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.

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.

Abstract. Small GTPases of the Rab family are key regulators of membrane trafficking. Monoclonal antibodies are useful tools for identifying proteins that interact with other proteins and for examining their tissue distribution. We selected a monoclonal antibody against Rab8 of Bombyx mori L. It specifically recognized amino acid residues 30-109, which are conserved among Rab8 proteins, and did not recognize any other Rab proteins. Western blotting using the antibody revealed one band in the brains of B. mori and rat. Far-Western blotting analysis detected three proteins interacting with Rab8. These results indicate that this antibody is useful for clarifying the physiological function of Rab8 of B. mori and other species. This is a report of a study on a monoclonal antibody against insect Rab protein.

Abstract. 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.