The Study of Protein-Nucleophosmin/B23 Interactions in Different Cell Growth Conditions

• Main Author: 翁千惠
• Other Authors: Yat-Ming Yung
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/36919118991869250519

碩士 === 長庚大學 === 基礎醫學研究所 === 90 === Abstract Nucleophosmin/B23 (B23), a nucleolar phosphoproteins, appears to be a multifunctional protein. B23 interacts with many proteins, and this protein-protein interaction may elucidate the function of B23. To isolate and characterize proteins that interact with B23, I established the GST-B23 and its deletion mutant fusion protein column to isolate B23-interacting proteins from HeLa cell extracts. SDS-PAGE was used to assay the fraction eluted by high salt buffer from GST-B23 or GST column, and the electrophoretic profiles of B23-interacting proteins could be visible by silver stain. In high salt-eluted fraction, I found that at least 9 proteins might bind with GST-B23 column, as compared with the fraction from GST column. Moreover, I screened these fractions with slot blot and Western blot. One of B23-interacting proteins was identified as RACK1 (receptor for activated C kinase). In the fractions eluted from B23 C-terminal deletion mutant columns, some protein bands disappeared compared to that from GST-B23 column. These results indicated that these proteins bound to the C-terminal domain of B23. Our previous studies have shown that B23 translocates from nucleoli to nucleoplasm during actinomycin D (Act-D) treatment. To study B23-interacting proteins during Act-D-induced-B23 translocation, the extracts of Act-D-treated cells were applied to GST-B23 column. The different electrophoretic profiles of B23-interacting proteins were observed, and this data may be valuable for B23 translocation study. In the complementary approach to analyze the interaction of B23 and RACK1 in vivo, immunofluorescence was observed by confocal microscop. Finally, proteomic analysis will also be applied to identify the other B23-interacting proteins.