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.
Highlights
Urokinase plasminogen activator1 is a serine protease and a key enzyme in the proteolytic cascade activating plasminogen and thereby leading to degradation of the extracellular matrix (Blasi and Verde, 1990)
Electrophoretic Mobility Shift Assays (EMSA) showed that the u-cooperation mediating (COM) region formed four retarded bands with a nuclear extract of HepG2 cells, uPA enhancer factors (UEF) 1 to 4 (Nerlov et al, 1992)
The activity of the uPA enhancer is dependent on the integrity of each of two protein binding sites: the upstream combined PEA3-AP-1 site and the downstream consensus AP-1 site (Rørth et al, 1990; Nerlov et al, 1991)
Summary
Urokinase plasminogen activator (uPA) is a serine protease and a key enzyme in the proteolytic cascade activating plasminogen and thereby leading to degradation of the extracellular matrix (Blasi and Verde, 1990). Two elements within the enhancer have been shown to be essential for its activity: a combined PEA3/AP-1 site similar to that of the collagenase gene, and a 74-base pair downstream consensus AP-1 site (Gutman et al, 1990; Nerlov et al, 1991; Rørth et al, 1990) (see Fig. 1A) These sites are important for both basal level and induced enhancer activity (Cassady et al, 1991; Nerlov et al, 1991; Rørth et al, 1990) and appear to cooperate to activate transcription. The activity of the NIP sites in these promoters appears to be that of modifying the efficiency of elements responding to the transcription-inducing signals In this view, and because of the sequence homologies, one or more UEF factor of the uPA enhancer might correspond to the NIPbinding proteins. In this paper we have purified and preliminarily characterized UEF3 and shown that it recognizes a TG(A/G)CAG sequence, common to both the uPA COM region and the NIP element
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
