Determination of phosphorylated amino acid residues of Rab8 from Bombyx mori |
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Authors: | Uno Tomohide Nakada Takuya Okamaoto Sota Nakamura Masahiko Matsubara Mamoru Imaishi Hiromasa Yamagata Hiroshi Kanamaru Kengo Takagi Michihiro |
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Institution: | Laboratory of Biological Chemistry, Department of Biofunctional Chemistry, Faculty of Agriculture, Kobe University, Kobe, Japan. unotom@kobe-u.ac.jp |
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Abstract: | The Rab family of small GTPases are key regulators of membrane trafficking. Partially purified Rab8 from Bombyx mori (BRab8) was phosphorylated by protein kinase C in mammalian cells in vitro. To determine which of the seven serines and four threonines are phosphorylated, we generated deletion and site-directed mutants of BRab8, inserted them in Escherichia coli, partially purified the encoded fusion proteins by affinity chromatography, and examined their phosphorylation by protein kinase C in vitro. We found that Ser-132 of BRab8 was specifically phosphorylated by protein kinase C. In addition, Western blotting using an antiserum against BRab8 and in-gel staining for phosphorylated proteins revealed that BRab8 is phosphorylated in vivo. |
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Keywords: | Bombyx mori Rab brain protein kinase |
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