The present study investigated human retinoid X receptor alpha (hRXR alpha) as a substrate for modification with small ubiquitin like modifier (SUMO) and how members of the protein inhibitor of activated STAT (PIAS) family may impact upon this process. In agreement with a previous study, we validate Ubc9 to facilitate SUMOylation of hRXR alpha at lysine 108 but note this modification to occur for all isoforms rather than specifically with SUMO1 and to preferentially occur with the unliganded form of hRXR alpha. SUMOylation of hRXR alpha is significantly enhanced through PIAS4-mediated activity with lysine 245 identified as a specific SUMO2 acceptor site modified in a PIAS4-dependent fashion. While individual mutations at lysine 108 or 245 modestly increase receptor activity, the combined loss of SUMOylation at both sites significantly potentiates the transcriptional responsiveness of hRXR alpha suggesting both sites may cooperate in a DNA element-dependent context. Our findings highlight combinatorial effects of SUMOylation may regulate RXR alpha-directed signalling in a gene-specific fashion. (C) 2015 Elsevier Inc. All rights reserved.