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 ERK2 binding 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.

Phenotypic Characterization of a Comprehensive Set of MAPK1 /ERK2 Missense Mutants

“…Consistent with this notion, mutations expected to disrupt ERK2 protein function, including charged (arginine, aspartate, glutamate and lysine) and rigid (proline) substitutions, were the most widely enriched across all ERK2 mutant proteins (Figure 1C). Additionally, variants encoding substitutions in functionally essential ERK2 protein domains, including a subset of residues in the ATP-binding pocket, regulatory phosphorylation sites, the glycine-rich loop, the catalytic loop, the activation loop and the F-helix were universally enriched and captured a number of previously-reported LOF ERK2 mutants (Goetz et al, 2014; Madhani et al, 1997; McReynolds et al, 2016; Shin et al, 2010) (Figure 1B, Figure S2D, Table S2). We generated a composite, phenotypic score for each residue that summarizes the impact of ERK2 mutation on phenotypic behavior.…”

Interference with ERK-dimerization at the nucleocytosolic interface targets pathological ERK1/2 signaling without cardiotoxic side-effects

“…The strong effect of EDI on gene regulation and thus cardiac remodeling as well as cancer cell proliferation may be due to direct or indirect activation of nuclear transcription factors by ERK1/2, but also kinase-independent ERK1/2 effects such as a direct binding of ERK to oligonucleotides, as shown for ERK2, activated ERK2, and ERK2 T188D (ref. 52). In line with the protection of cardiomyocyte cell death, EDI further preserved the functional integrity of cardiomyocyte mitochondria, i.e.…”

The Transcription Factor ETV5 Mediates BRAFV600E-Induced Proliferation and TWIST1 Expression in Papillary Thyroid Cancer Cells

“…Although PTC tumors and KTC1 cell proliferation following BRAF inhibition depend on both MAPK pathway and reactivated PI3K pathway [16] , [17] , [19] , we now demonstrate that ETV5 expression in PTC cells mainly depends on the MAPK pathway and ERK1/2 phosphorylation. It is presently not known if ERK1/2, a kinase with DNA binding ability [59] , directly associates with ETV5 or is inducing ETV5 expression through activation/phosphorylation of intermediates. Patients with advanced BRAF (V600E) PTCs respond well to vemurafenib and dabrafenib, which has been studied in clinical trials [60] .…”