Global profiling of human tyrosine kinase and phosphatase gene expression and of protein tyrosine phosphorylation
| Autor: | Michikawa, Yuichi, Umetsu, Atsushi, Watanabe, Naoko, Kouda, Masakazu, Saegusa, Kumiko, Ishikawa, Kenichi, Kawai, Seiko, Ban, Sadayuki, Iwakawa, Mayumi, Harada, Yoshinobu, Imai, Takashi |
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| Jazyk: | angličtina |
| Rok vydání: | 2003 |
| Popis: | Both protein kinases and phosphatases represent distinct large protein families, whose members are similar to each other in sequence, structure and biochemical properties, in the human genome. The human genome has been estimated to contain between 500 and 1000 protein kinases. The expression status of overall kinase and phosphatase genes and the phosphorylation status of their substrates in individual cells seems to represent the detailed character of the cells since these protein families play key roles in variety of intercellular and intracellular signaling pathways by transmitting, amplifying or integrating upstream signals. In the 22,000 spots on the Agilent Custom OligoDNA Microarray, there are spots corresponding to 68 tyrosine kinase genes and 74 tyrosine phosphatase genes. Thirty three human cancer cell lines and two normal human fibroblast cells were analyzed by the above microarray using a mixture of 11 human tissue RNAs (Clontech) as a reference for each sample. The RNA expression data of twenty one kinase genes and fourteen phosphatase genes were selected after elimination of spots with p-value of more than 0.05 in at least 12 cell lines. For the profiling of tyrosine phosphorylation substrates in individual cell lines, we have developed a qualitative and quantitative anti-phosphotyrosine antibody Western blotting (qqPYW) method. By this method, molecular weight and signal intensity of each band detected was normalized by two steps, using internal molecular weight markers constructed by chemically tyrosine-phosphorylated proteins, to make precise matching to the bands in different samples to be practical. By this study, both gene expression data and protein phosphorylation data have become to be obtained at the quality that they can be compared to see any possible correlation. It will be necessary to establish further systematic methodology, such as identification of phosphorylated substrate proteins and in vivo interaction assay, to confirm the correlation observed. 第２６回日本分子生物学会年会 |
| Databáze: | OpenAIRE |
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