Structural and functional characterization of a phosphatase domain within yeast general transcription factor IIIC

Abstract

Saccharomyces cerevisiae π55, a subunit of the RNA polymerase III-specific general transcription factor TFIIIC, comprises an N-terminal histidine phosphatase domain (π55-HPD) whose catalytic activity and cellular function is poorly understood. We solved the crystal structures of π55-HPD and its closely related paralogue Huf and used in silico docking methods to identify phosphoserine- and phosphotyrosine-containing peptides as possible substrates that were subsequently validated using in vitro phosphatase assays. A comparative phosphoproteomic study identified additional phosphopeptides as possible targets that show the involvement of these two phosphatases in the regulation of a variety of cellular functions. Our results identify π55-HPD and Huf as bona fide protein phosphatases, characterize their substrate specificities, and provide a small set of regulated phosphosite targets in vivo. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.