ERK phosphorylation of MED14 in promoter complexes during mitogen-induced gene activation by Elk-1

Matthew D Galbraith,Li Li,Joaquin M Espinosa,Hongmei Zhang,Peter E Shaw,Samuel J Shelton,Janice Saxton

doi:10.1093/nar/gkt837

Abstract

The ETS domain transcription factor Elk-1 stimulates expression of immediate early genes (IEGs) in response to mitogens. These events require phosphorylation of Elk-1 by extracellular signal-regulated kinase (ERK) and phosphorylation-dependent interaction of Elk-1 with co-activators, including histone acetyltransferases and the Mediator complex. Elk-1 also recruits ERK to the promoters of its target genes, suggesting that ERK phosphorylates additional substrates in transcription complexes at mitogen-responsive promoters. Here we report that MED14, a core subunit of the Mediator, is a bona fide ERK substrate and identify serine 986 (S986) within a serine-proline rich region of MED14 as the major ERK phosphorylation site. Mitogens induced phosphorylation of MED14 on S986 at IEG promoters; RNAi knockdown of MED14 reduced CDK8 and RNA polymerase II (RNAPII) recruitment, RNAPII C-terminal domain phosphorylation and impaired activation of IEG transcription. A single alanine substitution at S986 reduced activation of an E26 (ETS)-responsive reporter by oncogenic Ras and mitogen-induced, Elk-1-dependent transcription, whereas activities of other transcriptional activators were unaffected. We also demonstrate that Elk-1 can associate with MED14 independently of MED23, which may facilitate phosphorylation of MED14 by ERK to impart a positive and selective impact on mitogen-responsive gene expression.

Highlights

The specific and temporal co-ordination of gene expression is a fundamental process of cell-based life with patterns of gene expression controlling proliferation, differentiation and cell death
We have shown that extracellular signalregulated kinase (ERK) is recruited to the promoters of immediate early genes (IEGs) by Elk-1 and inferred that ERK may phosphorylate additional substrates in pre-initiation complexes (PICs) assembled on IEG promoters [19]
Our studies have revealed that active ERK is recruited by Elk-1 into PICs at mitogen-responsive promoters and phosphorylates MED14 on serine 986 (S986)

Summary

Introduction

The specific and temporal co-ordination of gene expression is a fundamental process of cell-based life with patterns of gene expression controlling proliferation, differentiation and cell death. Immediate early gene (IEG) expression has revealed how crucial regulatory events revolve around the interface between pathway-specific transcription factors and components of the transcription machinery, involving a plethora of protein interactions and modifications [1,2]. The ternary complex factor (TCF) Elk-1, an E26 (ETS) transcription factor family member, activates transcription of multiple IEGs in response to mitogens, a process initiated upon Elk-1 phosphorylation by the mitogenactivated protein kinases (MAPKs) extracellular signalregulated kinase (ERK) and ERK2 (ERK) [3,4,5]. Phosphorylation increases the affinity of Elk-1 for Serum Response Element (SRE)-containing promoters, which it engages in a complex with Serum Response Factor (SRF) [6], and correlates with desumoylation and derepression of Elk-1 to unleash its trans-activation potential [7,8]. Biochemical and structural analyses have revealed the mammalian Mediator to consist of up to 30 subunits with a mass of $1.5 MD in a head-body-tail arrangement plus auxiliary Cyclin-

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Conclusion