Studies On The Interaction Of CCDC106 Protein And P53 Protein

The tumour suppressor protein p53 maintains genomic integrity and cellular homeostasis. In response to stress signals such as DNA damage, oncogenic stimuli or hypoxia, p53 accumulates in the cell and becomes activated. It then initiates an anti-proliferation and pro-apoptotic program that prevents the multiplication of damaged and potentially pre-cancerous cells. Lack of p53 function is often connected with development of cancer. Inactivation of p53 by mutation occurs in over 50% of all human cancers. In cancer types that harbour wild-type p53, p53 function is frequently abolished by other factors. Some proteins can interact with p53, and then affect stability, transactivation or subcellular localization, leading to the loss of p53 function. Thus, identification of novel interacting partners of p53 will provide further insight into the regulation of p53 activity.The putative CCDC106 protein was previously identified as a p53-interacting partner by automated yeast two-hybrid screening, but its sequence and function have not been validated experimentally.Here, we cloned the full length cDNA sequence of CCDC106 gene and identified three variant transcripts of the CCDC106 gene; these transcripts differ in their second exons due to the use of different splice acceptor site, but these three variant tanscripts have an identical open reading frame of 843bp,which encodes a protein of 280 amino acids with a calculated molecular weight of 32 KDa.CCDC106 polyclonal antibody was raised and used for Western blot. we found that CCDC106 was highly expressed in cancer cell lines NCI-H292 and HeLa that harbour wild-type p53.We used fluorescence microscopy to examine EGFP-CCDC106 fusion protein and endogenous CCDC106 protein, and found that both proteins were located exclusively in the nucleus, indicating that CCDC106 is a nuclear protein. Using the LOCtree software, we found that CCDC106 contains two putative Nuclear localisation signal (NLS1, 151RRR153 and NLS2,162RRR164). Mutation of either NLS could lead to the change of localistation of CCDC106, indicating that two nuclear localization signals determine the nuclear localisation of CCDC106.Immunofluorescence revealed the colocalisation of endogenous CCDC 106 and p53 protein in nuclei. The in vivo interaction between CCDC 106 and p53 was confirmed by co-immunoprecipitation assay.We studied the effect of CCDC 106 overexpression on the transactivation activity of p53. The result showed that CCDC 106 overexpression decreased the luciferase activity of pp53-luc, suggesting CCDC 106 inhibits the transactivation of p53.To elucidate the mechanism for the inhibited transcription activity of p53 by CCDC 106, HeLa cells were co-transfected with p53 expression plasmid and increasing amounts of CCDC 106 expression plasmid in HeLa cells.The results showed that with an increase of CCDC106, the level of p53 protein decreased. To further determine whether the reduced level of the p53 protein resulted from p53 protein degradation,we examined the half-life of p53 protein. The result showed that p53 was rapidly degraded in cells transfected with CCDC106 expression plasmid. Then, we treated cells with the proteasomal inhibitor MG132, showing that the protein level of p53 was obviously rescued. These results suggest that CCDC106 can increase the proteasome-dependent degradation of p53.These studies suggest that CCDC106 protein is highly expressed in cancer cells that harbour wild-type p53. CCDC106 can promotes the degradation of p53 by interacting with p53,leading to the loss of p53 function. Thus, CCDC106 may be a oncogene; The high expression of CCDC106 may be one of causes that lead to development of tumor that harbour wild-type p53.