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.