| Co-Investigator(Kenkyū-buntansha) |
WATABE Akiko National Institute of Health Sciences, Division of Biological Chemistry and Biologicals, Researcher, 生物薬品部, 研究員 (50291117)
UCHIDA Eriko National Institute of Health Sciences, Division of Biological Chemistry and Biologicals, Section Chief, 生物薬品部, 室長 (80176685)
KAWAI Hiroshi National Institute of Health Sciences, Division of Biological Chemistry and Biologicals, Researcher, 生物薬品部, 研究員 (20321854)
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| Research Abstract |
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.
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