Isoform-Specific Regulation of a Steroid Hormone Nuclear Receptor by an E3 Ubiquitin Ligase inDrosophila melanogaster

Abstract

The steroid hormone 20-hydroxyecdysone (20E) regulates gene transcription through the heterodimeric nuclear receptor composed of ecdysone receptor (EcR) and Ultraspiracle (USP). The EcR gene encodes three protein isoforms-A, B1, and B2-with variant N-terminal domains that mediate tissue and developmental stage-specific responses to 20E. Ariadne-1a is a conserved member of the RING finger family of ubiquitin ligases first identified in Drosophila melanogaster. Loss-of-function mutations at key cysteines in either of the two RING finger motifs, as well as general overexpression of this enzyme, cause lethality in pupae, which suggests a requirement in metamorphosis. Here, we show that Ariadne-1a binds specifically the isoform A of EcR and ubiquitylates it. Coimmunoprecipitation experiments indicate that the full sequence of EcRA is required for this binding. Protein levels of EcRA and USP change in opposite directions when those of ARI-1a are genetically altered. This is an isoform-specific, E3-dependent regulatory mechanism for a steroid nuclear receptor. Further, qRT-PCR experiments show that the ARI-1a levels lead to the transcriptional regulation of Eip78C, Eip74EF, Eip75B, and Br-C, as well as that of EcR and usp genes. Thus, the activity of this enzyme results in the regulation of dimerizing receptors at the protein and gene transcription levels. This fine-tuned orchestration by a conserved ubiquitin ligase is required during insect metamorphosis and, likely, in other steroid hormone-controlled processes across species. STEROID hormones regulate multiple processes during development and adult life. In Drosophila, the steroid hormone 20-hydroxyecdysone (20E) triggers the transcriptional changes required for gonad development, larval molting, and the onset and completion of metamorphosis (Sliter and Gilbert 1992;Henrich et al. 1993;Carney and Bender 2000;Riddiford et al. 2000; Beckstead et al. 2005). Differentiation of imaginal structures and neuronal remodeling, two major features of metamorphosis, have been used as cellular systems to analyze the functional role of proteins that elicit morphogenetic changes at this phase of the life cycle (Schubiger et al. 2005;Brown et al. 2006;Santos et al. 2006). 20E regulates gene expression by binding to its nuclear receptor, EcR (King-Jones and Thummel 2005). To bind 20E and stimulate transcription, however, EcR must heterodimerize with Ultraspiracle (USP) to reconstitute specific activation domains (Yao et al. 1992). Different 20E levels activate transcription of different sets of genes (Champlin and Truman 1998;Li and White 2003;Schubiger et al. 2003). Like their vertebrate cognates (Chen and Evans 1995), unliganded EcR and USP act as repressors of transcription, whereas the liganded receptor stimulates expression of target genes (Tsai et al. 1999;Ghbeish et al. 2001;Schubiger et al. 2003).On the basis of sequence identities, it is considered that the mammalian orthologs of EcR are the group H of nuclear receptor subfamily 1 that include LXR and FXR, while US...

PIAS4 is associated with macro/microcephaly in the novel interstitial 19p13.3 microdeletion/microduplication syndrome

“…53 Third, PIAS4 may be involved in the WNT signaling pathway through its interaction with several transcription factors, 32,54 and WNT is important for several aspects of facial morphogenesis. Fourth, PIAS4 is an E3 ubiquitin ligase, and haploinsufficiency of E3 ubiquitin ligases in plants and Drosophila melanogaster [55][56][57] leads to organ overgrowth. Interestingly, individuals with haploinsufficiency of RNF135 or RNF125, which encode other RNF proteins, have been associated recently (via alteration of ubiquitin signaling pathways) by us and others with macrocephaly and overgrowth in patients with dysmorphic features and ID.…”

Ligand-independent requirements of steroid receptors EcR and USP for cell survival

“…6,7 Each EcR isoform is hypothesized to have specific functions based on their distinct spatial and temporal patterns and N-termini. [8][9][10] EcR and USP DNA-binding domains recognize ecdysone response sequences which are short palindromes. 11 Like its vertebrate counterparts, 12 the ligand-EcR/USP complex activates transcription, whereas the unliganded receptor act as a repressor.…”

PROTEIN PROFILES OF CHINESE WHITE WAX SCALE, Ericerus pela, AT THE MALE PUPAL STAGE BY HIGH‐THROUGHPUT PROTEOMICS

“…Three ubiquitins were identified in the E. pela proteome, including ubiquitin‐conjugating enzyme E2, ubiquitin‐like protein SMT3, and polyubiquitin. Protein degeneration is tagged by ubiquitin ligases and is crucial to many cellular processes (Gradilla et al., ). In addition, Annexin IX , which, in silkworm, is a 20E‐inducible gene that is involved in the determination of PCD timing (Li et al., ), was identified.…”