Phosphorylation of Rab proteins from the brain of Bombyx mori.
Rab proteins play fundamental roles in the regulation of membrane traffic. Previously, from the brain of Bombyx mori we isolated two cDNA clones (BRab1 and BRab14), each of which encoded a different member of Rab-protein family and was expressed in Escherichia coli and purified using an affinity chromatography. In this study, one cDNA clone (BRab8) was isolated from a cDNA library from the brain of B. mori. The recombinant protein was expressed in E. coli and purified. Next, the phosphorylations of these three purified BRab proteins were examined, using mammalian protein kinases in vitro. Protein kinase C (PKC) phosphorylated BRab8 and BRab14 proteins. Protein kinase A faintly phosphorylated BRab8 and BRab14 proteins. Calcium/calmodulin-dependent protein kinase faintly phosphorylated BRab8 protein. Next, brains of B. mori were dissected and homogenized. The homogenate showed a calcium-dependent protein kinase activity of BRab8 and BRab14 proteins. So PKC from the brain of B. mori was partially purified by a sequence of chromatographies on DEAE-Cellulofine and affinity chromatography. This PKC phosphorylated BRab8 and BRab14 proteins. These results suggest that the function of Rab proteins in the brain of B. mori is regulated by calcium-dependent protein kinases.
Uno
Tomohide
T
Laboratory of Biological Chemistry, Department of Biofunctional Chemistry, Faculty of Agriculture, Kobe University, Nada-ku Hyogo, Japan. unotom@kobe-u.ac.jp
Nakao
Atsushi
A
Katsurauma
Chisato
C
D000595:Amino Acid Sequence; D000818:Animals; D012831:Bombyx / Q000378:metabolism*; D001921:Brain / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002846:Chromatography, Affinity; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D015723:Gene Library; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D020691:rab GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D017871:Calcium-Calmodulin-Dependent Protein Kinases
D002118:Calcium; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D011494:Protein Kinases; D020691:rab GTP-Binding Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases