Modulation of Testicular Receptor 4 Activity by Mitogen-activated Protein Kinase-mediated Phosphorylation

Abstract

Testicular receptor 4 (TR4) is an orphan member of the nuclear receptor superfamily. Despite the lack of identified ligands, its functional role has been demonstrated both in animals and cell cultures. However, it remains unclear how the biological activity of TR4 is regulated without specific ligands. In this study, we showed that in the absence of specific ligands the activity of TR4 could be modulated by mitogen-activated protein kinase (MAPK)-mediated phosphorylation of its activation function 1 (AF-1) domain. A mass spectrometry-based proteome analysis of TR4 expressed in insect cells revealed three phosphorylation sites in its AF-1 domain, specifically on Ser Testicular receptor 4 (TR4)1 (also known as TAK1 and Nr2c2) remains an orphan member among the nuclear receptor superfamily (1, 2). Based upon sequence alignment, TR4 is closely related to some other orphan receptors such as retinoid X receptors, chicken ovalbumin upstream promotertranscription factor, and hepatocyte nuclear factor 4 (3). The expression of TR4 was found to be ubiquitous in both embryonic and adult stages (4 -6). Due to the lack of identified ligands, its physiological roles have been speculated. However, recent gene knock-out studies revealed its potential roles in animal behavior, coordination, and reproduction (7-9).In classical studies, it was shown that TR4 could bind to direct repeat (DR) AGGTCA with variable spacer nucleotides in the promoters of putative target genes (10 -13). These target genes could be activated or repressed. Among the potential target sequences, the DR1 sequence of the apolipoprotein E/C-I/C-II gene cluster was confirmed in the TR4 gene knock-out studies (13). In addition, TR4 was also shown to be an important regulator of other nuclear receptor signaling pathways, such as in pathways involving retinoic acid receptor, retinoid X receptor, peroxisome proliferator-activated receptor (PPAR), vitamin D 3 receptor, thyroid hormone receptor, estrogen receptor, and another closely related orphan member, TR2 (14). Together these studies suggested TR4 as an important member of nuclear receptors. Despite the widely demonstrated transcriptional regulatory activity of TR4, it remains unclear how the activity of TR4, as an activator or a repressor, is triggered without the involvement of specific ligands.Our recent proteomics studies of another orphan receptor, TR2, revealed interesting regulatory mechanisms mediated by receptor phosphorylation that could modulate its biological activities (15, 16). We thus set out to determine whether TR4 could also be regulated by protein modification such as phosphorylation. A systematic mass spectrometry-based proteomics analysis of TR4 was initiated by examining modifications of TR4 protein expressed in insect cells, a widely used eukaryotic system for protein expression. In this endeavor, we identified three mitogen-activated protein kinase-mediated phosphorylation sites on the activation function 1 (AF-1) domain of TR4. To validate the biological effects of these modified residue...

Conjugated Linoleic Acid Negatively Regulates TR4 Activity in 3T3-L1 Adipocytes

“…It has been reported that various signals such as MAPK/extracellular signal-related kinase (ERK) plays an important role as a signaling mediator for various nuclear receptors including TR4 (Chen et al, 2005;Huq et al, 2006). Early study showed that TR4 can be phosphorylated by MAPK and this phosphorylation reduces TR4 activity (Huq et al, 2006). Interestingly, CLA is involved in various signaling pathways including MAPK/ ERK (Chung et al, 2005).…”

“…However, we found that CLA consistently inhibited TR4 transcriptional activity when we used different reporter genes fused with different DR1 sequences, indicating that CLA is a functional modulator of TR4 activity. It has been reported that various signals such as MAPK/extracellular signal-related kinase (ERK) plays an important role as a signaling mediator for various nuclear receptors including TR4 (Chen et al, 2005;Huq et al, 2006). Early study showed that TR4 can be phosphorylated by MAPK and this phosphorylation reduces TR4 activity (Huq et al, 2006).…”

Differential roles of PPARγ vs TR4 in prostate cancer and metabolic diseases

“…TR4 functions as a repressor when MAPK mediates the phosphorylation of the AF1 domain through the preferential recruitment of corepressor RIP140. In contrast, TR4 functions as an activator when its AF1 domain is dephosphorylated due to its selective recruitment of the coactivator PCAF (Huq et al 2006). However, the potential physiological consequences of these MAPK phosphorylations of TR4 remain unclear.…”

“…TR4 could also be phosphorylated by MAPK at its AF1 domain in the absence of specific ligands (Huq et al 2006). MAPK activation by anisomycin causes a nearly complete loss of TR4 transactivation.…”

“…In contrast, PGC-1 utilizes a domain rich in proline residues to bind to a region that overlaps the DNA-binding and hinge region of PPARg (Puigserver et al 1998). There had been few identified coactivators for TR4 until Huq et al (2006) identified a classic steroid receptor family coactivator, p300/CREB-binding protein-associated factor (PCAF). Although originally characterized as a histone acetyltransferase, PCAF also acetylates non-histone transcriptionrelated proteins like p53 (Schiltz & Nakatani 2000).…”