Activation of TRAP/Mediator Subunit TRAP220/Med1 Is Regulated by Mitogen-Activated Protein Kinase-Dependent Phosphorylation

Abstract

The TRAP/Mediator coactivator complex serves as a molecular bridge between gene-specific activators and RNA polymerase II. TRAP220/Med1 is a key component of TRAP/Mediator that targets the complex to nuclear hormone receptors and other types of activators. We show here that human TRAP220/Med1 is a specific substrate for extracellular signal-regulated kinase (ERK) of the mitogen-activated protein kinase (MAPK) family. We demonstrate that ERK phosphorylates TRAP220/Med1 in vivo at two specific sites: threonine 1032 and threonine 1457. Importantly, we found that ERK phosphorylation significantly increases the stability and half-life of TRAP220/Med1 in vivo and correlates with increased thyroid hormone receptor-dependent transcription. Furthermore, ERK phosphorylates TRAP220/Med1 in a cell cycle-dependent manner, resulting in peak levels of expression during the G₂/M phase of the cell cycle. ERK phosphorylation of ectopic TRAP220/Med1 also triggered shuttling into the nucleolus, thus suggesting that ERK may regulate TRAP220/Med1 subnuclear localization. Finally, we observed that ERK phosphorylation of TRAP220/Med1 stimulates its intrinsic transcriptional coactivation activity. We propose that ERK-mediated phosphorylation is a regulatory mechanism that controls TRAP220/Med1 expression levels and modulates its functional activity.

ACKNOWLEDGMENTS

We thank Natalie Ahn and Melanie Cobb for providing the MKK and ERK expression constructs; Chih-Cheng Tsai for providing reagents and assistance with the fluorescent microscopy; Nagarajan Selvamarugan, Don Chen, Huizhou Fan, and Madesh Belakavadi for providing reagents and critically reading the manuscript; Ravi Vijayvargia for providing baculovirus-expressed TRAP220/Med1 protein; and Nicola Partridge for providing assistance with the real-time PCR studies.

This work was funded by NIH grant DK054030 awarded to J.D.F.