Characterization of a Novel Nuclear Protein Kinase RP-1

• Main Authors: Wu, Maan-Chaur, 吳滿潮
• Other Authors: Chen Chin-Chow
• Format: Others
• Language: zh-TW
• Published: 1998
• Online Access: http://ndltd.ncl.edu.tw/handle/67696758982318991709

碩士 === 國立臺灣大學 === 藥理學研究所 === 86 === 1.RP-1基因 (又名為G11基因) 位於人類主要組織相容抗原複合體第三 組基因群 (MHC class Ⅲ) 區域，介在補體C4A基因與補體Bf/C2基因間 。RP-1基因的長度約9.1 kb，轉 錄之mRNA含有9個exon，長度約為1.4 kb。依〝關聯研究〞(linkage study) 推測此基因 可能是Common Variable Immunodeficiency (CVID) 免疫疾病的潛在性致病基因。 2.在我們的實驗中，成功地以pGEX-3c-G11質體於細菌 (JM109) 中表現 GST-G11融合蛋 白，並以此融合蛋白為抗原免疫注射老鼠，由細胞融合瘤 技術製造了五個單株抗體 (4G3、1F3、1D10、5F9、6H9)。此五個抗 體均可用於西方點墨法專一地認知RP-1蛋白。 而其中四個 (1F3、1D10 、5F9、6H9) 屬於IgG1的單株抗體，則還直接可以應用於免疫沈 澱實驗 認知未變性的RP-1蛋白。 3.由於RP-1蛋白序列中具有nuclear localization signal (NLS)，因此 推測其可能分 佈在細胞核中。於COS-7細胞表現FLAG-G11融合蛋白亦發現 ，不論是以抗FLAG序列之抗體 或是抗RP-1之單株抗體作免疫螢光染色， 其分布均是位於細胞核中，證明RP-1為一種核 蛋白。 4.以RT-PCR方法偵測的結果，在人類的T細胞CEM、B細胞Ramos、單核球 U937、週邊血 液單核細胞 (PBMC)、上皮細胞A549均有RP-1基因表現， 且有alternative splicing forms。在A549細胞以TNF-α (10 ng/ ml)、 IL-1β (10 ng/ml)、IFN-γ (100 U/ml) 活化8個小時可以偵測 到RP-1 mRNA表現量的增加，而其中又以IL-1β 所增加的量最多。 5.西方點墨法及免疫沈澱實驗首次在A549細胞中偵測到RP-1胜。當以 IL-1β (10 ng/ml) 及thapsigargin (30 nM) 刺激A549細胞24小時， 可以增加RP-1蛋白的表現量。 6.以免疫沈澱得知thapsigargin除了可 以增加A549細胞RP-1蛋白量以外，還可以同時 促進RP-1蛋白的磷酸化， 而cytokines IL-1β 只會稍微增加磷酸化、IFN-γ 則不會促 進其磷酸 化。以PY20抗磷酸化酪胺酸 (phosphotyrosine) 抗體免疫沈澱的結果並 未偵測 到 RP-1蛋白，可能是其磷酸化的位置是在絲胺酸或是酥胺酸 (serine/threonine) 上， 而非在酪胺酸上。 7.總之，由我們所製備的抗RP-1單株抗體証實了RP-1為一個核蛋白，並且 在A549中偵 測到RP-1胜；IL-1β及thapsigargin可以增加A549細胞 RP-1蛋白的量，IL-1β只會稍 微增加RP-1的磷酸化，而thapsigargin則 會明顯地增加RP-1的磷酸化。此磷酸化的位置 可能在絲胺酸或酥胺酸上 而非在酪胺酸上。 1.RP-1 gene (also called G11 gene) is located in the class Ⅲ of the Major Histocompatibility Complex (MHC), between complement C4A gene and complement Bf/C2 gene. RP-1 gene spans about 9.1 kb, and transcripts about 1.4 kb mRNA splitting into 9 exons. Suggesting by "linkage study", it is a susceptible gene for Common Variable Immunodeficiency (CVID). 2.In this study, we have successfully expressed GST-G11 fusion protein by pGEX-3c-G11 plasmid in bacteria (JM109), and generated five monoclonal antibodies (4G3, 1F3, 1D10, 5F9, and 6H9) by this fusion protein. All of these antibodies can specifically recognize RP-1 protein by Western blot. Four of these monoclonal antibodies (1F3, 1D10, 5F9, and 6H9) are IgG1 type, and can directly recognize non- denatured RP-1 protein in immunoprecipitation experiment. 3.RP-1 protein sequence contains nuclear localization signal (NLS), suggesting its location in nucleus. Indeed, immunofluorescence staining with either anti-FLAG or anti-RP-1 antibody indicates that the transfected FLAG-G11 fusion protein locates in the nucleus of COS-7 cells. 4.RP-1 mRNAs , as detected by RT-PCR, are expressed in human T- cell CEM, B-cell Ramos, monocyte U937, peripheral blood mononuclear cells (PBMC), and pulmonary A549 epithelial cells, where several alternative splicing forms are present. A549 cells treated with TNF-α (10 ng/ml), IL-1β (10 ng/ml), or IFN-γ (100U/ml) for 8 hours expressed greater amount of RP-1 mRNA, with the highest increase after IL-1β treatment. 5.RP-1 protein can also be detected in A549 cells by Western blot and immunoprecipitation. When treated with IL-1β (10 ng/ml) or thapsigargin (30 nM) for 24 hours, the RP-1 protein level was increased. 6.In addition to the increase in RP-1 protein level, thapsigargin treatment can also promote RP-1 phosphorylation. IL-1β only slightly increased RP-1 phosphorylation, while IFN-g had no such effect. Further study with phosphotyrosine antibody (PY20), indicates that the phosphorylation was not on tyrosine residue. 7.Altogether, by using the RP-1 monoclonal antibodies we have prepared, we conclude that RP-1 is a nuclear protein, and can be expressed in A549 cells. Both IL-1β and thapsigargin can increase RP-1 protein in A549 cells. As compared to IL-1β, thapsigargin can elicit a more obvious phosphorylation of RP-1 protein. The phosphorylation site(s) may be on serine/threonine, but not tyrosine residue.