Phos-tag固定化ポリマーを利用したシグナル伝達解析における選択的な蛋白質リン酸化検出のための新技術

抄録

In signal-transduction research, phosphoproteins are usually detected by using anti-phosphoprotein antibodies. However, low levels of phosphoproteins in various cell lysates frequently escape detection, even by immunoblotting with anti-phosphoprotein antibodies. If high concentrations of the antibody and long exposure times are employed as means of circumventing this problem, a lack of specificity can be a problem. Optimization of the conditions for specific detection of individual phosphoproteins by using anti-phosphoprotein antibodies is generally performed empirically and is time consuming.
Here, we introduce two techniques using Phos-tag-immobilized polymers for selective detection of protein phosphorylation. One is based on the efficient separation of phosphoproteins from complex mixtures containing solubilized cellular proteins together with more-specific immunoblot detection using Phos-tag agarose phosphate-affinity beads. We demonstrated improved detection of phosphorylated Shc and Erk isoforms in A431 cell lysates after EGF stimulation by pretreatment of the lysates with Phos-tag agarose as an immunoblotting enhancer. The other is based on the mobility shift of larger phosphoproteins with molecular masses of more than 200 kDa in agarose-polyacrylamide composite phosphate-affinity gel electrophoresis using Phos-tag acrylamide. We demonstrated reliable determination of the phosphoisotypes of high-molecular-mass proteins, such as mTOR (289 kDa), ATM kinase (350 kDa), and 53BP1 (213 kDa), as clear up-shifted migration bands in HeLa cell lysates after extracellular stimulation by subsequent immunoblotting without anti-phosphoprotein antibodies.
We believe that these solid techniques could assist in mapping low-abundance phosphorylation events on proteins with a wide range of molecular masses and should increase the utility for detection of hierarchical phosphorylation and dephosphorylation in the phosphoproteome study of the complicated kinase-phosphatase network.