Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
10536321 | Analytical Biochemistry | 2005 | 10 Pages |
Abstract
p38 mitogen-activated protein kinase α (MAPKα) belongs to the MAPK subfamily, which plays a pivotal role in cell signal transduction, where it mediates responses to cell stresses and, to a lesser extent, growth factors. Although its cellular function has been under intense scrutiny since its initial discovery, little progress has been made in understanding its kinetic mechanism. A contributory factor has been the lack of a fast and rigorous method for the purification of activated p38 MAPKα in sufficient quantity and purity for biophysical studies. Here we present a method for the preparation of milligram quantities of activated p38 MAPKα, specifically phosphorylated on Thr180 and Tyr182. Purification of the inactive (unphosphorylated) p38 MAPKα is facilitated by an N-terminal hexahistidine tag. Removal of this tag from His6-p38 MAPKα, prior to its activation, is essential to ensure preparation of high yields of homogeneous, dually phosphorylated enzyme. Activation is achieved on incubation with a glutathione S-transferase (GST) fusion of the constitutively active mutant of the upstream activator, MKK6b (GST-MKK6b S207E T211E), in the presence of MgATP2â. Notably, we show that specific formation of activated p38 MAPKα can be quantified by following the formation of the bis-phosphorylated tryptic peptide, 173-HTDDEMT*GY*VATR-186, using [γ-32P]adenosine triphosphate (ATP) as the phosphate source and reverse-phase high-performance liquid chromatography (HPLC) to separate the phosphopeptides. This approach offers the only means to specifically determine both stoichiometry and specificity of p38 MAPKα phosphorylation.
Related Topics
Physical Sciences and Engineering
Chemistry
Analytical Chemistry
Authors
Anna E. Szafranska, Xuemei Luo, Kevin N. Dalby,