title: Phosphorylation or Mutation of the ERK2 Activation Loop Alters Oligonucleotide Binding.
abstract: The mitogen-activated protein kinase ERK2 is able to elicit a wide range of context-specific responses to distinct stimuli, but the mechanisms underlying this versatility remain in question. Some cellular functions of ERK2 are mediated through regulation of gene expression. In addition to phosphorylating numerous transcriptional regulators, ERK2 is known to associate with chromatin and has been shown to bind oligonucleotides directly. ERK2 is activated by the upstream kinases MEK1/2, which phosphorylate both tyrosine 185 and threonine 183. ERK2 requires phosphorylation on both sites to be fully active. Some additional ERK2 phosphorylation sites have also been reported, including threonine 188. It has been suggested that this phospho form has distinct properties. We detected some ERK2 phosphorylated on T188 in bacterial preparations of ERK2 by mass spectrometry and further demonstrate that phosphomimetic substitution of this ERK2 residue impairs its kinase activity toward well-defined substrates and also affects its DNA binding. We used electrophoretic mobility shift assays with oligonucleotides derived from the insulin gene promoter and other regions to examine effects of phosphorylation and mutations on the binding of ERK2 to DNA. We show that ERK2 can bind oligonucleotides directly. Phosphorylation and mutations alter DNA binding and support the idea that signaling functions may be influenced through an alternate phosphorylation site.
authors.affiliation: Department of Pharmacology, The University of Texas Southwestern Medical Center , Dallas, Texas 75390, United States.
authors.forename: Andrea C
authors.initials: AC
authors.lastname: McReynolds
authors.affiliation: Department of Pharmacology, The University of Texas Southwestern Medical Center , Dallas, Texas 75390, United States.
authors.forename: Aroon S
authors.initials: AS
authors.lastname: Karra
authors.affiliation: Department of Pharmacology, The University of Texas Southwestern Medical Center , Dallas, Texas 75390, United States.|Protein/Peptide Sequencing Facility, National Institute of Neurological Disorders and Stroke , Bethesda, Maryland 20824, United States.
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
authors.affiliation: Laboratory of Structural Bioinformatics, Department of Chemical Biology, University of Buenos Aires , Buenos Aires, Argentina.
authors.forename: Elias Daniel
authors.initials: ED
authors.lastname: Lopez
authors.affiliation: Laboratory of Structural Bioinformatics, Department of Chemical Biology, University of Buenos Aires , Buenos Aires, Argentina.
authors.forename: Adrian G
authors.initials: AG
authors.lastname: Turjanski
authors.affiliation: Department of Pharmacology, The University of Texas Southwestern Medical Center , Dallas, Texas 75390, United States.
authors.forename: Elhadji
authors.initials: E
authors.lastname: Dioum
authors.affiliation: Leibniz-Institut für Analytische Wissenschaften-ISAS-e.V. , Dortmund, Germany.
authors.forename: Kristina
authors.initials: K
authors.lastname: Lorenz
authors.affiliation: Department of Pharmacology, The University of Texas Southwestern Medical Center , Dallas, Texas 75390, United States.
authors.forename: Elma
authors.initials: E
authors.lastname: Zaganjor
authors.affiliation: Department of Pharmacology, The University of Texas Southwestern Medical Center , Dallas, Texas 75390, United States.
authors.forename: Steve
authors.initials: S
authors.lastname: Stippec
authors.affiliation: Department of Pharmacology, The University of Texas Southwestern Medical Center , Dallas, Texas 75390, United States.
authors.forename: Kathleen
authors.initials: K
authors.lastname: McGlynn
authors.affiliation: Department of Pharmacology, The University of Texas Southwestern Medical Center , Dallas, Texas 75390, United States.
authors.forename: Svetlana
authors.initials: S
authors.lastname: Earnest
authors.affiliation: Department of Pharmacology, The University of Texas Southwestern Medical Center , Dallas, Texas 75390, United States.
authors.forename: Melanie H
authors.initials: MH
authors.lastname: Cobb
mesh_terms: D000818:Animals; D019950:Mitogen-Activated Protein Kinase 1 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D009154:Mutation / Q000502:physiology; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010766:Phosphorylation / Q000502:physiology; D011485:Protein Binding / Q000502:physiology; D017433:Protein Structure, Secondary; D051381:Rats
chemical_list: D009841:Oligonucleotides; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1