Determination of phosphorylated amino acid residues of Rab8 from Bombyx mori.
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.
Uno
Tomohide
T
Laboratory of Biological Chemistry, Department of Biofunctional Chemistry, Faculty of Agriculture, Kobe University, Kobe, Japan. unotom@kobe-u.ac.jp
Nakada
Takuya
T
Okamaoto
Sota
S
Nakamura
Masahiko
M
Matsubara
Mamoru
M
Imaishi
Hiromasa
H
Yamagata
Hiroshi
H
Kanamaru
Kengo
K
Takagi
Michihiro
M
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D012831:Bombyx / Q000201:enzymology / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D012694:Serine / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000378:metabolism*
D012694:Serine; D011493:Protein Kinase C; D020691:rab GTP-Binding Proteins