Mechanism of Smad2/3 nucleocytoplasmic shuttling
Mechanism of Smad2/3 nucleocytoplasmic shuttling
Disciplines
Biology (100%)
Keywords
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TGF-beta,
Photoactivatable GFP,
Smad,
Karopherin,
Nucleocytoplasmic,
Mechanisms of Signal Transduction
The growth factor TGF-beta plays a key role in tumour formation. Binding of TGF-beta to specific cell surface receptors leads to phosphorylation of intracellular proteins, termed Smad2 and Smad3. This modification allows them to bind the co-Smad Smad4 and to move into the nucleus. Smad proteins not only transmit the TGF-beta induced signal from the cell membrane into the cell nucleus, but they concomitantly participate in the regulation of gene transcription, thereby mediating the cell`s reaction to the incoming TGF-beta signal. Notably, Smad proteins do not remain statically within the nucleus during active signalling, but rather shuttle between the nucleus and the cell membrane thereby constantly monitoring the intensity of the incoming signal. However, the exact mechanism of Smad shuttling is poorly understood, as critical regulatory components of the nucleocytoplasmic transport machinery remain still unidentified. A detailed knowledge of the exact molecular mechanisms of Smad nuclear accumulation and shuttling is crucial for the development of potential chemotherapeutic agents that are able to interfere with the pathway. This project aims at a close characterisation of the exact mechanism by which TGF-ß leads to nuclear accumulation of R-Smads. Moreover, nuclear transport receptors (karyopherins) or other proteins involved in Smad import and export will be identified by an siRNA library-based screen. Initially, GFP photoactivation experiments will allow detailed studies of nucleocytoplasmic transport kinetics in real time in vivo. Permeabilised cells depleted of cytoplasm will be used to directly compare the import/export characteristics of recombinant full length phosphorylated and unphosphorylated Smad2 under different conditions. The same assay and a quantitative in vivo export assay will be used to study proteins found in the RNAi screen in greater detail.