Specific Recognition and Detection of Phosphorylated Proteins Using Characteristics of Metal Ion

Abstract

Protein phosphorylation is one of the most important post-translational modifications. Organisms utilize this reversible reaction of proteins to control many cellular activities, including signal transduction, apoptosis, gene expression, cell cycle progression, cytoskeletal regulation, and energy metabolism. Abnormal protein phosphorylation is deeply related to carcinogenesis and neuropathogenesis. 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. The Phos-tag has a vacancy on two metal ions that is suitable for the access of a phosphomonoester dianion (R-OPO(2)3*) as a bridging ligand. The resulting 1:1 phosphate-binding complex, R-OPO(2)3*-(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 SO(2)4*, CH3COO*, Cl-, and bisphenyl phosphate monoanion at 25 degrees C are 5.2 x 10(3), 1.6 x 10(4), 8.0 x 10(5), and>2 x 10(6), respectively. A manganese(II) homologue (Mn2+-Phos-tag) can also capture the R-OPO(2)3* anion, such as phosphoserine, phosphotyrosine, and phosphohistidine, at an alkaline pH. By utilizing the Phos-tag molecule and its derivatives, we developed convenient and reliable methods for the detection of phosphorylated proteins. We believe that our Phos-tag technology will result in great progress in phosphoproteomics.

Aquaporin OsPIP2;2 links the H2O2 signal and a membrane-anchored transcription factor to promote plant defense

“…The above total protein samples were incubated with calf intestinal alkaline phosphatase (Cat#9001-78-9; Solarbio, Beijing, China) at 37°C for 30 min, and then were separated by a 12% w/v SDS-PAGE gel contained biotinylated Mn 2+ -Phos-tag complex AAL-107 ( Kinoshita et al, 2007 ) purchased from Wako ( http://www.Phos-tag.com ). The OsPIP2;2 protein was further detected by western blot with His antibody.…”

Mn2+‐Phos‐Tag Polyacrylamide for the Quantification of Protein Phosphorylation Levels

“…Assuming it is similar to that for Zn 2+ complex binding to the phenyl phosphate di‐anion, the binding affinity would be ∼ K d = 2.5 × 10 −8 M under neutral pH. The high binding affinity and compromised protein transfer efficiency from Phos‐tag gels prompted the exploration of adding SDS in the transfer buffer (Kinoshita et al., 2009b; Kinoshita, Kinoshita‐Kikuta, & Koike, 2007; Kinoshita‐Kikuta et al., 2014).…”