Indentification And Characterization Of Proteins Interacting With Transcription Factor AP-2 Alpha

As a member of an important transcription factor family, AP-2 a expresses in specific tissue and binds with specific DNA sequence. By controlling the transcription of a number of downstream genes, AP-2 a plays a vital role in embryonic development and cell diffrenciation and tumor genesis. In order to figure out the machanism of transcription control by AP-2 a and to find the partner proteins which derectly binding to AP-2 a , a yeast two-hybrid system by using AP-2 a as a bait was performed to screen the HeLa cell cDNA library. Two positive clones were obtained and sequenced. Using the BLAST against NCBI gene database, they were comfirmed to be the cDNA fragments of TESTIN and ACTN1. At present, there is not much study with respect to the functional analysis of TESTIN gene. What we know about is that TESTIN may be a cancer supressing gene. The protein contains three LIM domains at C-terminus. While ACTM gene is well known to code a-actinin 1 protein which involes in cell skeleten and cell signals. The two cDNA fragments , AP-2a full length cDNA and AP-2 a cDNA fragments were inserted in frame into GST gene fusion system. With an AP-2a monoclonal antibody we detected the expression of GST- AP-2a . There were some degration in the purified protein, but GST- AP-2 a still had the DNA binding activity in the gel shift assay. The GST-TESTIN and GST-ANTN1 were used for immunolize rabbits. With the polyclonal antiserum against TESTIN and ACTN1 , and the monoclonal antibody against AP-2a , the expression of TESTIN, ACTN1 and AP-2a in HeLa cell was determined. The results indicates that the expression of AP-2 a is concentrated in nuclear extract, whereas the major expression of TESTIN is in cytoplasmic extract, and the expression of ACTN1 is in both nuclear and cytoplasmic extract. The interaction between AP-2a and TESTIN or ACTNl should be comfirmed by further test. Their real interaction maycontribute to the explaination of AP-2α localization shift between cytoplasm and nuclear, and be a complementary mechanism to regulate for the transcriptional activity of AP-2α.