Abstract
Post-translational modifications have crucial roles in the regulation of many physiological processes such as development, differentiation, and response to extracellular signals. Currently, protein phosphorylation is considered as an important trigger for many signal transductions. Protein ubiquitination initially was thought to be a tag for protein degradation; however, recent studies demonstrated that protein ubiquitination also functions as a key regulator of signal transductions. In particular, proteomics analysis using high-sensitivity mass spectrometry provides a large number of modification sites for phosphorylation or ubiquitination of protein in the cells. Although the information from these modifications allows us to image the regulatory mechanism of the protein, it is difficult to know the protein kinase or ubiquitin ligase responsible for these modifications. Recently, we developed a protein array technology based on a wheat germ cell-free protein production system for biochemical analysis. Here, we introduce the procedure of the protein array construction and novel methods using the protein array to identify the responsible enzymes for phosphorylation or ubiquitination of target protein.
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Acknowledgments
This work was supported by the Program to Disseminate Tenure Tracking System (H.T.), a Grant-in-Aid for Scientific Research (B) (T.S.) and Platform for Drug Discovery, Informatics, and Structural Life Science (T.S.), and a Grant-in-Aid for Scientific Research on Innovative Areas (T.S.) from the Ministry of Education, Culture, Sports, Science, and Technology in Japan. The pictures in Fig. 4.4d,f were reproduced from Takahashi et al. 2009 (BMC Plant Biology 9:39). The pictures in Fig. 4.3a,b, and Fig. 4.5 were reproduced from Sawasaki et al. 2013 (Seikagaku 85:438–446). We thank the Applied Protein Research Laboratory of Ehime University for technical assistance, and also thank K. Takeda for proofreading the manuscript.
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Takahashi, H., Nemoto, K., Abdelaziz, R., Uematsu, A., Sawasaki, T. (2015). Technology of Wheat Cell-Free-Based Protein Array for Biochemical Analyses of Protein Kinases and Ubiquitin E3 Ligases. In: Inoue, Ji., Takekawa, M. (eds) Protein Modifications in Pathogenic Dysregulation of Signaling. Springer, Tokyo. https://doi.org/10.1007/978-4-431-55561-2_4
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DOI: https://doi.org/10.1007/978-4-431-55561-2_4
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