Purification and Characterization of UEF3, A Novel Factor Involved in the Regulation of the Urokinase and Other AP-1 Controlled Promoters

doi:10.1074/jbc.271.7.3822

Purification and Characterization of UEF3, A Novel Factor Involved in the Regulation of the Urokinase and Other AP-1 Controlled Promoters (*)

From the (1)Department of Genetics and Biology of Microorganisms, University of Milano, Via Celoria 26, 20135 Milano, Italy, the DIBIT (Department of Biological and Technological Research), S. Raffaele Scientific Institute, the
(2)Department of Molecular Cell Biology, University of Copenhagen, Øster Farimagsgade 2A, 1353 Copenhagen, Denmark, and the
(3)Laboratorium voor Genetica, Rijksuniversiteit Gent, B-9000 Gent, Belgium

^§To whom correspondence should be addressed:
DIBIT H.S. Raffaele, via Olgettina 60, 20132 Milan, Italy.
Tel.: 39-2-2643-4832; Fax: 39-2-2643-4844.

Abstract

Basal as well as induced transcription from the human urokinase-type plasminogen activator gene requires an enhancer containing two elements, a combined PEA3/AP-1 and a consensus AP-1 site. The integrity of each of these binding sites as well as their cooperation is required for activating transcription. The two elements are separated by a 74-base pair cooperation mediating (COM) region required for the cooperation between the transactivating sites. The COM region contains binding sites for four different unidentified urokinase-type plasminogen activator enhancer factors (UEF 1 to 4), all four required for correct COM activity. We have purified UEF3 from HeLa nuclear extracts by several chromatographic steps including DNA affinity purification. Purification and UV cross-linking data showed that UEF3 is a complex of three polypeptides (p40, p50, and p64). Amino acid sequence from one peptide of p64 was obtained, which showed no homology to other known proteins. Both crude and purified UEF3 specifically bound to the sequence TGACAG as shown by electrophoretic mobility shifts and methylation interference studies. DNA-binding specificity of purified UEF3 was identical to that of NIP, a non-characterized factor binding and regulating multiple AP-1-regulated promoters like stromelysin and interleukin-3. Thus UEF3 appears to be a general DNA-binding factor involved in modulating the transcriptional response of AP-1 containing promoters.

Footnotes

↵* This work was supported by grants of the Italian Association for Cancer Research (AIRC), Consiglio Nazionale delle Ricerche (PF ACRO), the AIDS Grant of Italian Ministry of Health, the Danish Cancer Society, and the Human Capital and Mobility Program of the European Union. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

uPA

urokinase plasminogen activator

DTT

dithiothreitol

COM

cooperation mediating region

PMSF

phenylmethylsulfonyl fluoride

UEF

uPA enhancer factor(s)

EMSA

electrophoretic mobility shift assay

PAGE

polyacrylamide gel electrophoresis

IL-3

interleukin 3

UC

upper complex, LC, lower complex.
↵²M. Palazzolo and D. De Cesare, personal communication.
↵³D. De Cesare, personal communication.
- Received August 18, 1995.
- Revision received November 28, 1995.