title: Proteomic identification of p38 MAP kinase substrates using in vitro phosphorylation.
abstract: Protein phosphorylation is a major mechanism that regulates many basic cellular processes. Identification and characterization of substrates for a given protein kinase can lead to a better understanding of signal transduction pathways. However, it is still difficult to efficiently identify substrates for protein kinases. Here, we propose an integrated proteomic approach consisting of in vitro dephosphorylation and phosphorylation, phosphoprotein enrichment, and 2D-DIGE. Phosphatase treatment significantly reduced the complexity of the phosphoproteome, which enabled us to efficiently identify the substrates. We employed p38 mitogen-activated protein kinase (p38 MAP kinase) as a model kinase and identified 23 novel candidate substrates for this kinase. Seven selected candidates were phosphorylated by p38 MAP kinase in vitro and in p38 MAP kinase-activated cells. This proteomic approach can be applied to any protein kinase, allowing global identification of novel substrates.
authors.affiliation: Division of Biochemistry, School of Pharmaceutical Sciences, Kitasato University, Tokyo, Japan.
authors.forename: Naoyuki
authors.initials: N
authors.lastname: Iida
authors.forename: Masayuki
authors.initials: M
authors.lastname: Fujita
authors.forename: Kohtaro
authors.initials: K
authors.lastname: Miyazawa
authors.forename: Michimoto
authors.initials: M
authors.lastname: Kobayashi
authors.forename: Seisuke
authors.initials: S
authors.lastname: Hattori
mesh_terms: D002846:Chromatography, Affinity; D057075:Enzyme Assays; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D010750:Phosphoproteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D020543:Proteome / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D057825:Two-Dimensional Difference Gel Electrophoresis; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*
chemical_list: D010750:Phosphoproteins; D020543:Proteome; D048051:p38 Mitogen-Activated Protein Kinases
keywords: 2D-DIGE; Immobilized metal affinity chromatography; In vitro kinase assay; Phosphoproteomics; p38 mitogen-activated protein kinase