title: A Phos-tag SDS-PAGE method that effectively uses phosphoproteomic data for profiling the phosphorylation dynamics of MEK1.
abstract: MEK1, an essential component of the mitogen-activated protein kinase (MAPK) pathway, is phosphorylated during activation of the pathway; 12 phosphorylation sites have been identified in human MEK1 by MS-based phosphoproteomic methods. By using Phos-tag SDS-PAGE, we found that multiple variants of MEK1 with different phosphorylation states are constitutively present in typical human cells. The Phos-tag-based strategy, which makes effective use of existing information on the location of phosphorylation sites, permits quantitative time-course profiling of MEK1 phosphospecies in their respective phosphorylation states. By subsequent immunoblotting with an anti-HaloTag antibody, we analyzed a HaloTag-fused MEK1 protein and 12 potential phosphorylation-site-directed mutants of the protein transiently expressed in HEK 293 cells. This strategy revealed that MEK1 is constitutively and mainly phosphorylated at the Thr-292, Ser-298, Thr-386, and Thr-388 residues in vivo, and that combinations of phosphorylations at these four residues produce at least six phosphorylated variants of MEK1. Like the levels of phosphorylation of the Ser-218 and Ser-222 residues by RAF1, which have been well studied, the phosphorylation statuses of Thr-292, Ser-298, Thr-386, and Thr-388 residues vary widely during activation and deactivation of the MAPK pathway. Furthermore, we demonstrated inhibitor-specific profiling of MEK1 phosphospecies by using three MEK inhibitors: TAK-733, PD98059, and U0126.
authors.affiliation: Department of Functional Molecular Science, Institute of Biomedical and Health Sciences, Hiroshima University, Japan.
authors.forename: Eiji
authors.initials: E
authors.lastname: Kinoshita
authors.affiliation: Department of Functional Molecular Science, Institute of Biomedical and Health Sciences, Hiroshima University, Japan.
authors.forename: Emiko
authors.initials: E
authors.lastname: Kinoshita-Kikuta
authors.affiliation: Division of Cell Signaling and Molecular Medicine, Institute of Medical Science, The University of Tokyo, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Kubota
authors.affiliation: Division of Cell Signaling and Molecular Medicine, Institute of Medical Science, The University of Tokyo, Japan.
authors.forename: Mutsuhiro
authors.initials: M
authors.lastname: Takekawa
authors.affiliation: Department of Functional Molecular Science, Institute of Biomedical and Health Sciences, Hiroshima University, Japan.
authors.forename: Tohru
authors.initials: T
authors.lastname: Koike
mesh_terms: D000596:Amino Acids / Q000032:analysis / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004815:Epidermal Growth Factor / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D048369:MAP Kinase Kinase 1 / Q000737:chemistry / Q000378:metabolism*; D020935:MAP Kinase Signaling System; D010766:Phosphorylation; D040901:Proteomics / Q000379:methods*
chemical_list: D000596:Amino Acids; D004815:Epidermal Growth Factor; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human
keywords: HaloTag; MAPK pathway; MEK1; Phos-tag SDS-PAGE; PhosphoSitePlus; Technology