PHOSPHORYLATION ENHANCES THE TARGET GENE SEQUENCE-DEPENDENT DIMERIZATION OF THYROID-HORMONE RECEPTOR WITH RETINOID-X RECEPTOR

To understand the molecular basis of the phosphorylation-enhanced tran scriptional activity of human thyroid hormone nuclear receptor subtype beta 1 (hTR beta 1), we studied the effect of phosphorylation on the interaction of hTR beta 1 with the retinoid X receptor beta (RXR beta) . In vitro, the extent of hTR beta 1.RXR beta heterodimer bound to var ious thyroid hormone response elements (TREs) was compared before and after phosphorylation of hTR beta 1. Without phosphorylation, hTR beta 1.RXR beta heterodimer was barely detectable under the experimental c onditions. After phosphorylation of hTR beta 1, heterodimer bound to ( i) the chicken lysozyme gene TRE, (ii) a TRE consisting of direct repe ats of half-site binding moths separated by four gaps, and (iii) a pal indromic TRE was enhanced by approximately 10-, 7-, and 6-fold, respec tively. The effect of phosphorylation on hTR beta 1.RXR beta heterodim erization was reversible. Dephosphorylation of the phosphorylated hTR beta 1 by alkaline phosphatase led to loss of the ability of hTR beta 1 to form a heterodimer with RXR beta in either the absence or the pre sence of DNA. These results indicate that the heterodimerization is en hanced by phosphorylation, To evaluate the effect of phosphorylation o n the interaction of hTR beta 1 with RXR beta in vivo, we cotransfecte d hTR beta 1, RXR beta and TRE-chloramphenicol acetyltransferase (CAT) expression plasmids into CV-1 cells. CAT activity was assessed in the presence or absence of okadaic acid. Okadaic acid is a potent inhibit or of phosphatases 1 and 2A and increases the in vivo phosphorylation of hTR beta 1 by approximate to 10-fold. Using the CAT reporter gene u nder control of the TRE from the mafic enzyme gene, we found that RXR beta increased the okadaic acid-enhanced hTR beta 1-mediated CAT activ ity by 2- to 3-fold in the presence of 3,3',5-triiodo-L-thyronine, How ever, 9-cis-retinoic acid did not enhance the effect of okadaic acid. Our results indicate that phosphorylation is essential for the interac tion of hTR beta 1 with RXR beta. Thus, phosphorylation plays a pivota l role in the gene-regulating activity of hTR beta 1.