An alternatively spliced mRNA from the AP-2 gene encodes a negative regulator of transcriptional activation by AP-2.
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.
Buettner
R
R
Department of Tumor Biology, University of Texas M. D. Anderson Cancer Center, Houston 77030.
Kannan
P
P
Imhof
A
A
Bauer
R
R
Yim
S O
SO
Glockshuber
R
R
Van Dyke
M W
MW
Tainsky
M A
MA
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002454:Cell Differentiation; D002455:Cell Division; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004330:Drosophila; D029721:Drosophila Proteins; D005260:Female; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014212:Tretinoin / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; C493674:TfAP-2 protein, Drosophila; D050656:Transcription Factor AP-2; D014157:Transcription Factors; D014212:Tretinoin; D004247:DNA