%0 Generic
%A Schilling, Marcel
%D 2007
%F heidok:7383
%K systems biology , erythropoiesis , MAP-kinase , quantitative immunoblotting , data processing
%R 10.11588/heidok.00007383
%T Strategies for High Quality Quantitative Data Generation and Dynamic Modeling of the MAP-Kinase Signaling Cascade
%U https://archiv.ub.uni-heidelberg.de/volltextserver/7383/
%X Systems biology aims at understanding how living organisms function in health and fail in disease by studying how new properties arise from dynamic interactions. Beyond qualitatively analyzing static data of individual components, dynamic quantitative data are combined with mathematical modeling to elucidate systems properties that determine cellular decisions. Such cell fate decisions are taken during erythropoiesis, when erythroid progenitor cells mature to erythrocytes by tightly regulated proliferation and differentiation processes, which are dependent on the cytokine erythropoietin (Epo) and the signaling transduction network activated by its receptor (EpoR). A major bottleneck in systems biology is the lack of high-quality quantitative data. Therefore, we developed strategies for error reduction and algorithms for automated data processing, establishing the widely used techniques of immunoprecipitation and immunoblotting as highly precise methods for the quantification of protein levels and modifications. By randomized gel-loading we prevented correlated errors and further improved our data using housekeeping proteins or adding purified proteins to immunoprecipitation in combination with criteria-based normalization, enabling the generation of large and accurate sets of quantitative data. Dysfunctional signaling in erythroid progenitor cells is associated with diseases such as anemia and leukemia, but the effects of interfering with the MAP-kinase signaling network are unknown. To causatively understand cell fate decisions and be able to predictably manipulate growth and maturation of erythroid progenitor cells, we applied a systems biology approach. We monitored components of the Epo-induced MAP-kinase network after stimulation of primary murine erythroid progenitor cells by quantitative immunoblotting. A dynamic mathematical model was compiled and kinetic parameters were estimated by multi-parameter fitting algorithms. We predicted that an increase in expression of a single ERK isoform would lead to feedback-mediated rerouting of signaling, which was confirmed by isoform-specific protein overexpression. The model was extended based on two hypotheses of negative feedback mechanisms. We experimentally confirmed feedback inhibition by phosphorylation as expressing a kinase-defective ERK isoform resulted in similar phenotypes as overexpression of the wild-type isoform. We demonstrated the influence of the integrated response of activated ERK on erythroid proliferation and differentiation, demonstrating that hyperactivation of the MAP-kinase signaling network leads to accelerated erythropoiesis but surprisingly to reduced hemoglobinization. The input for signaling is critically dependent on the receptor presence on the cell surface. Endocytosis of cell surface receptors was thought to be responsible for long-term adaptation of a cell to a continuous stimulus. However, it remained to be identified what induces the rapid decline in signal transduction after activation of a cell surface receptor. We performed dynamic modeling of EpoR endocytosis, showing that the majority of internalized Epo is recycled to the medium. Sensitivity analysis revealed that the constant turnover of the receptor on the plasma membrane and ligand-induced internalization determine the sharp peak of EpoR activation. Furthermore, we predicted that the binding kinetics, but not the binding affinity determine the strength of EpoR signaling. Surprisingly, receptor internalization is crucial for rapid activation and deactivation of signaling, but irrelevant for long-term desensitization of cells. In conclusion we employed mathematical modeling based on high-quality quantitative data, providing computational models of Epo-induced receptor endocytosis and MAP-kinase activation. Our systems biology approaches provided counterintuitive results that could not be obtained by conventional methods. For example, overexpression of an ERK isoform leads to rerouting of signaling and endocytosis of the EpoR is not required for long-term termination, but for rapid activation and deactivation of signaling. Furthermore, the mathematical models enable the identification of general systems properties and the sensitivity analyses predict targets for efficient interventions. In the future, this information can be used for drug design, opening new possibilities for treatments of anemia and leukemia.