Novel type of phosphate-affinity electrophoresis for protein kinase profiling.

Abstract

SUMMARYWe developed a novel type of phosphate-affinity polyacrylamide gel electrophoresis using an alkoxide-bridged dinuclear manganese(II) complex as a phosphate-binding tag, Phos-tag. The phosphate-affinity site is a polyacrylamide-bound Mn 2+ -Phos-tag that enables the mobility shift detection of phosphorylated proteins from their nonphosphorylated counterpart in an SDS-PAGE gel and the quantitative analysis of kinase reactions. Herein, we describe three applications of protein kinase profiling using the phosphate-affinity electrophoresis (Mn 2+ -Phos-tag SDS-PAGE). The first application is in vitro kinase activity profiling for the analysis of varied phosphoprotein status. The activity profiles of six kinds of kinases were determined using a substrate, Tau, which has a number of phosphorylation sites. Each kinase demonstrated characteristic multiple bands up-shifted from the nonphosphorylated Tau due to differences in the phosphorylation sites and stoichiometry. The second is in vivo kinase activity profiling for the analysis of protein phosphorylation involved in intracellular signal transduction. The time-course changes in the EGF-induced phosphorylation levels of Shc and MAPK in A431 cells were visualized as highly up-shifted migration bands by subsequent immunoblotting analysis. The final application is in vitro kinase inhibition profiling for the quantitative screening of kinase-specific inhibitors. The inhibition profile of a tyrosine kinase, Abl, was determined using the substrate Abltide-GST and the approved inhibitor Glivec. The dose-dependent inhibition of Glivec was determined by a change in the ratio of the monophosphorylated and nonphosphorylated Abltide-GST bands.