The transactivation function of the Pea3 subfamily Ets transcription factors is regulated by sumoylation.

Pea3, an Ets transcriptional factor, comprises multiple regulatory domains that affect its DNA binding and transcriptional activation. The aim of this work is to uncover the mechanism of action of negative regulatory regions flanking the transactivation domain. Mutagenesis of amino acid residues in the C-terminal negative regulatory region for transactivation revealed critical residues, including a lysine residue, K96, required for its function. Corresponding mutations in the closely related Pea3 subfamily members, Erm and Er81, also dramatically increased the transactivation capacity of their activation domains. Interestingly, all three proteins are sumoylated at this conserved lysine residue. Pea3 contains four other lysines, K222, K256, K318, and K437, embedded in a perfect SUMO consensus motif. The contribution of these lysine residues to the regulation of Pea3 activity and their sumoylation pattern was explored using a GAL4-PEA3 chimera devoid of the ETS DNA-binding domain and by analyzing the native protein. All four candidate SUMO sites included in the GAL4-PEA3 chimera were modified by sumoylation, and their simultaneous mutation dramatically increased the transactivation potential of Pea3. Similar analysis of full-length Pea3 confirmed K96, K222, and K256 as major SUMO modification sites. Collectively, these observations suggest that the activity of Pea3 and its paralogs, Erm and Er81, is negatively regulated by sumoylation.