An alternatively spliced mRNA from the AP-2 gene encodes a negative regulator of transcriptional activation by AP-2.

Abstract

AP-2 is a retinoic acid-inducible and developmentally regulated activator of transcription. We have cloned an alternative AP-2 transcript (AP-2B) from the human teratocarcinoma cell line PA-1, which encodes a protein differing in the C terminus from the previously isolated AP-2 protein (AP-2A). This protein contains the activation domain of AP-2 and part of the DNA binding domain but lacks the dimerization domain which is necessary for DNA binding. Analysis of overlapping genomic clones spanning the entire AP-2 gene proves that AP-2A and AP-2B transcripts are alternatively spliced from the same gene. Both transient and stable transfection experiments show that AP-2B inhibits AP-2 transactivator function, as measured by an AP-2-responsive chloramphenicol acetyltransferase reporter plasmid. Furthermore, constitutive AP-2B expression in PA-1 cells causes a retinoic acid-resistant phenotype, anchorage-independent growth in soft agar, and tumorigenicity in nude mice, in a fashion similar to transformation of these cells by oncogenes. To determine the mechanism by which AP-2B exerts its inhibitory function, we purified bacterially expressed AP-2A and AP-2B proteins. While bacterial AP-2B does not bind an AP-2 consensus site, it strongly inhibits binding of the endogenous AP-2 present in PA-1 cell nuclear extracts. However, DNA sequence-specific binding of bacterially expressed AP-2A cannot be inhibited by bacterially expressed AP-2B. Therefore, inhibition of AP-2 activity by the protein AP-2B may require an additional factor or modification supplied by nuclear extracts.

Different mechanisms are implicated in ERBB2 gene overexpression in breast and in other cancers

“…Some authors described the loss of AP-2a expression early in the development of prostate adenocarcinoma (Ruiz et al, 2001). Moreover, AP-2B, a dominant-negative variant of AP-2a, was detected by RT -PCR in LNCaP cells (Buettner et al, 1993). Others did detect AP-2a by immunohistochemistry in some prostate cancer specimens (Lipponen et al, 2000).…”

Cloning and characterization of the Drosophila homologue of the AP-2 transcription factor

“…A failure to splice at the splice site between exons 5 and 6 of the hAP-a gene results in hAP-2aB, a dominant negative form of hAP-2a (Buettner et al, 1993;Bauer et al, 1994). Thus, the AP-2aB protein, made from AP-2a apparently required a di erent exon organization, which may re¯ect a function acquired during evolution.…”

“…In addition, an intron located between exons 5 and 6 of the hAP-2a gene is missing from DAP-2. Interestingly, the alternative hAP-2aB mRNA, which is a dominant negative regulator of hAP-2 (Buettner et al, 1993), is generated by failure to splice after the ®fth exon. Thereby a continuous transcript is processed that encodes for an alternative C-terminal open reading frame (Bauer et al, 1994).…”

“…We refer to this construct as AP-2-622 CAT. AP-2hMtII CAT contains a trimerized hMtIIa 7180 site in front of a minimal TK promoter and is described elsewhere (Buettner et al, 1993;Kannan et al, 1994). Transient transfection of the PA-1 human teratocarcinoma cell line clone 9117 (Buettner et al, 1991: Tainsky et al, 1988 was performed using calcium phosphate precipitations (Graham and van der Eb, 1973).…”

“…Additionally, a number of splice variants exist (Buettner et al, 1993;Meier et al, 1995) that generate more diversity in AP-2 isoforms. The three AP-2 proteins share common structural features and exhibit an overall identity between 63% and 66%.…”