| Project Overview: | Although in very general terms the core TGF?/Smad pathway is well understood, there are a number of key processes for which the factors responsible are unknown. For example, we know very little about the phosphatases that remove the activating phosphates from the receptor-regulated Smads. We have recently shown that the phosphatase (PPM1A) that is published to have this activity with respect to Smad2/3, actually does not and moreover is not even in the correct cellular compartment. We understand very little about receptor regulation. In addition, a recent study in my lab of the dynamics of the signalling pathway has revealed further levels of regulation that were previously unsuspected, in particular refractory behaviour in the pathway such that a prior stimulation with TGF? renders cells completely refractory to further stimulation, until the ligand is withdrawn and signalling-competent receptors are allowed to reaccumulate at the plasma membrane, which takes 12-24 h. We have shown that this behaviour is common to different cell types, and is fundamental to TGF? signalling in vivo, being of great importance to the activities of tumour cells, which are constantly exposed to high levels of TGF? secreted from the stroma. To identify components required for regulating Smad activation and deactivation, for establishing refractory behaviour and for recovery from this state, we want to perform whole genome siRNA screens. We previously carried out several smaller scale siRNA screens with focused libraries using a transcriptional readout. However, although we obtained the E3 ubiquitin ligases, Arkadia and TIF1 gamma from these screens, which have turned out to be exceptionally important regulators in this pathway, we obtained a very large number of false positives. For these genomic screens we will therefore use a more direct readout, which involves only endogenous pathway components, specifically Smad2/3 subcellular localisation and Smad2 phosphorylation. We have adapted established lab assays using well-characterised reagents to a 384-well format. We will use three different conditions for the screens: cells stimulated for 1 h with TGF? cells stimulated for 1 h with TGF? followed by a 1 h treatment of cells with SB-431542 (inhibitor of the type I receptor) to terminate receptor activity; cells pre-treated with TGF?f or 30 h followed by a 1 h stimulation with TGF?. With regard to this third condition, we have shown that after treatment of cells with TGF? for 30 h, the ligand is completely depleted by the cells and surface receptor levels have incompletely recovered, and thus the cells are partially responsive. This contrasts with cells that have been treated with three 10 h pulses of TGF? (chronic signalling), which are completely unresponsive. We will use HaCaT cells that are efficiently transfected with siRNAs using interferin. This has been optimized. The antibody for Smad2/3 is from BD (Catalogue number 610842). The phospho-Smad2 antibody is from Millipore (Clone A5S - Catalogue number 04-953). We have optimized the immunostaining with these antibodies in the 384 well format. We will also screen for cell number and viability as well as cell shape using the Cellomics ArrayScan automated microscope. |
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| Screener: | Debbie Das (Hill Lab). Extension:2936, Rm 4 |
| Project Status: | Published |
| Publications: | The Dynamics of TGF-β Signaling Are Dictated by Receptor Trafficking via the ESCRT Machinery.. Miller et al., 2018 |
| Project Start Date: | May 2012 |
| Keywords: | Smad2/3 TGFB TGFbeta TGFß |
| Libraries Screened: |
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