title: Click conjugation of a binuclear terbium(III) complex for real-time detection of tyrosine phosphorylation.
abstract: Phosphorylation of proteins is closely associated with various diseases, and, therefore, its detection is vitally important in molecular biology and drug discovery. Previously, we developed a binuclear Tb(III) complex, which emits notable luminescence only in the presence of phosphotyrosine. In this study, we conjugated a newly synthesized binuclear Tb(III) complex to substrate peptides by using click chemistry. Using these conjugates, we were able to detect tyrosine phosphorylation in real time. These conjugates were superior to nonconjugated Tb(III) complexes for the detection of tyrosine phosphorylation, especially when the substrate peptides used were positively charged. Luminescence intensity upon phosphorylation was enhanced 10-fold, making the luminescence intensity of this system one of the largest among lanthanide luminescence-based systems. We also determined Michaelis-Menten parameters for the phosphorylation of various kinase/peptide combinations and quantitatively analyzed the effects of mutations in the peptide substrates. Furthermore, we successfully monitored the inhibition of enzymatic phosphorylation by inhibitors in real time. Advantageously, this system detects only the phosphorylation of tyrosine (phosphorylated serine and threonine are virtually silent) and is applicable to versatile peptide substrates. Our study thus demonstrates the applicability of this system for the analysis of kinase activity, which could lead to drug discovery.
authors.affiliation: †Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan.|‡Department of Bioengineering, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan, and.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Akiba
authors.affiliation: †Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan.|§Life Science Center of Tsukuba Advanced Research Alliance, University of Tsukuba, 1-1-1 Ten-noudai, Tsukuba, Ibaraki 305-8577, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Sumaoka
authors.affiliation: ‡Department of Bioengineering, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan, and.
authors.forename: Kouhei
authors.initials: K
authors.lastname: Tsumoto
authors.affiliation: †Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan.|§Life Science Center of Tsukuba Advanced Research Alliance, University of Tsukuba, 1-1-1 Ten-noudai, Tsukuba, Ibaraki 305-8577, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Komiyama
mesh_terms: D057930:Click Chemistry; D000069439:Dasatinib; D049449:Luminescence; D015394:Molecular Structure; D009942:Organometallic Compounds / Q000138:chemical synthesis / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D010770:Phosphotransferases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000494:pharmacology; D011743:Pyrimidines / Q000737:chemistry / Q000494:pharmacology; D013725:Terbium / Q000737:chemistry*; D013844:Thiazoles / Q000737:chemistry / Q000494:pharmacology; D013997:Time Factors; D014443:Tyrosine / Q000032:analysis* / Q000378:metabolism*
chemical_list: D009942:Organometallic Compounds; D010455:Peptides; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; D013844:Thiazoles; D013725:Terbium; D014443:Tyrosine; D010770:Phosphotransferases; D000069439:Dasatinib