Final Activity Report Summary - SMAD SHUTTLING (TGF-beta signal transduction:mechanisms of smad2/3 nucleocytoplasmic transport) TGF-beta is a multipotent growth factor involved in embryogenesis and tissue homeostasis. TGF-beta-type ligands signal through Smad proteins, a subset of which are phosphorylated in response to TGF-beta, which creates the interaction interface enabling Smads to form complexes that then accumulate in the nucleus, where they are directly involved in target gene expression. Perturbation of this pathway has severe implications on embryogenesis and can cause cancer in adult organisms. Nuclear Smad accumulation is a key feature of the pathway, and we have aimed to elucidate quantitative aspects, in particular how the intensity and duration of the active signal is relayed into the nucleus.To this end, we have created human cell lines expressing Smads fused to Green fluorescent protein (GFP), allowing as to study the kinetics of Smad nucleocytoplasmic dynamics in vivo by fluorescence perturbation approaches such as photobleaching and photoactivation on a laser scanning confocal microscope. We have found that nuclear accumulation of Smads in response to TGF-beta is not static, but is dynamically maintained by continuous Smad phosphorylation in the cytoplasm by active receptors and dephosphorylation in the nucleus. These two processes are coupled by Smad nucleocytoplasmic cycling. Importantly, we find that nuclear accumulation is caused by a pronounced export deficiency of complexed Smad, superseding earlier theories of a release of Smads from cytoplasmic retention in response to TGF-beta as the driving force of nuclear accumulation.The quantitative information obtained by fluorescence perturbation was used together with other time course data to establish a mathematical model of the nucleocytoplasmic dynamics of Smads. We were able to recreate the behaviour of the biological system using a system of ordinary differential equations. The model provided novel insights into mechanistic details of the biological system. It became now clear that nuclear retention of complexed Smads is not sufficient to explain the observed accumulation kinetics, but that Smad complexes also need to be imported into the nucleus faster than monomeric Smads. Most importantly, the model demonstrates convincingly that Smad nucleocytoplasmic dynamics are able to faithfully translate intensity and duration of the extracellular signal into a corresponding amount of nuclear Smad complexes, and hence appropriate target gene expression. We thus explain how the quantitative features of the signal are transmitted into the nucleus, which is a key insight of huge relevance for the understanding of how morphogenic gradients are interpreted during embryonic development.The second part of the work carried out during the funding period was the identification of PPP2R2D, a regulatory B-subunit of the protein phosphatase PP2A, as a novel factor limiting the TGF-beta response to signal intensities above a critical threshold. We also identified the closely related B-subunit PPP2R2A as a protein protecting the type I receptors ALK4 and ALK5 from lysosomal degradation, thus augmenting TGF-beta signalling. These findings are of particular interest, first, because manipulation of the levels of these proteins has extreme phenotypic effects on early Xenopus development, second, because we demonstrate for the first time specific, non-redundant functions of PPP2R2A and PPP2R2D, and, third, because parts of these functions are conserved from fly to man.
