Identification of N-terminal Eps8 binding proteins by Yeast Two-Hybrid Method

• Main Authors: Tsai-Hwa Jong, 鐘彩華
• Other Authors: Tzeng-Horng Leu
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/15479452975835535937

碩士 === 國立成功大學 === 藥理學研究所 === 91 === Previously, we demonstrated that cells overexpressing p97Eps8 not only exhibited the ability of focus formation in cell culture but also promoted the tumor formation in mice as compared to controls. However the N-terminal truncated 261-p97Eps8 has lost such transforming ability. Thus, the N-terminal domain may play an important role in Eps8-mediated cellular transformation. Protein sequence analysis revealed the following domains existing in the N-terminal p97Eps8 that might contribute to its cellular functions : a putative nuclear targeting sequence, a split PH domain and two proline-rich regions. In this study we utilized yeast two-hybrid method to screen a human brain cDNA expression library to search for N-terminal p97Eps8 binding proteins (NEBP). We have obtained 246 cDNA clones encoding the potential N-terminal p97Eps8 binding proteins. Sequence comparison with the information derived from GenBank, NEBP1 and NEBP2 were identified BAIAP2 and TLE2 respectively. How these proteins interact with p97Eps8 and play a role in Eps8-induced cellular transformation needs further investigation. Previously, there was evidence indicating that Src can phosphorylate p97Eps8 and the proline rich regions of p97Eps8 can interact with the Src SH3 doamin in an in vitro binding assay. To substantiate this interaction in vivo, we also utilize yeast two-hybrid method to define the Src SH3 domain binding sequence of p97Eps8. We find that SrcSH3SH2 domain can interact with wt-p97Eps8 and 261-p97Eps8, but not the other truncated p97Eps8.