Development of Real-time Imaging Method of Protein Tyrosine Phosphorylation

我们已经尝试开发荧光探针成像蛋白酪氨酸磷酸化。首先，将Grb2/Ash和plc - γ1中编码SH2结构域的基因与pEGFP-N和-C载体（Clonetech）融合，得到表达EGFP-SH2的pEGFP-SH2载体。在大肠杆菌中表达了4种与EGFP具有相同荧光光谱的共轭蛋白。这些蛋白可在Hela细胞、RBL-2H3细胞、HegG2细胞等中表达。然而，在酪氨酸磷酸化刺激剂处理后，荧光定位没有可重复的变化。其次，研制了荧光探针，其荧光波长在ECFP和EYFP之间通过FRET变化。获得了表达CFP-SH2 (Grb2/Ash) -YFP、YFP-SH2 (Grb2/Ash) -CFP和YFP-SH2 (plc - γ1) -CFP的质粒。被EGF受体和PDGF受体β的SH2结构域识别的肽和酪氨酸被磷酸化的肽都是化学合成的。YFP-SH2 (plc - γ1) -CFP与合成肽孵育后的荧光光谱与磷酸化肽孵育后的荧光光谱存在差异。从EGF受体和PDGF受体的n端和c端设计表达酪氨酸磷酸化的多肽融合蛋白的质粒，并制备了ECFP或EYFP。与HeLa细胞、RBL-2H3细胞等的匀浆碎片孵育后，在ATP和EGF （PDGF）存在下，融合蛋白的一个（CFP- （PDFG受体磷酸化的酪氨酸残基周围的20个氨基酸残基）- yfp）的荧光光谱发生了变化。不幸的是，这些探针在酪氨酸磷酸化刺激后荧光的显著变化尚未在细胞中检测到。

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.