title: Purification and activation of recombinant p38 isoforms alpha, beta, gamma, and delta.
abstract: p38 is a proline-directed serine/threonine kinase that is activated by inflammatory cytokines and cellular stress. At present, four isoforms of p38 have been identified and termed alpha, beta, gamma, and delta. We expressed each p38 homolog in Escherichia coli and purified the recombinant isoforms. p38alpha and C-terminal Flag-tagged p38beta were purified by Q-Sepharose fast flow, hydroxyapatite, and Q-Sepharose high-performance chromatography. His-tagged p38gamma was purified using Ni2+-NTA resin followed by Mono Q chromatography. Glutathione S-transferase-Flag p38delta was purified using M2 affinity agarose and gel-filtration chromatography. Upstream activators of p38, constitutively active (ca) MKK3 and MKK6, were also cloned, purified, and used to activate each p38 isoform. p38 alpha, gamma, and delta were phosphorylated by both MKK6 and caMKK3. p38beta was phosphorylated only by MKK6. Mass spectrometry analysis and kinase assays showed that MKK6 was the superior reagent for phosphorylating and activating all p38 isoforms.
authors.affiliation: Amgen Inc., Boulder, Colorado, 80301, USA. gkeesler@amgen.com
authors.forename: G A
authors.initials: GA
authors.lastname: Keesler
authors.forename: J
authors.initials: J
authors.lastname: Bray
authors.forename: J
authors.initials: J
authors.lastname: Hunt
authors.forename: D A
authors.initials: DA
authors.lastname: Johnson
authors.forename: T
authors.initials: T
authors.lastname: Gleason
authors.forename: Z
authors.initials: Z
authors.lastname: Yao
authors.forename: S W
authors.initials: SW
authors.lastname: Wang
authors.forename: C
authors.initials: C
authors.lastname: Parker
authors.forename: H
authors.initials: H
authors.lastname: Yamane
authors.forename: C
authors.initials: C
authors.lastname: Cole
authors.forename: H S
authors.initials: HS
authors.lastname: Lichenstein
mesh_terms: D051698:Activating Transcription Factor 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry* / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein / Q000737:chemistry; D004789:Enzyme Activation / Q000502:physiology; D004926:Escherichia coli / Q000235:genetics; D007527:Isoenzymes / Q000302:isolation & purification*; D048371:MAP Kinase Kinase 3; D048669:MAP Kinase Kinase 6; D013058:Mass Spectrometry; D020929:Mitogen-Activated Protein Kinase Kinases; D020928:Mitogen-Activated Protein Kinases; D009842:Oligopeptides; D010455:Peptides / Q000276:immunology; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry*; D014157:Transcription Factors / Q000737:chemistry; D048051:p38 Mitogen-Activated Protein Kinases
chemical_list: D051698:Activating Transcription Factor 2; D017362:Cyclic AMP Response Element-Binding Protein; D007527:Isoenzymes; D009842:Oligopeptides; D010455:Peptides; D011994:Recombinant Proteins; D014157:Transcription Factors; C085706:FLAG peptide; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D048371:MAP Kinase Kinase 3; D048669:MAP Kinase Kinase 6; D020929:Mitogen-Activated Protein Kinase Kinases