Mapping the interactive domain between p12^CDK2-AP1 and MBD3

Objectives: Previously we demonstrated that p12CDK2-AP1 (p12, cyclin-dependent kinase-2-associating protein) is frequently downregulated in oral/head and neck squamous cell carcinomas. We observed the deregulation of DNA methylation and histone acetylation in p12-/- in ES cell line. Moreover, the p12 protein physically interacts with the methyl-CpG-binding domain protein (MBD3) which is a key molecule in DNA methylation. We hypothesize that disrupted p12 causes altered interaction with MBD3, resulting in deregulated epigenetic regulation, which eventually leads to cellular transformation. The objective of this study is to further understand the physical interaction between p12 and MBD3 by identifying responsible domain(s) of p12 for binding to MBD3.

Methods: Full-length GST-p12, various deleted recombinant GST-p12 and full-length His-MBD3 proteins were generated for in vitro binding analysis. His-tagged MBD3 was immobilized to Talon metal affinity resin and reacted with serial deletion mutants of GST-p12. The final binding complex was analyzed by western analysis with anti-p12 and anti-His antibody.

Results: The in vitro binding assay revealed that C-terminal deletion mutants of p12 (amino acids 1-15, 1-35, 1-55, 1-75, 1-95, 1-103 and 1-111) showed comparable level of interaction to the full-length p12 protein. However, the deletion of amino acid 1-61 in p12 abolished its interaction with MBD3, indicating that the amino acid residue (1-61) of p12 is critical for its binding to MBD3.

Conclusions: From this study, we concluded that p12 interacts with MBD3 protein and MBD3 binding domain of p12 resides at the N-terminus of the protein. Currently, we are investigating the role of p12 in DNA methylation and histone acetylation by utilizing the MBD3-binding defective p12 mutant.

Supported by 2007 AADR Student Research Grant GlaxoSmithKline (JJK), T32 Training Grant NIDCR DE 07296 (JJK), and R01 DE 14857 (DTW)