The Interaction Of AP-2α And TES

AP-2 is a critical transcriptional family temporally and spatially regulated in mammal embryo development including neural crest, cranial face, limbs, heart and kidney. To date, five members of the AP-2 family of transcription factors, AP-2α, AP-2β, AP-2γ, AP-2δand AP-2εhave been identified and all of them can bind as homo- or hetero-dimers to the typical consensus sequence of 5'-GCCNNNGGC-3'. AP-2αis the first gene cloned and the relatively best characterized among the AP-2 gene family. It has been shown that AP-2αis an important regulator that mediates essential events in embryo development and also participate in many biological processes such as cell growth, differentiation, carcinogenesis, or apoptosis through the transcriptional control of multiple target downstream genes. To be a functional regulator, AP-2αis co-operator with a number of partners. To identify more of the interacting co-activators of AP-2α, a HeLa eDNA library was screened by yeast two-hybrid assay using the full length sequence of AP-2αas a bait protein. One of the positive clones was identified by the yeast two-hybrid assay as the human TESTIN (TES) gene and the other was actinin (ACTN). Both proteins can bind with the actin involving in the cytoskeleton. The TES has a PET domain in the NH₂-terminus and 3 tandem LIM domains in the COOH-terminus, which has been reported as a candidate of tumor suppressor. In this study, we put emphasis to the TES function research and the relevance between TES AP-2α. The full-length or truncations of AP-2αand TES proteins were expressed and purified, and then corresponding antiserums are prepeared. The interaction between TES and AP-2αproteins was confirmed by pull-down assay in vitro and co-immunoprecipitation in vivo. Furthermore, we found that the interaction domains between TES and AP-2αlie in the COOH-termini of both proteins. In addition, we also detected the intramolecular interaction between the NH₂- and COOH-terminal of the TES proteins, and found the detailed region responsible for binding with the NH₂-terminus was LIM3 in the COOH-terminus of TES. In the luciferase reporter analysis, overexpression of TES suppressed the transcriptional activity of AP-2αover the promoter of ERBB2 gene. It is a puzzle that the two termini of TES also had some function upon the transcriptional activity of AP-2α. In the cellular immunofluorescence assays, we showed a variable endogenous TES localization especially in the nucleus and ER. Further, we demonstrated that TES and AP-2αwere partially co-localized in thenucleus. Moreover, it is interesting that the TES overexpression leads to a decreased level of AP-2αprotein. While transfection of siRNA specifically against TES gene resulted in a significant increase of ERBB2 transcription. Taken together, our studies revealed that AP-2αis physically associated with TES in vitro and formed a complex with TES in vivo. And their interaction may be involving an inhibitory mechanism over AP-2-mediated transactivation of ERBB2 gene. At the same time, we also suggest a new role of TES as a tumor suppressor by suppressing the transcriptional activity of AP-2α. Whereas the novel cellular localization of TES protein implicates that TES functions more than a cytoskeleton associated protein, and its first physical and functional partner in nucleus may be AP-2α.