Following in vitro activation of mitogen-activated protein kinases by mass spectrometry and tryptic peptide analysis: purifying fully activated p38 mitogen-activated protein kinase α

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 MgATP2−. 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.