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

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.

Figure 1

Specificity and duration of RNAi in S2 cell culture. (A) S2 cells were incubated with the indicated concentrations of DSH3PX1 or DACK dsRNAs. Cellular extracts were prepared after 3 days and subjected to Western analysis as described in Materials and Methods. (B) S2 cells were incubated with 37 nM DSH3PX1 dsRNA for 10–240 min. (Left) Samples undergoing the standard recovery procedure. (Right) Samples rinsed to remove the unincorporated dsRNA before addition of serum containing media. Western analysis on extracts prepared after a 3-day recovery period was performed as described using antibodies generated against DSH3PX1 and Dock. (C) S2 cells were exposed to 37 nM DSH3PX1 dsRNA as described in Materials and Methods. Cellular extracts were prepared on the indicated days and subjected to Western analysis using antibodies against DSH3PX1 and Dock. (D) The specified amounts of dsRNAs corresponding to DSH3PX1 coding sequence were added to either BG2-C6 or KC cells. Western analysis was performed as described using antibodies directed against DSH3PX1. In each panel, an arrow indicates the relative mobility of DACK, DSH3PX1, or Dock.

Figure 2

Dissection of the insulin signal transduction pathway in S2 cell culture. (A) Schematic representation of the ERK-A and PI3K branches of the insulin signal transduction pathway. The mammalian homologues of the Drosophila proteins are in parentheses. (B) S2 cells were incubated in the absence (NT) or presence of dsRNAs representing DSOR1 or ERK-A (dsRNA DSOR1/ERK-A). After 3 days, the cells were either left untreated or treated with 10 μg/ml insulin for 5 min. Cellular extracts were prepared and subjected to Western analysis using antibodies directed against phospho-ERK-A (αP-ERK-A), ERK-A (αERK-A), phospho-DSOR1 (αP- DSOR1), or DSOR1 (α- DSOR1). (C) S2 cells were treated with dsRNAs directed against the expression of CHICO, PTEN, and DPTP61F as indicated. Cellular extracts were prepared and DAKT/PKB activity measured as described in Materials and Methods.

Figure 3

DACK is upstream of DSH3PX1 phosphorylation. S2 cells stably expressing the 6xHis-tagged SH2 domain of Dock were treated with dsRNAs representing DACK and DSH3PX1. The protein complex associated with Dock's SH2 domain was purified as described in Materials and Methods and subjected to Western blot analysis using antibodies recognizing phosphotyrosine (pTyr) or DSH3PX1. The relative mobilities of DACK and DSH3PX1 are indicated.