title: Non-canonical Activation of Akt in Serum-Stimulated Fibroblasts, Revealed by Comparative Modeling of Pathway Dynamics.
abstract: The dynamic behaviors of signaling pathways can provide clues to pathway mechanisms. In cancer cells, excessive phosphorylation and activation of the Akt pathway is responsible for cell survival advantages. In normal cells, serum stimulation causes brief peaks of extremely high Akt phosphorylation before reaching a moderate steady-state. Previous modeling assumed this peak and decline behavior (i.e., "overshoot") was due to receptor internalization. In this work, we modeled the dynamics of the overshoot as a tool for gaining insight into Akt pathway function. We built an ordinary differential equation (ODE) model describing pathway activation immediately upstream of Akt phosphorylation at Thr308 (Aktp308). The model was fit to experimental measurements of Aktp308, total Akt, and phosphatidylinositol (3,4,5)-trisphosphate (PIP3), from mouse embryonic fibroblasts with serum stimulation. The canonical Akt activation model (the null hypothesis) was unable to recapitulate the observed delay between the peak of PIP3 (at 2 minutes), and the peak of Aktp308 (at 30-60 minutes). From this we conclude that the peak and decline behavior of Aktp308 is not caused by PIP3 dynamics. Models for alternative hypotheses were constructed by allowing an arbitrary dynamic curve to perturb each of 5 steps of the pathway. All 5 of the alternative models could reproduce the observed delay. To distinguish among the alternatives, simulations suggested which species and timepoints would show strong differences. Time-series experiments with membrane fractionation and PI3K inhibition were performed, and incompatible hypotheses were excluded. We conclude that the peak and decline behavior of Aktp308 is caused by a non-canonical effect that retains Akt at the membrane, and not by receptor internalization. Furthermore, we provide a novel spline-based method for simulating the network implications of an unknown effect, and we demonstrate a process of hypothesis management for guiding efficient experiments.
authors.affiliation: Computational Systems Biology Programme, Singapore-MIT Alliance, Singapore.|Systems Biology Institute (SBI), Clayton, Victoria, Australia.|Australian Regenerative Medicine Institute and Faculty of IT, Monash University, Clayton, Victoria, Australia.
authors.forename: Tri Hieu
authors.initials: TH
authors.lastname: Nim
authors.affiliation: Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
authors.forename: Le
authors.initials: L
authors.lastname: Luo
authors.affiliation: Computational Systems Biology Programme, Singapore-MIT Alliance, Singapore.|Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America.
authors.forename: Jacob K
authors.initials: JK
authors.lastname: White
authors.affiliation: Systems Biology Institute (SBI), Clayton, Victoria, Australia.|Graduate School of Integrative Sciences and Engineering, National University of Singapore, Singapore.
authors.forename: Marie-Véronique
authors.initials: MV
authors.lastname: Clément
authors.affiliation: Computational Systems Biology Programme, Singapore-MIT Alliance, Singapore.|Duke-NUS Graduate Medical School Singapore, National University of Singapore, Singapore.
authors.forename: Lisa
authors.initials: L
authors.lastname: Tucker-Kellogg
mesh_terms: D000818:Animals; D002460:Cell Line; D019295:Computational Biology; D005347:Fibroblasts / Q000378:metabolism*; D051379:Mice; D008954:Models, Biological; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*
chemical_list: D018129:Phosphatidylinositol Phosphates; C060974:phosphatidylinositol 3,4,5-triphosphate; D051057:Proto-Oncogene Proteins c-akt