GAS41 interacts with transcription factor AP-2β and stimulates AP-2β-mediated transactivation

Xiaofeng Ding 1 Changzheng Fan 1 Jianlin Zhou 1 Yingli Zhong 1 Rushi Liu 1 Kaiqun Ren 1 Xiang Hu 1 Chang Luo 1 Shunyong Xiao 1 Yeqi Wang 1 Du Feng 1 Jian Zhang 0 1 0 Model Organism Division, E-Institutes of Shanghai Universities, Shanghai Second Medical University , Shanghai 200025, China 1 Key Laboratory of Protein Chemistry and Developmental Biology of State Education Ministry of China, College of Life Science, Hunan Normal University , Changsha, Hunan 410081, China Transcription factor AP-2 regulates transcription of a number of genes involving mammalian development, differentiation and carcinogenesis. Recent studies have shown that interaction partners can modulate the transcriptional activity of AP-2 over the downstream targets. In this study, we reported the identification of GAS41 as an interaction partner of AP-2b. We documented the interaction both in vivo by co-immunoprecipitation as well as in vitro through glutathione S-transferase (GST) pull-down assays. We also showed that the two proteins are co-localized in the nuclei of mammalian cells. We further mapped the interaction domains between the two proteins to the C-termini of both AP-2b and GAS41, respectively. Furthermore, we have identified three critical residues of GAS41 that are important for the interaction between the two proteins. In addition, by transient co-expression experiments using reporter containing three AP-2 consensus binding sites in the promoter region, we found that GAS41 stimulates the transcriptional activity of AP-2b over the reporter. Finally, electrophoretic mobility shift assay (EMSA) suggested that GAS41 enhances the DNA-binding activity of AP-2b. Our data provide evidence for a novel cellular function of GAS41 as a transcriptional co-activator for AP-2b. - To date, five members of the AP-2 family of transcription factors, AP-2a, AP-2b, AP-2g, AP-2d and AP-2e, have been identified. The AP-2a, AP-2b, AP-2g genes are relatively well characterized (16). The AP-2 protein forms a unique modular structure consisting of an N-terminal proline- and glutamine-rich transactivational domain and a complex helix-span-helix motif necessary and sufficient for dimerization and site-specific DNA binding (7,8). A number of genes that mediate cell growth, cell shape, cell movement, cell fate and cell communication frequently possess the AP-2 binding site in their cis-regulatory sequences (915). Several genes related to cancers have also been shown to be regulated by AP-2, such as erbB-2 (3,10,16,17), ERa (12) and IGF IR (16,18) in breast cancer, and MUC18 and c-KIT genes in melanoma (19,20). The AP-2 family of genes also plays important roles in mammalian development. AP-2 genes show overlapping but distinct patterns of expression during vertebrate embryogenesis, and function in the development and differentiation of the neural tube, neural crest derivatives, heart, skin, urogenital tissues and extraembryonic trophoblasts (2123). The importance of AP-2 genes is highlighted by knockout experiments of AP-2a, AP-2b and AP-2g. Mice lacking both copies of AP-2a gene die perinatally and exhibit at least six major defects during embryogenesis: morphogenesis of the neural tube, face, eye, body-wall, cardiovascular system and forelimbs (2427). Mice lacking AP-2b display fewer gross phenotypic defects but die shortly after birth due to the disruption of terminal kidney differentiation (28). The AP-2g-null mice die around E7.5, shortly after implantation due to the defects within the extraembryonic cell lineages (23,29). The AP-2 family of transcription factors plays a broad range of roles from cell growth, tissue morphogenesis and cancers. One of mechanisms for the AP-2 family fulfills their roles is to activate or suppress various downstream target genes at transcriptional levels. A number of studies demonstrated that AP-2-interacting proteins can affect the transcription of AP-2 downstream targets by modulating the transcriptional activity of AP-2. In fact, several AP-2ainteracting partners have been identified. For example, the transactivation of p21WAF1 by AP-2a was augmented while activation of laminin receptor by AP-2a was reduced through a direct interaction with p53 (30,31). AP-2a represses the transactivation by Myc through associating with Myc and competing the binding site with Myc (11). Other AP-2ainteracting proteins include Yin Yang factor 1 (YY1) (32), retinoblastoma protein (RB) (33,34) and oncogene DEK (35). Wwox tumor suppressor protein was also identified as an AP-2g interacting partner, and Wwox protein triggers redistribution of nuclear AP-2g to the cytoplasm, hence suppressing AP-2g-mediated transactivation (36). To date, no AP-2b-interacting factor has been reported yet. To search for AP-2b-interacting proteins, we used AP-2b as the bait and screened a HeLa cDNA library in yeast twohybrid system. We identified GAS41 as a protein partner of AP-2b. The interaction between the two proteins was confirmed in vivo by co-immunoprecipitation and co-localization assays, and demonstrated in vitro by glutathione S-transferase (GST) pull-down assay. The interaction domains between the two proteins were mapped to the C-terminus of AP-2b and C-terminus of GAS41. Furthermore, we demonstrated that GAS41 resulted in enhancement of transcriptional activity of AP-2b over AP-2 response element reporter by, at least in part, enhancing the DNA-binding activity of AP-2b. MATERIALS AND METHODS For yeast two-hybrid screening, full-length cDNA of AP-2b was ligated in frame with the GAL4 DNA-binding domain of the pDBLeu vector resulting in pDBLeu/AP-2b. For immunoprecipitation and colocalization assays, the fulllength cDNA of AP-2b was cloned into the mammalian expression plasmid pCMV-Myc vector (Clontech), forming a Myc tagged AP-2b expression vector pCMV-Myc-AP-2b, while full-length cDNA and the mutations of GAS41 were inserted into pCMV-HA vector (Clontech), forming a HA tagged GAS41 expression vector pCMV-HA-GAS41, and full-length cDNA of GAS41 was also cloned into pCMV-Myc vector. Vector pGEX-4T-2 (Amersham) was used to construct vectors expressing GST-AP-2b fusion proteins. The cDNA fragments encoding full-length and subdomains (Figure 3G) of AP-2b were cloned in frame with respect to GST into pGEX-4T-2 individually. Plasmid pQE-N3 (Qiagen) was used to generate vectors expressing His-tagged GAS41 fusion proteins. The cDNAs encoding full-length subdomains (Figure 4F) and point mutations of GAS41 were fused in frame to His tag of pQE-N3 individually. Reporter plasmid A2-Luc was constructed by replacing CAT gene with luciferase gene in pA2BCAT vector (generous gift of T. Williams) which contains three copies of AP-2 binding site in human metallothionein IIa gene in the promoter region (7). Vector pCMV-LacZ was constructed by fusing LacZ gene into pCMV-Myc. Yeast two-hybrid screens The pro yeast two-hybrid system was obtained from GIBCO/ BRL. A HeLa cDNA library cloned in frame with the GAL4 activation (...truncated)