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BioAssay: AID 686943

MAPK Signaling Pathway Screen

Receptor tyrosine kinase (RTK) signalling through extracellular signal-regulated kinases (ERKs) has pivotal roles during metazoan development, underlying processes as diverse as fate determination, differentiation, proliferation, survival, migration and growth. Abnormal RTK/ERK signalling has been extensively documented to contribute to developmental disorders and disease, most notably in more ..
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 Tested RNAi Reagents
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Active(1643)
 
 
Inactive(20890)
 
 
 Screened Genes
 Screened Genes
AID: 686943
Data Source: Drosophila RNAi Screening Center (DRSC) (DRSC-P28)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: Assay Vendor
Deposit Date: 2013-04-17

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Description:
Title: A functional RNAi screen for regulators of receptor tyrosine kinase and ERK signalling

Abstract:
Receptor tyrosine kinase (RTK) signalling through extracellular signal-regulated kinases (ERKs) has pivotal roles during metazoan development, underlying processes as diverse as fate determination, differentiation, proliferation, survival, migration and growth. Abnormal RTK/ERK signalling has been extensively documented to contribute to developmental disorders and disease, most notably in oncogenic transformation by mutant RTKs1 or downstream pathway components such as Ras and Raf2. Although the core RTK/ERK signalling cassette has been characterized by decades of research using mammalian cell culture and forward genetic screens in model organisms, signal propagation through this pathway is probably regulated by a larger network of moderate, context-specific proteins. The genes encoding these proteins may not have been discovered through traditional screens owing, in particular, to the requirement for visible phenotypes. To obtain a global view of RTK/ERK signalling, we performed an unbiased, RNA interference (RNAi), genome-wide, high-throughput screen in Drosophila cells using a novel, quantitative, cellular assay monitoring ERK activation. Here we show that ERK pathway output integrates a wide array of conserved cellular processes. Further analysis of selected components - in multiple cell types with different RTK ligands and oncogenic stimuli - validates and classifies 331 pathway regulators. The relevance of these genes is highlighted by our isolation of a Ste20-like kinase and a PPM-family phosphatase that seem to regulate RTK/ERK signalling in vivo and in mammalian cells. Novel regulators that modulate specific pathway outputs may be selective targets for drug discovery.
Protocol
Drosophila haemocytes express the insulin receptor (InR), which activates both the Akt and ERK pathways when stimulated with insulin. S2R1 cells expressing yellow fluorescent protein (YFP)-tagged Rolled were seeded in 384-well plates aliquoted with a genome-wide dsRNA collection targeting 20,420 predicted unique genes. Following knockdown, unstimulated or insulin-stimulated cells were stained with fluorescently-conjugated dpERK antibodies, and dpERK fluorescence was converted to ERK-YFP normalized Z-scores.
Comment
The activity scores are given using the standard DRSC scoring system for hits:
1 = weak hit
2 = medium hit
3 = strong hit
In many assays, only 2 is used.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Stim Insulin MAPK dpERK replicate 1Float
2Stim Insulin MAPK dpERK replicate 2Float
3Stim Insulin MAPK dpERK replicate 3Float
4Stim Insulin MAPK dpERK replicate 4Float
5Stim Insulin MAPK dpERK replicate 5Float
6Stim Insulin MAPK dpERK replicate 6Float
7Stim Insulin MAPK ERK replicate 1Float
8Stim Insulin MAPK ERK replicate 2Float
9Stim Insulin MAPK ERK replicate 3Float
10Stim Insulin MAPK ERK replicate 4Float
11Stim Insulin MAPK ERK replicate 5Float
12Stim Insulin MAPK ERK replicate 6Float
13Stim Insulin MAPK Normalized dpERK Z-score replicate 1Float
14Stim Insulin MAPK Normalized dpERK Z-score replicate 2Float
15Stim Insulin MAPK Normalized dpERK Z-score replicate 3Float
16Stim Insulin MAPK Normalized dpERK Z-score replicate 4Float
17Stim Insulin MAPK Normalized dpERK Z-score replicate 5Float
18Stim Insulin MAPK Normalized dpERK Z-score replicate 6Float
19Stim Insulin MAPK YFP Z-score replicate 1Float
20Stim Insulin MAPK YFP Z-score replicate 2Float
21Stim Insulin MAPK YFP Z-score replicate 3Float
22Stim Insulin MAPK YFP Z-score replicate 4Float
23Stim Insulin MAPK YFP Z-score replicate 5Float
24Stim Insulin MAPK YFP Z-score replicate 6Float
25Unstim Insulin MAPK dpERK replicate 1Float
26Unstim Insulin MAPK dpERK replicate 2Float
27Unstim Insulin MAPK dpERK replicate 3Float
28Unstim Insulin MAPK dpERK replicate 4Float
29Unstim Insulin MAPK dpERK replicate 5Float
30Unstim Insulin MAPK dpERK replicate 6Float
31Unstim Insulin MAPK ERK replicate 1Float
32Unstim Insulin MAPK ERK replicate 2Float
33Unstim Insulin MAPK ERK replicate 3Float
34Unstim Insulin MAPK ERK replicate 4Float
35Unstim Insulin MAPK ERK replicate 5Float
36Unstim Insulin MAPK ERK replicate 6Float
37Unstim Insulin MAPK Normalized dpERK Z-score replicate 1Float
38Unstim Insulin MAPK Normalized dpERK Z-score replicate 2Float
39Unstim Insulin MAPK Normalized dpERK Z-score replicate 3Float
40Unstim Insulin MAPK Normalized dpERK Z-score replicate 4Float
41Unstim Insulin MAPK Normalized dpERK Z-score replicate 5Float
42Unstim Insulin MAPK Normalized dpERK Z-score replicate 6Float
43Unstim Insulin MAPK YFP Z-score replicate 1Float
44Unstim Insulin MAPK YFP Z-score replicate 2Float
45Unstim Insulin MAPK YFP Z-score replicate 3Float
46Unstim Insulin MAPK YFP Z-score replicate 4Float
47Unstim Insulin MAPK YFP Z-score replicate 5Float
48Unstim Insulin MAPK YFP Z-score replicate 6Float
49Insulin Hits Average normalized baseline dpERK Z-scoreFloat
50Insulin Hits Average normalized stimulated dpERK Z-scoreFloat
51Insulin Hits Average baseline YFP Z-scoreFloat
52Insulin Hits Average stimulated YFP Z-scoreFloat
53Predicted Target GeneString

RNAi Target.
Additional Information
Substance Type: Nucleotide

Data Table (Concise)
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