Two partial cDNA clones (Protein kinase C alpha and Protein kinase C iota), each of which encoded a different member of PKC-protein family, were isolated using RT-PCR from mRNA of Bombyx mori. The full-length cDNAs were isolated using SMART-RACE. The cDNAs were expressed in HepG2 cells and the recombinant proteins were partially purified using an affinity chromatography. Protein kinase C alpha (BPKC alpha) showed a calcium-dependent kinase activity of histones. Whereas protein kinase C iota (BPKC iota) showed a calcium-independent activity. Bisindolyl maleimide I, a PKC inhibitor, inhibited these kinase activities. Furthermore, in vitro BPKC alpha interacted and phosphorylated two proteins expressed in the brain of Bombyx mori: Rab protein, which plays important roles in the vesicle transport in the brain, and bMBD2/3, which is a methyl DNA-binding protein and regulates transcription. These results suggest that these PKCs phosphorylate various substrate proteins and function in 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.