Monitoring of extracellular signal-regulated kinase (ERK) activity with capillary electrophoresis

Abstract

Extracellular signal-regulated kinase (ERK), which is an isoform of mitogen-activated protein kinases (MAPKs), is an important component of various signaling pathways. ERK phosphorylates numerous substrates including myelin basic protein (MBP) and is one of the key components in linking growth factor receptor activation to serine/threonine protein phosphorylation processes. We investigated a rapid and safe method for determining ERK activity using capillary electrophoresis (CE). ERK converts MBP substrate to phosphorylation MBP product. The phosphorylation reactions of MBP by ERK were studied under varied experimental conditions. Reaction mixtures were carried out using MBP substrate and by adding ERK as well as adenosine triphosphosphate (ATP). The CE analysis method was performed in untreated fused-silica capillary columns of 37 cm X 75 mum i.d. and 185 nm wavelength using the 150 mM tris-phosphate buffer (pH 2.5) as a run buffer. The method of CE provided a very rapid analysis time of less than 10 min and high reproducibility within 5% of RSD% for MBP substrate detection. In this article, ERK reactions were determined from the decrease in MBP substrates using the CE method while the formation of phosphorylated MBP product by ERK reaction was monitored using the matrix-assisted laser desorption/ionization time-of-flight mass spectrum (MALDI-TOF MS) method. These results suggest that CE can be applied to many other enzymatic assays because of its various advantages such as nonradiolabeled substrates usage, short analysis time, and inexpensive expenses. (C) 2001 John Wiley & Sons, Inc.