Designing optimal experiments to discriminate interaction graph models.

Modern methods for the inference of cellular networks from experimental data often express nondeterminism through an ensemble of candidate models. To discriminate among these candidates new experiments need to be carried out. Theoretically, the number of possible experiments is exponential in the number of possible perturbations. In praxis, experiments are expensive and there exist several limiting constraints. Limiting factors exist on the combinations of perturbations that are technically possible, which components can be measured, and on the number of affordable experiments. Further, not all experiments are equally well suited to discriminate model candidates. The goal of optimal experiment design is to determine those experiments that discriminate most of the candidates while minimizing the costs. We present an approach for experiment planning with interaction graph models and sign consistency methods. This new approach can be used in combination with methods for network inference and consistency checking. We applied our method to study the Erythropoietin signal transduction in human kidney cells HEK293. We first used simulated experiment data from an ODE model to demonstrate in silico that our experimental design results in the inference of the gold standard model. Finally, we used the approach to plan in vivo experiments that discriminate model candidates for the Erythropoietin signal transduction in this cell line.

The multi-site docking protein Gab1 is constitutively phosphorylated independent from its recruitment to the plasma membrane in Jak2-V617F-positive cells and mediates proliferation of human erythroleukaemia cells.

The constitutively active Janus kinase 2 mutant Jak2-V617F is responsible for cytokine-independent growth of hematopoietic cells and the development of myeloproliferative neoplasms, such as polycythaemia vera and essential thrombocythaemia. Cells expressing Jak2-V617F exhibit constitutive STAT, MAPK, and PI3K signalling, and constitutive association of the multi-site docking protein Gab1 to PIP3 at the plasma membrane. Here, we demonstrate the crucial role of Gab1 for the proliferation of Jak2-V617F-positive human erythroleukaemia (HEL) cells. In Jak2-V617F-expressing cells Gab1 is constitutively phosphorylated by Erk1/2 on serine residue 552, which regulates binding to PIP3. Additionally, Gab1 is constitutively phosphorylated on tyrosine residue 627. Tyrosine 627 is a SHP2 binding site and required for Gab1-dependent Erk1/2 activation. As previously shown, Jak2-V617F-dependent Erk1/2 and PI3K activation act synergistically on the proliferation of Jak2-V617F-positive cells. Here, we examined whether constitutive membrane association of Gab1 explains cytokine-independent Gab1 phosphorylation in Jak2-V617F-expressing cells. Although we could demonstrate Jak2-V617F-dependent constitutive serine 552 and tyrosine 627 phosphorylation of Gab1, interestingly, both phosphorylations do not require binding of Gab1 to PIP3 at the plasma membrane. Instead, we observed a constitutive interaction of Gab1 with the erythropoietin receptor in Jak2-V617F-expressing cells, which depends on Janus kinase activity. Thus, constitutive Gab1-dependent signalling in Jak2-V617F-expressing cells does not occur due to the constitutive association of Gab1 with PIP3 at the plasma membrane.