Analysis of Androgen Receptor SUMOylation

Androgen receptor (AR) is a ligand-controlled transcription factor that is deregulated and therefore targeted in prostate cancer. In addition to androgens, AR is regulated by post-translational modifications (PTMs). SUMOylation, conjugation of small ubiquitin-related modifier (SUMO) protein 1, 2, or 3, is a bulky PTM regulating several important physiological processes. We have shown that AR is modified by SUMO-1 at two conserved lysine residues in its N-terminal domain. This agonist-enhanced modification represses the transcriptional activity of the receptor in a reversible and target gene-selective fashion. Acceptor sites for SUMOs are also found in several other nuclear receptors. Since the cellular steady-state level of SUMO modifications of most substrates, including AR, is very low, transfection- and SUMO overexpression-based protocols are often needed to render the modifications clearly detectable. This chapter describes protocols for analyzing AR SUMOylation in cultured cells by immunoblotting, gel mobility shift assays, and immunoprecipitation. These methodologies are generally applicable for determining whether a particular protein is SUMOylated and for identifying the lysine residue(s) modified.