title: Following in vitro activation of mitogen-activated protein kinases by mass spectrometry and tryptic peptide analysis: purifying fully activated p38 mitogen-activated protein kinase alpha.
abstract: p38 mitogen-activated protein kinase alpha (MAPKalpha) 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 MAPKalpha in sufficient quantity and purity for biophysical studies. Here we present a method for the preparation of milligram quantities of activated p38 MAPKalpha, specifically phosphorylated on Thr180 and Tyr182. Purification of the inactive (unphosphorylated) p38 MAPKalpha is facilitated by an N-terminal hexahistidine tag. Removal of this tag from His6-p38 MAPKalpha, 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 MAPKalpha can be quantified by following the formation of the bis-phosphorylated tryptic peptide, 173-HTDDEMT*GY*VATR-186, using [gamma-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 MAPKalpha phosphorylation.
authors.affiliation: Division of Medicinal Chemistry, University of Texas at Austin, Austin, TX 78712, USA.
authors.forename: Anna E
authors.initials: AE
authors.lastname: Szafranska
authors.forename: Xuemei
authors.initials: X
authors.lastname: Luo
authors.forename: Kevin N
authors.initials: KN
authors.lastname: Dalby
mesh_terms: D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology; D006639:Histidine; D048669:MAP Kinase Kinase 6; D009842:Oligopeptides; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D013912:Threonine / Q000737:chemistry; D014357:Trypsin; D014443:Tyrosine / Q000737:chemistry; D048051:p38 Mitogen-Activated Protein Kinases / Q000302:isolation & purification*
chemical_list: C471213:His-His-His-His-His-His; D009842:Oligopeptides; D013912:Threonine; D014443:Tyrosine; D006639:Histidine; D048051:p38 Mitogen-Activated Protein Kinases; D048669:MAP Kinase Kinase 6; D014357:Trypsin