Yeast two-hybrid analysis and truncational study of an oncofetal-like gene - LRRC16B

• Main Authors: Pei-Shan Hsieh, 謝佩珊
• Other Authors: Chung-Liang Ho
• Format: Others
• Language: zh-TW
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/85385407813207603403

碩士 === 國立成功大學 === 醫學檢驗生物技術學系碩博士班 === 97 === Oncofetal proteins, such as alpha-fetoprotein (AFP) and glypican-3 (GPC-3), are proteins expressed in embryonic stage, down-regulated in adult normal tissues but re-expressed in tumors. Recent studies showed that these proteins may be useful in cancer diagnosis and prognosis. Previously, we found LRRC16B (EGRT-U5, Embryonic Gene Re-express in Tumor-Unknown 5) presenting oncofetal-like patterns that was expressed in many tumor and fetal tissues including liver and colon but down-regulated in normal and non-tumor tissues. According to the bioinformatics analysis, EGRT-U5 has several leucine rich repeats (LRR) in N-terminus and nuclear localization signals in C-terminus. A number of recent studies revealed that the LRR domains probably were related to protein–protein interactions and the protein containing LRR domain may be associated with many biologically important processes, morphology and dynamics of the cytoskeleton and early mammalian development. In order to further determine protein domains of EGRT-U5 which are responsible for interaction, we had cloned two truncated constructs pGBKT7-U5 (1-991) and pGBKT7-U5 (991-1370). By using these truncated constructs as baits, we screened a testis cDNA library to sift protein-protein interaction by yeast two-hybrid system. Presently, we found 4 proteins may interact with U5 (1-991) and 5 proteins may interact with U5 (991-1370), including beta-actin, Protein phosphatase 3 (PPP3), c-Src tyrosine kinase and importin alpha among others. Using immunoprecipitation and immunofluorescence to verify these results, we observed the EGRT-U5 could interact with c-Src. Combined with the data before, it revealed the ability of cell proliferation and tumor formation may associated with the interaction between EGRT-U5 and c-Src. Moreover, we also detected the c-Src downstream signaling pathway expression levels, only the JNK pathway showed the dramatic change. In conclusion, we suggested the oncofetal-like protein, LRRC16B (EGRT-U5) may play a role in tumorgenesis and medieated by its interaction with c-Src to regulate the JNK pathway which results in tumor formation.