Functional Dissection of the α and β Subunits of Transcription Factor PEBP2 and the Redox Susceptibility of Its DNA Binding Activity

doi:10.1074/jbc.271.51.33074

Functional Dissection of the α and β Subunits of Transcription Factor PEBP2 and the Redox Susceptibility of Its DNA Binding Activity*

From the Laboratory of Biochemistry, Department of Genetics and Molecular Biology, and the
^¶ Laboratory of Cell Regulation, Department of Viral Oncology, Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto 606, Japan

^∥To whom correspondence should be addressed. Tel.: 075-751-4018; Fax: 075-751-3992; E-mail: kshigesa{at}virus.kyoto-u.ac.jp

↵^‡ Present address: Biozentrum, Der Universitat Basel, Abteilung Zellbiologie, Klingelbergstrasse 70, CH-4056 Basel, Switzerland.

Abstract

The mouse transcription factor PEBP2 is a heterodimer of two subunits: a DNA binding subunit α and its partner subunit β. The α subunit shares a region of high homology, termed the Runt domain, with the products of the Drosophila melanogaster segmentation gene runt and the human acute myeloid leukemia-related gene AML1. To study the molecular basis for the DNA binding and heterodimerization functions of this factor, we constructed series of deletions of the α and β subunits and examined their activities by electrophoretic mobility shift and affinity column assays. The minimal functional region of the α subunit for DNA binding and dimerization was shown to coincide with the Runt domain. On the other hand, the region of the β subunit required for heterodimerization was localized to the N-terminal 135 amino acids. Furthermore, it was found that the DNA binding activity of the Runt domain is regulated by a reduction/oxidization (redox) mechanism and that its reductively activated state, which is extremely labile, is stabilized by the β subunit. These findings add a new layer to the mechanism and significance of the regulatory interplay between the two subunits of PEBP2.

Footnotes

↵^§ Supported by a research fellowship from the Japan Society for the Promotion of Science for Young Scientists. Present address: Dept. of Molecular Biology, Massachusetts General Hospital, 50 Blossom St., Boston, MA 02214.
↵* This study was supported in part by Ministry of Education, Science, Culture and Sports of Japan Research Grants 06280215 and 07272221 (to K. S.) and by a grant from the Human Frontier Science Program Organization. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

Ni-NTA

nickel nitrilotriacetic

DTT

dithiothreitol

PAGE

polyacrylamide gel electrophoresis

EMSA

electrophoretic mobility shift assay

DHFR

dihydrofolate reductase

RD

Runt domain

ADF

adult T cell leukemia-derived factor.
↵² Y. Akamatsu, unpublished observation.
↵³ Y. Akamatsu, unpublished data.
↵⁴ T. Ohno, Y. Akamatsu, J. Yodoi, and K. Shigesada, unpublished observation.
- Received July 22, 1996.
- Revision received October 4, 1996.