Development of Real-time Imaging Method of Protein Tyrosine Phosphorylation

我们试图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PLCγ-1中编码SH2结构域的基因与pEGFP-N和-C载体(Clonetech)进行融合，构建表达载体pEGFP-SH2。已在大肠杆菌中表达了四种结合蛋白，其荧光光谱与绿色荧光蛋白几乎相同。该蛋白可在Hela细胞、RBL-2H3细胞、HegG2细胞等细胞中表达。然而，在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性变化。其次，研制了荧光探针，其荧光波长可通过FRET在ECFP和EYFP之间进行转换。构建了CFP-SH2(Grb2/Ash)-YFP、YFP-SH2(Grb2/Ash)-CFP和YFP-SH2(PLCγ1)-CFP的表达载体。由表皮生长因子受体和血小板衍生生长因子受体β的SH2结构域识别的多肽和酪氨酸被磷酸化的多肽都是化学合成的。YFP-SH2(PLCγ1)-CFP与合成肽孵育和与磷酸化多肽孵育的荧光光谱不同。构建了酪氨酸磷酸化的EGF受体和PDGF受体N-端和C-端的融合蛋白表达载体，并产生ECFP或EYFP。其中一种融合蛋白(CFP-(PDFG受体酪氨酸残基周围的20个氨基酸残基)-YFP)与HeLa细胞、RBL-2H3等细胞的匀浆碎屑孵育后，在三磷酸腺苷(ATP)和表皮生长因子(EGF)的存在下，荧光光谱发生了变化。不幸的是，这些探针在刺激酪氨酸磷酸化后荧光的显著变化还没有在细胞中检测到。

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.