Transcriptional ERRγ2-mediated activation is regulated by sentrin-specific proteases

Modification with SUMOs (small ubiquitin-related modifiers) has emerged as an important means of regulating the activity of transcription factors, often by repressing their activity. The ERRγ [oestrogen receptor-related receptor γ; ERR3 or NR3B3 (nuclear receptor subfamily 3, group B, gene3)] is a constitutively active orphan nuclear receptor. A PDSM, (phosphorylation-dependent sumoylation motif) is located in the close vicinity of the N-terminally located ERRγ2-specific AF-1 (activation function-1). Its function can be replaced by an NDSM (negatively charged amino acid-dependent sumoylation motif). A mutational analysis reveals that ERRγ2 activity is modulated through sumoylation of a lysine residue at position 40, which in turn is regulated by phosphorylation. Phosphorylation at the +5 position relative to the sumoylation target is directly visualized by a high-resolution EMSA (electrophoretic mobility-shift assay). Sumoylation represses the activity of ERRγ both with and without forced expression of the PGC-1β (peroxisome-proliferator-activated receptor γ co-activator-1β). Fusion proteins of a heterologous DNA-binding domain with the ERRγ2 N-terminus demonstrate the function of the PDSM as the RF-1 (repression function-1) for the neighbouring AF-1. De-repression is achieved by co-expression of sentrin/SENP (sentrin-specific protease) family members. Together, our results demonstrate reversible phosphorylation-dependent sumoylation as a means to regulate the activity of an orphan nuclear receptor.