UCSF

Steroidogenic Factor-1 (SF-1) is a constitutively active nuclear hormone receptor, thus its post-translational modifications play an even more important role in its regulation. SF-1 is sumoylated at two sites, K119 and K194 of which the former has been shown to determine SF-1 binding specificity to sumo-sensitive response elements. The basic enzymology behind sumoylation of SF-1 K119 sumoylation is not fully understood nor optimized with the reaction taking well over 8 hours while still not reaching completion. Similarly, sensitive visualization assays are not currently available in order to facilitate kinetic studies of this in vitro reaction. This graduate work focuses on sumoylation of the SF-1 DBD with the goal of developing a more robust and sensitive in vitro system in order to assay new potential SF-1 binding sequences and apply the revamped protocol to other sumoylation substrates. Our work demonstrates that the rate-limiting step of the enzymatic reaction is the loading of the E2 enzyme, Ubc9, and pre-loading this enzyme prior to incubation with substrate dramatically enhances the rate of sumoylation. Finally, we attempt to determine how the putative E3 ligase, PIASγ, functions to enhance SF-1 sumoylation in vitro.