NDLI: Use of double-stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways.

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Use of double-stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways.

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Use of double-stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Dixon, J. E. Maehama, T. Simonson-leff, N. Muda, M. Hemmings, B. A. Clemens, J. C. Worby, C. A.
Description	Author Affiliation: Clemens JC ( Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109-0606, USA.);
Abstract	We demonstrate the efficacy of double-stranded RNA-mediated interference (RNAi) of gene expression in generating 'knock-out' phenotypes for specific proteins in several Drosophila cell lines. We prove the applicability of this technique for studying signaling cascades by dissecting the well-characterized insulin signal transduction pathway. Specifically, we demonstrate that inhibiting the expression of the DSOR1 (mitogen-activated protein kinase kinase, MAPKK) prevents the activation of the downstream ERK-A (MAPK). In contrast, blocking ERK-A expression results in increased activation of DSOR1. We also show that Drosophila AKT (DAKT) activation depends on the insulin receptor substrate, CHICO (IRS1-4). Finally, we demonstrate that blocking the expression of Drosophila PTEN results in the activation of DAKT. In all cases, the interference of the biochemical cascade by RNAi is consistent with the known steps in the pathway. We extend this powerful technique to study two proteins, DSH3PX1 and Drosophila ACK (DACK). DSH3PX1 is an SH3, phox homology domain-containing protein, and DACK is homologous to the mammalian activated Cdc42 tyrosine kinase, ACK. Using RNAi, we demonstrate that DACK is upstream of DSH3PX1 phosphorylation, making DSH3PX1 an identified downstream target/substrate of ACK-like tyrosine kinases. These experiments highlight the usefulness of RNAi in dissecting complex biochemical signaling cascades and provide a highly effective method for determining the function of the identified genes arising from the Drosophila genome sequencing project.
ISSN	00278424
e-ISSN	10916490
Journal	Proceedings of the National Academy of Sciences of the United States of America
Issue Number	12
Volume Number	97
Language	English
Publisher	National Academy of Sciences
Publisher Date	2000-07-01
Publisher Place	United States
Access Restriction	Open
Subject Keyword	RNA, Double-Stranded Physiology Signal Transduction Animals Cell Line Drosophila MAP Kinase Signaling System Phosphorylation Rabbits Cdc42 GTP-Binding Protein Metabolism Src Homology Domains Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S. Multidisciplinary
Content Type	Text
Resource Type	Article
Subject	Multidisciplinary

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