Journal
BIOCHEMISTRY
Volume 42, Issue 24, Pages 7571-7579Publisher
AMER CHEMICAL SOC
DOI: 10.1021/bi0273967
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In CV-1 cells transfected with wild-type (wt) nuclear thyroid hormone receptor TRbeta1 (TR), L-thyroxine (T-4) causes activation and nuclear translocation of mitogen-activated protein kinase (MAPK, ERK1/2). co-immunoprecipitation of MAPK and TR, and MAPK-dependent serine phosphorylation of TR. In the present studies, we have identified (1) the likely site of TR serine phosphorylation in the TR DNA-binding domain (DBD) by T-4-activated MAPK, (2) the site of MAPK docking on TR induced by T-4, and (3) functional consequences of TR docking site and serine phosphorylation site mutations on co-repressor and co-activator binding and on transcriptional activation by wt and mutant receptors in T-4-treated cells. Plasmids containing TRwt, serine 142-substituted TR (TRS142A or TRS142E), TRK128A, TRR132A, or TRR133A were transfected into CV-1 cells, and the cells were treated with 10(-7) M T-4 for 30 min. Activated MAPK was present in nuclear fractions of all T-4-treated cells and co-immunoprecipitated prominently with TRwt, TRS142A, and TRS142E. TRK128A complexing with activated MAPK was minimally detectable, but no association of MAPK with TRR132A or TRR133A was seen in cells treated with T-4. Serine phosphorylation of TRwt, but not of any mutants, occurred with T-4. In in vitro phosphorylation studies, constitutively activated MAPK phosphorylated only TRwt. We concluded that serine 142 of the TR DBD is the likely site of phosphorylation by T-4-activated MAPK and that the docking site on TR for activated MAPK includes residues 128-133 (KGFFRR), a basic amino acid-enriched motif novel for MAPK substrates. TR mutations in the proposed MAPK docking domain and at residue 142 modulated T-4-conditioned shedding of co-repressor and recruitment of co-activator proteins by the receptor, and they altered transcriptional activity of TR in a thyroid hormone response element-luciferase reporter assay.
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