リン酸化プロテオーム研究の新戦略　～リン酸基認識素子としての二核錯体とその応用～

抄録

Phosphorylation is a major post-translational modification that regulates the function, localization, and binding specificity of target proteins. Abnormal protein phosphorylations are deeply related to various pathogenesis. Methods for monitoring the phosphorylation status of proteins are thus very important with respect to the evaluation of diverse biological and pathological processes.
Recently, we reported that a dinuclear metal complex of 1,3-bis[bis(pyridin-2-ylmethyl)-amino]propan-2-olato acts as a novel phosphate-binding tag molecule, Phos-tag, in an aqueous solution under physiological conditions (Fig. 1). The Phos-tag has a vacancy on two metal ions that is suitable for the access of a phosphomonoester dianion (R-OPO₃^2-) as a bridging ligand. The resulting 1:1 phosphate-binding complex, R-OPO₃^2--(Phos-tag)³⁺, has a total charge of +1. A dinuclear zinc(II) complex (Zn²⁺-Phos-tag) strongly binds to phenyl phosphate dianion (K_d = 2.5 x 10^-8 M) at a neutral pH. The anion selectivity indexes against SO₄^2-, CH₃COO^-, Cl^-, and the bisphenyl phosphate monoanion at 25 °C are 5.2 x 10³, 1.6 x 10⁴, 8.0 x 10⁵, and > 2 x 10⁶, respectively. A manganese(II) homologue (Mn²⁺-Phos-tag) can also capture R-OPO₃^2- anion, such as phosphoserine, phosphotyrosine, or phosphohistidine, at an alkaline pH. By utilizing the Phos-tag molecule, we here introduce convenient and reliable methods for the detection of phosphorylated proteins, such as phosphate-affinity chromatography or phosphate-affinity electrophoresis. We believe that our Phos-tag technology would result in great progress in phosphoproteomics.