Abstract
The signal transduction pathway wherein mTOR regulates cellular growth and proliferation is an active target for drug discovery. The search for new mTOR inhibitors has recently yielded a handful of promising compounds that hold therapeutic potential. This search has been limited by the lack of a high-throughput assay to monitor the phosphorylation of a direct rapamycin-sensitive mTOR substrate in cells. Here we describe a novel cell-based chemical genetic screen useful for efficiently monitoring mTOR signaling to 4E-BPs in response to stimuli. The screen is based on the nuclear accumulation of eIF4E, which occurs in a 4E-BP-dependent manner specifically upon inhibition of mTOR signaling. Using this assay in a small-scale screen, we have identified several compounds not previously known to inhibit mTOR signaling, demonstrating that this method can be adapted to larger screens.
Copyright 2009 Elsevier Ltd. All rights reserved.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Adaptor Proteins, Signal Transducing
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Animals
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Carrier Proteins / genetics
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Carrier Proteins / metabolism
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Cell Cycle Proteins
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Cell Line
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Cell Nucleus / metabolism
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Chromones / pharmacology
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Drug Discovery / methods*
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Enzyme Inhibitors / pharmacology
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Eukaryotic Initiation Factor-4E / genetics
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Eukaryotic Initiation Factor-4E / metabolism*
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Eukaryotic Initiation Factors / deficiency
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Eukaryotic Initiation Factors / genetics
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Eukaryotic Initiation Factors / metabolism
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HeLa Cells
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Humans
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Intracellular Signaling Peptides and Proteins / antagonists & inhibitors
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Intracellular Signaling Peptides and Proteins / metabolism*
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Mice
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Mice, Knockout
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Morpholines / pharmacology
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Phosphoproteins / deficiency
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Phosphoproteins / genetics
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Phosphoproteins / metabolism
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Phosphorylation
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Protein Serine-Threonine Kinases / antagonists & inhibitors
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Protein Serine-Threonine Kinases / metabolism*
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Signal Transduction*
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Sirolimus / pharmacology
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TOR Serine-Threonine Kinases
Substances
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Adaptor Proteins, Signal Transducing
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Carrier Proteins
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Cell Cycle Proteins
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Chromones
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Eif4ebp1 protein, mouse
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Eif4ebp2 protein, mouse
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Enzyme Inhibitors
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Eukaryotic Initiation Factor-4E
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Eukaryotic Initiation Factors
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Intracellular Signaling Peptides and Proteins
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Morpholines
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Phosphoproteins
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2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
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MTOR protein, human
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mTOR protein, mouse
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Protein Serine-Threonine Kinases
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TOR Serine-Threonine Kinases
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Sirolimus