Eur. J. Entomol. 106 (4): 499-506, 2009 | DOI: 10.14411/eje.2009.063

Phosphorylation of small GTPase Rab proteins from Bombyx mori (Lepidoptera: Bombycidae)

Tomohide UNO1, Keisuke HATA1, Dieu Trang LE THI2, Susumu HIRAGAKI2, Takuya NAKADA1, Masahiko NAKAMURA3, Yuichi UNO4, Hiroshi YAMAGATA1, Kengo KANAMARU1, Makio TAKEDA2, Mamoru MATSUBARA3
1 Laboratory of Biological Chemistry, Department of Biofunctional Chemistry, Faculty of Agriculture, Kobe University, Nada-ku Hyogo 657-8501, Japan; e-mail: unotom@kobe-u.ac.jp
2 Division of Molecular Science, Graduate School of Science and Technology, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657 8501, Japan
3 Department of Bioscience and Biotechnology, Faculty of Bioenvironmental Science, Kyoto Gakuen University, 1-1 Nanjo, Sogabe, Kameoka, Kyoto 621-8555, Japan
4 Department of Plant Resource Science, Faculty of Agriculture, Kobe University, Nada-ku Hyogo 657-8501, Japan

Small GTPases of the Rab family act as essential regulators of vesicle transport pathways. Five Rab cDNA clones (BRab1, 7, 8, 11 and 14) from Bombyx mori were expressed in Escherichia coli as a thioredxin or glutathione sulfotransferase fusion protein. After purification, the fusion protein was tested for phosphorylation using protein kinase C (PKC). Results indicate that all of them were phosphorylated in vitro. The phosphorylation site of BRab1 was determined by mass-spectrometric analysis, which identified that Ser-17 of BRab1 was phosphorylated by PKC. Deletion and site-directed mutagenesis indicated that Ser-111of BRab8, in addition to Ser-17, was newly phosphorylated. Further immunohistochemical analysis using antibodies against Rab8 indicated that there are some Rab8 immunoreactive cells close to the neuropeptide secreting cells. This result suggests that in insects Rab proteins are regulated by phosphorylation and at least some of them are involved in neuropeptide secretion.

Keywords: Small GTP binding protein, Rab, Bombyx mori, PKC, PTTH

Received: February 10, 2009; Accepted: June 15, 2009; Published: November 20, 2009

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