title: FHL2 mediates p53-induced transcriptional activation through a direct association with HIPK2.
abstract: To understand the molecular mechanism underlying HIPK2 regulation of the transcriptional activation by p53, we sought to identify the protein that interacts with HIPK2. From our yeast two-hybrid screen, we found that four and a half LIM domains 2 (FHL2) could bind to the C-terminal half of HIPK2. Further assays in yeast mapped the minimal interaction domain to amino acids 812-907 in HIPK2. The interaction was confirmed using a GST pull-down assay in vitro, and an immunoprecipitation (IP) assay and fluorescence microscopy in vivo. FHL2 alone spread throughout both the cytoplasm and nucleus but was redistributed to dot-like structures in the nucleus when HIPK2 was coexpressed in HEK293 cells. When tethered to the Gal4-responsive promoter through the Gal4 DBD fusion, FHL2 showed autonomous transcriptional activity that was enhanced by wild-type HIPK2, but not by the kinase-defective mutant. In addition, FHL2 increased the p53-dependent transcriptional activation and had an additive effect on the activation when coexpressed with HIPK2, which was again not observed with the kinase-defective mutant of HIPK2. Finally, we found a ternary complex of p53, HIPK2, and FHL2 using IP, and their recruitment to the p53-responsive p21Waf1 promoter in chromatin IP assays. Overall, our findings indicate that FHL2 can also regulate p53 via a direct association with HIPK2.
authors.affiliation: Department of Bioscience and Biotechnology/Institute of Bioscience, Sejong University, Seoul, Republic of Korea.
authors.forename: Sang-Wang
authors.initials: SW
authors.lastname: Lee
authors.forename: Eun-Joo
authors.initials: EJ
authors.lastname: Kim
authors.forename: Soo-Jong
authors.initials: SJ
authors.lastname: Um
mesh_terms: D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D006367:HeLa Cells; D018398:Homeodomain Proteins / Q000378:metabolism*; D006801:Humans; D060850:LIM-Homeodomain Proteins; D009124:Muscle Proteins / Q000378:metabolism*; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation / Q000502:physiology*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; C105987:FHL2 protein, human; D018398:Homeodomain Proteins; D060850:LIM-Homeodomain Proteins; D009124:Muscle Proteins; D014157:Transcription Factors; D016159:Tumor Suppressor Protein p53; C115114:HIPK2 protein, human; D017346:Protein Serine-Threonine Kinases