Structural characterization of the PIT-1/ETS-1 interaction: PIT-1 phosphorylation regulates PIT-1/ETS-1 binding

doi:10.1073/pnas.192693499

Structural characterization of the PIT-1/ETS-1 interaction: PIT-1 phosphorylation regulates PIT-1/ETS-1 binding

^*Department of Physiological Chemistry, University Medical Center Utrecht and Center for Biomedical Genetics, Universiteitsweg 100, 3584 CG, Utrecht, The Netherlands; ^‡Department of Medicine, Biochemistry, and Molecular Genetics, University of Colorado Health Sciences Center, 4200 East 9th Avenue, Box B-151 Denver, CO 80262; and ^§Bijvoet Center for Biomolecular Research, Padualaan 8, 3584 CH, Utrecht, The Netherlands

Edited by John W. Kappler, National Jewish Medical and Research Center, Denver, CO, and approved July 26, 2002 (received for review December 21, 2001)

Abstract

The POU-domain transcription factor Pit-1 and Ets-1, a member of the ETS family of transcription factors, can associate in solution and synergistically activate the prolactin promoter by binding to a composite response element in the prolactin promoter. We mapped the minimal region of Ets-1 required for the interaction with the Pit-1 POU-homeodomain. Here, we describe a detailed NMR study of the interaction between the POU-homeodomain of Pit-1 and the minimal interacting region of Ets-1. By using heteronuclear single quantum coherence titration experiments, we were able to map exact residues on the POU-homeodomain that are involved in the interaction with this minimal Ets-1 interaction domain. By using our NMR data, we generated point mutants in the POU-homeodomain and tested their effect on the interaction with Ets-1. Our results show that phosphorylation of Pit-1 can regulate the interaction with Ets-1.

transcription factor‖POU domain‖NMR‖heteronuclear single quantum coherence‖titration

Footnotes

↵ † K.D.A. and D.L.D. contributed equally to this work.
↵ ¶ To whom reprint requests should be addressed. E-mail: p.c.vandervliet{at}med.uu.nl.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations:

PKA,

protein kinase A;

HSQC,

heteronuclear single quantum coherence