Abstract
Steroidogenic factor 1 (NR5A1/SF1) is a well-known master regulator in controlling adrenal and sexual development, as well as regulating numerous genes involved in adrenal and gonadal steroidogenesis. Several studies including ours have demonstrated that NR5A1 can be SUMOylated on lysine 194 (K194, the major site) and lysine 119 (K119, the minor site), and the cycle of SUMOylation regulates NR5A1’s transcriptional activity. An extended consensus negatively charged amino acid-dependent SUMOylation motif (NDSM) enhances the specificity of substrate modification by SUMO has been reported; however, the mechanism of NDSM for NR5A1 remains to be clarified. In this study, we investigated the functional significance of the acidic residue located downstream from the core consensus SUMO site of NR5A1. Here we report that E199A (glutamic acid was replaced with alanine) of NR5A1 reduced, but not completely abolished, its SUMOylation level. We next characterized the functional role of NR5A1 E199A on target gene expression and protein levels. We found that E199A alone, as well as combination with K194R, increased Mc2r and Cyp19a1 reporter activities. Moreover, E199A alone as well as combination with K194R enhanced NR5A1-mediated STAR protein levels in mouse adrenocortical cancer Y1 cells. We also observed that E199A increased interaction of NR5A1 with CDK7 and SRC1. Overall, we provide the evidence that the acidic residue (E199) located downstream from the core consensus SUMO site of NR5A1 is, at least in part, required for SUMOylation of NR5A1 and for its mediated target gene and protein expression.
Highlights
Steroidogenic factor 1 (NR5A1/SF1) is a nuclear receptor and plays a crucial role in the regulation of steroid hormone biosynthesis, as well as in the endocrine development and differentiation of both the gonads and adrenal glands [1,2,3,4]
We demonstrate that the acidic residue (E199) located downstream from the core consensus small ubiquitin-related modifier (SUMO) site of NR5A1 is, at least in part, required for SUMOylation of NR5A1 and for NR5A1-mediated target gene and protein expression
As negatively charged amino acid-dependent SUMOylation motif (NDSM) has been shown to play an important role in regulation of SUMOylation and NR5A1 has one acidic residue, E199, downstream from the consensus SUMO (K194) site (Figure 1A), we decided to examine the role of E199 on SUMO modification of NR5A1
Summary
Steroidogenic factor 1 (NR5A1/SF1) is a nuclear receptor and plays a crucial role in the regulation of steroid hormone biosynthesis, as well as in the endocrine development and differentiation of both the gonads and adrenal glands [1,2,3,4]. Recent studies on SUMO substrates suggest that SUMOylation is regulated by the acidic residues located downstream from the core consensus SUMO site of the target proteins [50]. We demonstrate that the acidic residue (E199) located downstream from the core consensus SUMO site of NR5A1 is, at least in part, required for SUMOylation of NR5A1 and for NR5A1-mediated target gene and protein expression
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