Analysis of protein phosphatase function in Drosophila cells using RNA interference.
Double stranded RNA-mediated RNA interference is an effective method to downregulate the levels of protein phosphatases in Drosophila S2 cells. In many cases, nearly complete ablation of the targeted protein can be achieved. RNAi-mediated knockdown of protein phosphatases is akin to pharmacological inhibition with drugs and can be used to determine the roles of specific protein phosphatases in intact cells. RNAi can avoid the problems associated with less than adequate specificity of phosphatase inhibitors. Although information about the signaling pathways present in Drosophila S2 cells is not as well developed as many mammalian cell lines, the Drosophila system is particularly attractive for the study of oligomeric phosphatases like PP2A. Drosophila has far fewer isoforms for the phosphatases we have examined. This is especially true of the genes for PP2A regulatory subunits where over 50 isoforms are present in mammals but only four are present in Drosophila. Once hypotheses regarding phosphatase function have been generated from RNAi experiments in S2 cells, they can potentially be tested utilizing recent advances in the use of siRNAs to conduct RNAi experiments in mammalian cell lines. RNAi in Drosophila S2 cells has proven to be a powerful technique for identifying physiological functions of signaling proteins. The RNAi method is straightforward and works routinely with almost all proteins. RNAi in S2 cells can be used to assess the role of signaling proteins in specific pathways and as a screening tool to identify new roles for signaling molecules. For example, results from RNAi analysis of PP2A show that regulation of MAP kinase signaling involves the R2/B regulatory subunit and that the R5/B56 subunits play a previously unidentified role in apoptosis. While RNAi in Drosophila S2 cells is a powerful tool for analyzing protein function, the method does have limitations. Foremost, cells may exhibit an RNAi response to any nonspecific dsRNA, even in the absence of interferon. Therefore, physiological processes that respond to nonspecific dsRNA will be difficult to study. A second limitation is the need to produce antibodies that react with Drosophila isoforms. We have found that many antibodies to mammalian protein phosphatases do not cross-react with the corresponding Drosophila proteins. Finally, the physiology and signaling pathways of S2 cells have not been extensively studied. This lack of information limits the number of available readouts that can be used when assessing the effects of protein knockdowns.
Silverstein
Adam M
AM
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9041, USA.
Mumby
Marc C
MC
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers; D018076:DNA, Complementary; D029721:Drosophila Proteins / Q000378:metabolism; D004331:Drosophila melanogaster / Q000196:embryology / Q000201:enzymology* / Q000235:genetics; D004625:Embryo, Nonmammalian / Q000201:enzymology; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008322:Mammals; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012330:RNA, Double-Stranded / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection / Q000379:methods
D017931:DNA Primers; D018076:DNA, Complementary; D029721:Drosophila Proteins; D007527:Isoenzymes; D012330:RNA, Double-Stranded; D034741:RNA, Small Interfering; D010749:Phosphoprotein Phosphatases