SUMO-1 Modification of PIASy, an E3 Ligase, Is Necessary for PIASy-Dependent Activation of Tcf-4

Abstract

We have previously shown that modification of Tcf-4, a transcription factor in the Wnt pathway, with SUMO by PIASy, a SUMO E3 ligase, enhances its transcriptional activity. Since PIASy itself was also modified with SUMO-1, we studied the role of sumoylation of PIASy in the regulation of Tcf-4. Lys³⁵ was found to be a sumoylation site of PIASy. PIASy^K35R, in which Lys³⁵ was mutated to Arg, did not enhance sumoylation of Tcf-4, although this PIASy mutant did not lose the ligase activity of sumoylation for other proteins. Wild-type PIASy and PIASy^K35R showed a distinct distribution in the nucleus, although both were colocalized with Tcf-4. Promyelocytic leukemia protein, which is involved in transcriptional regulation, was associated with PIASy^K35R more frequently than wild-type PIASy in the nucleus. PIASy^K35R could not stimulate the transcriptional activity of Tcf-4 under the conditions in which wild-type PIASy enhanced it. Conjugation of SUMO-1 to the amino terminus of PIASy^K35R neither enhanced sumoylation of Tcf-4 nor stimulated the transcriptional activity of Tcf-4. These results suggest that sumoylation of Lys³⁵ in PIASy determines the nuclear localization of PIASy and that it is necessary for PIASy-dependent sumoylation and transcriptional activation of Tcf-4.

ACKNOWLEDGMENTS

We thank H. Yasuda, K. Shuai, A. Nagafuchi, T. Akiyama, and H. Clevers for donating proteins and plasmids. We thank K. Igarashi, S. Tashiro, and J. Sun (Hiroshima University) for helpful discussions.

This study was supported by Grants-in-Aid for Scientific Research and for Scientific Research on priority areas from the Ministry of Education, Science, and Culture of Japan (in 2002, 2003, and 2004) and by grants from the Yamanouchi Foundation for Research on Metabolic Disorders (in 2002 and 2003) and the Uehara Memorial Foundation (in 2003).