B6_SMAD Signalling

Switching Towards Termination-Modullation of the Dynamic Behavior of the SMAD Signaling Cascade

DKFZ (Heidelberg)
PD Dr. Ursula Klingmüller (PL), Dr. Peter Nickel (PhD), Dipl.- Biol. Sebastian Bohl (PhD student)

Center for Data Analysisi and Modelling (University of Freiburg):
Prof. Dr. Jens Timmer (PL)

Project summary:

Based on time-resolved quantitative data for the activation of the SMAD signaling cascade in primary hepatocytes (Klingmüller et al., IEE Proc Systems Biology) we established a mathematical model indicating that cytoplasmic retention factors constitute negative feed-back loops and critically determine the dynamic behavior of the signaling cascade. To extent the model of TGF-β mediated signal transduction, we will examine in addition to SMAD2/3 phosphorylation the activation of the TGF-β- activated kinase (TAK)1 and the Nemo-like kinase (NLK). To understand mechanisms promoting the switch towards termination of hepatocyte regeneration, we will monitor cross-talk with other signaling cascades that could influence signal integration by the TGF-β stimulated signaling. Since recent evidence suggests that serine phosphorylation of STAT3 contributes to TGF-β-mediated developmental processes, potentially including termination of hepatocyte regeneration, we will compare the induction of target genes in response to TGF-β stimulation in normal versus hepatocytes obtained from S727A STAT3 knock-in mice (collaboration with Project B1). Furthermore, we will examine the induction of the phosphoinositide-5-phosphatase SHIP-1 that is a target gene of SMAD2/3. Since SHIP-1 is an important negative regulator of PI3 kinase signaling, a signaling cascade that is activated by the c-Met receptor during the proliferative phase, we will focus in collaboration with Project B5 on the suppressive effects of SMAD signaling on c-Met receptor signaling. The stoichiometry of pathway components will be monitored by quantitative proteomics in collaboration with Project A2 and systems-wide analysis will be performed in collaboration with Project A3. Target gene induction will be examined initially by microarray analysis and later on by qRT-PCR. To address spatial effects, fluorescently labeled SMAD and TAK1 will be expressed at endogenous levels by applying the Tet-inducible system developed in Project B5. The dynamics of nuclearcytoplasmic cycling of SMAD will be quantitatively monitored by live cell imaging and the kinetics of the TAK1 and STAT3 interaction will be analyzed by fluorescence resonance energy transfer (FRET). The acquired data will be used to establish a spatio-temporal model. The comprehensive model of SMAD signaling will be employed to identify mechanisms promoting the coordinated induction of termination of hepatocytes.

Former coworkers: Thomas Maiwald