FLI-1 Functionally Interacts with PIASxα, a Member of the PIAS E3 SUMO Ligase Family

doi:10.1074/jbc.M502938200

FLI-1 Functionally Interacts with PIASxα, a Member of the PIAS E3 SUMO Ligase Family*

^‡Institut Curie, CNRS UMR 146, 91405 Orsay, France, the ^§Institute of Biomedical Sciences, Academia Sinica, 11529 Taipei, Taiwan, the ^¶Department of Medical Biochemistry, University of Kuopio, FIN 70211 Kuopio, Finland, the ^∥Institute of Biomedicine, Biomedicum Helsinki, University of Helsinki, FIN 00014 Helsinki, Finland, and the ^**Institut Pasteur, INSERM U579, 75015 Paris, France

3 To whom correspondence should be addressed: Institut Curie, Centre Universitaire, CNRS UMR 146, Bat. 110, 91405 Orsay, France. Tel.: 33-1-6986-3152; Fax: 33-1-6907-4525; E-mail: Jacques.Ghysdael{at}curie.u-psud.fr.

Abstract

FLI-1 is a transcription factor of the ETS family that is involved in several developmental processes and that becomes oncogenic when overexpressed or mutated. As the functional regulators of FLI-1 are largely unknown, we performed a yeast two-hybrid screen with FLI-1 and identified the SUMO E3 ligase PIASxα/ARIP3 as a novel in vitro and in vivo binding partner of FLI-1. This interaction involved the ETS domain of FLI-1 and required the integrity of the SAP domain of PIASxα/ARIP3. SUMO-1 and Ubc9, the ubiquitin carrier protein component in the sumoylation pathway, were also identified as interactors of FLI-1. Both PIASxα/ARIP3 and the closely related PIASxβ isoform specifically enhanced sumoylation of FLI-1 at Lys⁶⁷, located in its N-terminal activation domain. PIASxα/ARIP3 relocalized the normally nuclear but diffusely distributed FLI-1 protein to PIASxα nuclear bodies and repressed FLI-1 transcriptional activation as assessed using different ETS-binding site-dependent promoters and different cell systems. PIASxα repressive activity was independent of sumoylation and did not result from inhibition of FLI-1 DNA-binding activity. Analysis of the properties of a series of ARIP3 mutants showed that the repressive properties of PIASxα/ARIP3 require its physical interaction with FLI-1, identifying PIASxα as a novel corepressor of FLI-1.

Footnotes

↵4 The abbreviations used are: EBS, ETS-binding site; STAT, signal transducer and activator of transcription; E3, ubiquitin-protein isopeptide ligase; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; ERK2, extracellular signal-regulated kinase-2; HA, hemagglutinin; Gal4AD, Gal4 activation domain; GST, glutathione S-transferase; wt, wild-type; Luc, luciferase; m, mouse; PBS, phosphate-buffered saline.
↵5 E. van den Akker, S. Ano, and J. Ghysdael, unpublished data.
↵* This work was supported in part by CNRS, the Institut Curie, the Ligue Nationale contre le Cancer (Equipe Labellisée La Ligue 2004), and the Association for International Cancer Research (to J. G.) and by the Academy of Finland (to J. J. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵1 Both authors contributed equally to this work.
↵2 Supported by a predoctoral fellowship from the Ministère de l' Education Nationale et de la Recherche and the Association pour la Recherche contre le Cancer.
- Received March 17, 2005.
- Revision received August 23, 2005.
The American Society for Biochemistry and Molecular Biology, Inc.