Thyroxine Promotes Association of Mitogen-activated Protein Kinase and Nuclear Thyroid Hormone Receptor (TR) and Causes Serine Phosphorylation of TR

Paul J Davis,Ai Shih,Hung-Yun Lin,Leon J Martino,Faith B Davis

doi:10.1074/jbc.m002560200

Abstract

Activated nongenomically by l-thyroxine (T(4)), mitogen-activated protein kinase (MAPK) complexed in 10-20 min with endogenous nuclear thyroid hormone receptor (TRbeta1 or TR) in nuclear fractions of 293T cells, resulting in serine phosphorylation of TR. Treatment of cells with the MAPK kinase inhibitor, PD 98059, prevented both T(4)-induced nuclear MAPK-TR co-immunoprecipitation and serine phosphorylation of TR. T(4) treatment caused dissociation of TR and SMRT (silencing mediator of retinoid and thyroid hormone receptor), an effect also inhibited by PD 98059 and presumptively a result of association of nuclear MAPK with TR. Transfection into CV-1 cells of TR gene constructs in which one or both zinc fingers in the TR DNA-binding domain were replaced with those from the glucocorticoid receptor localized the site of TR phosphorylation by T(4)-activated MAPK to a serine in the second zinc finger of the TR DNA-binding domain. In an in vitro cell- and hormone-free system, purified activated MAPK phosphorylated recombinant human TRbeta1 (). Thus, T(4) activates MAPK and causes MAPK-mediated serine phosphorylation of TRbeta1 and dissociation of TR and the co-repressor SMRT.

Highlights

We have demonstrated in cultured cells that L-thyroxine (T4)1 can nongenomically activate signal transduction proteins such as mitogen-activated protein kinase (MAPK) [1] and, through serine phosphorylation by MAPK, can enhance the activity of several nuclear transactivator proteins
Complexing, of thyroid hormone receptor (TR)␤1 and MAPK, nuclear fractions were prepared from 293T cell lysates of control samples and of cells treated with T4 (10Ϫ7 M) for 10 –90 min and were immunoprecipitated with antibody to the carboxyl-terminal half of the AB domain of TR␤1
There is an increase in nuclear MAPK complexed with TR␤1 immunoprecipitates that reaches a peak in 40 min (22-fold increase over control in the figure, decreasing to 12-fold in 90 min)

Summary

The abbreviations used are

T4, L-thyroxine; T3, 3,5,3Ј-triiodo-Lthyronine; tetrac, tetraiodothyroacetic acid; triac, triiodothyroacetic acid; rT3, 3,3Ј,5Ј-triiodo-L-thyronine; TR, thyroid hormone receptor ␤1; DBD, DNA-binding domain; LBD, ligand-binding domain; MAPK, mitogen-activated protein kinase; MEK, MAPK kinase; PD, PD 98059; PTU, 6-n-propyl-2-thiouracil; MBP, myelin basic protein; STAT, signal transducer and activator of transcription; PKA, cyclic AMP-dependent protein kinase; 8-Br-cAMP, 8-bromo-cyclic AMP; GR, glucocorticoid receptor; PAGE, polyacrylamide gel electrophoresis; GTP␥S, guanosine 5Ј-O-(3-thio)triphosphate. In the absence of T3, TR exists in the transcriptionally inactive (repressed) state This state is imposed by the binding to unliganded TR of the co-repressor proteins, SMRT (silencing mediator of retinoid and thyroid hormone receptors) and NCoR (nuclear co-repressor) [8]. Using a serine/threonine kinase inhibitor, H7, Jones et al [16] showed a reduction in T3-induced transcriptional activity of both TR␣1 and TR␤1. In some of these studies [12], the results were highly cell line-specific. In the study reported here, we describe a signal transduction mechanism by which T4, acting at the cell surface, promotes MAPK-dependent serine phosphorylation of TR␤1 and resultant dissociation of TR and SMRT. MAPK, and TR region of TR␤1 that is required for MAPK binding to the receptor

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION