The RISC component VIG is a target for dsRNA-independent protein kinase activity in Drosophila S2 cells

Research Article

J RNAi Gene Silenc (August 2005), 1(1), 12-20

doi: jrgsxx

Published online: 27 July 2005

Full Text: (html | pdf ~814kb | refs)

The RISC component VIG is a target for dsRNA-independent protein kinase activity inDrosophila S2 cells

Konstantin I Ivanov †*, Timofey V Tselykh ‡¥, Tapio I Heino ‡§ and Kristiina Mäkinen †*

† Department of Applied Biology, ‡ Institute of Biotechnology, Developmental Biology Program and § Department of Biological and Environmental Sciences, University of Helsinki, FIN-00014, Finland

¥ Present address: Molecular and Cancer Biology Research Program, Institute of Biomedicine, Biomedicum Helsinki, University of Helsinki, FIN-00014, Finland

Correspondence to: Konstantin I Ivanov or Kristiina Mäkinen, Email : Konstantin.Ivanov@helsinki.fi;Kristiina.Makinen@helsinki.fi, Tel: +358-9-191-58349 (KI); +358-9-191-58342 (KM) , Fax: +358-9-191-58633

Received: 16 May 2005, Revised: 06 July 2005, Accepted: 07 July 2005

© Copyright The Authors

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ABSTRACT

RNA interference (RNAi) is mediated by a multicomponent RNA-induced silencing complex (RISC). Here we examine the phosphorylation state of three Drosophila RISC-associated proteins, VIG, R2D2 and a truncated form of Argonaute2 devoid of the nonconserved N-terminal glutamine-rich domain . We show that of the three studied proteins, only VIG is phosphorylated in cultured Drosophila cells. We also demonstrate that the phosphorylation state of VIG remains unchanged after cell transfection with exogenous dsRNA. A sequence similarity search revealed that VIG shares significant similarity with the human phosphoprotein Ki-1/57, a known in vivo substrate for protein kinase C (PKC). In vitro kinase assays followed by tryptic phosphopeptide mapping showed that PKC could efficiently phosphorylate VIG on multiple sites, suggesting PKC as a candidate kinase for VIG phosphorylationin vivo. Taken together, our results identify the RISC component VIG as a novel kinase substrate in cultured Drosophila cells and suggest a possible involvement of PKC in its phosphorylation.

KEYWORDS: RNA interference, RNAi, RISC, Vasa Intronic Gene, VIG, Argonaute, R2D2

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