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 G 2 /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.Mediator is a multisubunit transcriptional coactivator complex evolutionarily conserved from yeasts to mammals that serves as a functional interface between DNA-bound transcription factors and RNA polymerase II (pol II) (5, 32, 39). In humans, the complex was originally isolated as a thyroid hormone receptor (TR)-associated protein (TRAP) complex that facilitates ligand-dependent gene expression by TR in vitro (12) and was thus termed TRAP/Mediator. More recent studies have established TRAP/Mediator and other highly related human Mediator complexes (ARC, CRSP, PC2, and DRIP) as essential coactivators for a broad range of nuclear hormone receptors (NRs) and other types of transcriptional activators, including SREBP, p53, NF-B, VP16, and E1A (reviewed in references 28, 32, 44, and 53). In contrast to coactivators that function by covalently modifying histones (56) or rearranging higher-ordered chromatin (3), TRAP/Mediator appears to function by facilitating the recruitment of RNA pol II and by subsequently activating the basal transcription apparatus (4, 60). TRAP220/Med1 (also termed PBP, ARC/DRIP205, or Med220) is a peripherally associated subunit of the human TRAP/Mediator complex (31, 54, 55) that facilitates direct ligand-dependent interactions with NRs by virtue of its two signature LXXLL motifs (45,46,67,71) shown previously in other types of coactivators to mediate NR binding (16). Indeed, liganddependent recruitment of TRAP/Mediator to NR target genes via TRAP220/Med1 is believed to facilitate an essential activation step in NR-regulated gene expression (19,34). TRAP220/Med1 gene knockout in mice is embryonic lethal (21, 72), and primary embryonic fibroblasts derived from TRAP220/Me...

Regulation of Androgen Receptor-dependent Transcription by Coactivator MED1 Is Mediated through a Newly Discovered Noncanonical Binding Motif

“…Plasmids-The pSG5-HA-MED1-wt, -C⌬454, -C⌬690, -C⌬918, -C⌬1215, -N⌬1233, -ERK mutant (T1032A and T1457A), -LXXAA(A), and -LXXAA(B) mammalian expression vectors were described previously (24,28). The pGEX-2TK-RBD, -RBD-LXXAA, -RBD1, and -RBD2 bacterial expression vectors were described previously (24).…”

“…Immunoprecipitation of Endogenous AR-Mediator-Smart pool small interfering RNA (siRNA) specific for MED1 was from Dharmacon Research as previously described (28). A scrambled siRNA smart pool (Dharmacon) was used as a control.…”

“…In Vitro Phosphorylation Assay-50 ng of purified baculovirus-expressed MED1 (bv-MED1-FL) or MED1-Erk mut (bv-MED1-Erk mut) protein was incubated with ATP (0.1 mM) plus 10 ng of ERK1 (Upstate) in kinase buffer exactly as described previously (28).…”

“…We previously discovered that mitogen-activated protein kinase ERK1/2 phosphorylates human MED1 (28). We subsequently found that ERK1/2 phosphorylation promotes MED1 association with the core Mediator complex and further enhances its ability to coactivate ligand-dependent transcription by the thyroid hormone receptor (27).…”

“…Despite its functional importance as the binding target for most NRs, MED1 is only variably associated with Mediator existing in a small subpopulation (ϳ20%) of steady-state Mediator complexes (25,26). Notably in this regard, the extracellular signal-regulated kinase 1/2 (ERK1/2) regulates MED1 activity via phosphorylation by promoting its association with Mediator and enhancing its NR-dependent transcriptional coactivator activity (27,28).…”