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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. Fil: McReynolds, Andrea C.. The University of Texas Southwestern Medical Center; Estados Unidos Fil: Karra, Aroon S.. The University of Texas Southwestern Medical Center; Estados Unidos Fil: Li, Yan. The University of Texas Southwestern Medical Center; Estados Unidos. National Institute of Neurological Disorders and Stroke; Estados Unidos Fil: Lopez, Elias Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires; Argentina Fil: Turjanski, Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires; Argentina Fil: Dioum, Elhadji. The University of Texas Southwestern Medical Center; Estados Unidos Fil: Lorenz, Kristina. Leibniz-Institut für Analytische Wissenschaften-ISAS-e.V; Alemania Fil: Zaganjor, Elma. The University of Texas Southwestern Medical Center; Estados Unidos Fil: Stippec, Steve. The University of Texas Southwestern Medical Center; Estados Unidos Fil: McGlynn, Kathleen. The University of Texas Southwestern Medical Center; Estados Unidos Fil: Earnest, Svetlana. The University of Texas Southwestern Medical Center; Estados Unidos Fil: Cobb, Melanie H.. The University of Texas Southwestern Medical Center; Estados Unidos