Characterization of the MN/CA 9 promoter proximal region: a role for specificity protein (SP) and activator protein 1 (AP1) factors

MN/CA IX (MN) is a tumour-associated isoenzyme of the carbonic anhydrase fa mily. Previous deletion analysis of the MN promoter established that protec ted regions (PRs) 1 and 2 are crucial for its transcriptional activity. Com puter-assisted searching indicated putative binding sites for activator pro tein (AP) 2 and specificity protein (SP) 1 transcription factors, plus a CA CCC box in PR1 and an AP1 site in PR2. PR1 produced four complexes in elect rophoretic mobility-shift assay (EMSA) with HeLa nuclear extracts. Of these , three were completely competed with the SP1 and transforming growth facto r-beta retinoblastoma control-element CACCC box (RCE) probes, whereas the A P2 probe competed against the same three complexes partially. Supershift EM SA identified SP I in the complex 1 and SP3 in the complexes 2 and 4. Point mutations in the SP1 site abrogated the PR1 function, while mutations affe cting the overlapping CACCC box/AP2 site in PR1 had minor effect on MN prom oter activity. Block-replaced MN promoter mutants that had a consensus bind ing site (SP1 or AP2) or the RCE in place of PR I demonstrated the stringen t selectivity of the PR1 position as only the SP1 mutant reconstituted the MN promoter activity. The consensus SP1 probe generated the same SP1 and SP 3 complexes as PR1 in EMSA; therefore we conclude that SP activity is both necessary and sufficient in the PR1 position. The critical role of AP1 in t he PR2 position was confirmed by supershift of the PR2 complex with c-Fos a ntibody and markedly decreased activity of the construct with a mutated AP1 site. Detailed deletion analysis proved that PR1 + PR2 account for 90% of the MN promoter activity, while neither PR I nor PR2 on their own are suffi cient for trans activation. Thus, synergistic co-operation between SP and A PI factors bound to the adjacent PR1 and PR2, respectively, is necessary fo r MN transcriptional activity. The PR1 + PR2 module also stimulated transcr iption from a heterologous promoter. The modulation of API activity with PM A stimulated MN expression and activated the MN promoter, whereas inhibitio n of protein kinase C activity had no effect on MN expression in HeLa cells .