teshima,Hideharu Ikebuchi,Hisayuki Ohata,Kazutaka Momose,Takao Hayakawa and Michihito Takahashi: "Suppression of Calcium Oscillation by Tributyltin Chloride in Cultured Rat Hepa

Toru kawanishi、Hiroki Asoh、Takashi Kato、Chikako Uneyama、Kazuhiro Toyoda、Reiko Teshima、Hideharu Ikebuchi、Hisayuki Ohata、Kazutaka Momose、Takao Hayakawa 和 Michihito Takahashi：“三丁基氯化锡对培养大鼠肝脏中钙振荡的抑制