The mammalian target of rapamycin (mTOR) is a serine-threonine kinase that acts as a central mediator of translation and plays important roles in cell growth, synaptic plasticity, cancer, and a wide range of developmental disorders. The signaling cascade linking lipid kinases (phosphoinositide 3-kinases), protein kinases (AKT), and translation initiation complexes (EIFs) to mTOR has been extensively modeled, but does not fully describe mTOR system behavior. Here, we use quantitative multiplex coimmunoprecipitation to monitor a protein interaction network (PIN) composed of 300+ binary interactions among mTOR-related proteins. Using a simple model system of serum-deprived or fresh-media-fed mouse 3T3 fibroblasts, we observed extensive PIN remodeling involving 27+ individual protein interactions after 1 h, despite phosphorylation changes observed after only 5 min. Using small molecule inhibitors of phosphoinositide 3-kinase, AKT, mTOR, MEK and ERK, we define subsets of the PIN, termed "modules", that respond differently to each inhibitor. Using primary fibroblasts from individuals with overgrowth disorders caused by pathogenic PIK3CA or MTOR variants, we find that hyperactivation of mTOR pathway components is reflected in a hyperactive PIN. Our data define a "modular" organization of the mTOR PIN in which coordinated groups of interactions respond to the activation or inhibition of distinct nodes, and demonstrate that kinase inhibitors affect the modular network architecture in a complex manner, inconsistent with simple linear models of signal transduction.
Signal Transduction , TOR Serine-Threonine Kinases , Animals , Mice , Phosphorylation , Protein Interaction Maps , TOR Serine-Threonine Kinases/antagonists & inhibitors , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolism , Protein Kinase Inhibitors/pharmacology , NIH 3T3 Cells , Cells, Cultured , Humans , MAP Kinase Signaling System/drug effects , Mutation
The mammalian target of rapamycin (mTOR) is a serine-threonine kinase that acts as a central mediator of translation, and plays important roles in cell growth, synaptic plasticity, cancer, and a wide range of developmental disorders. The signaling cascade linking lipid kinases (PI3Ks), protein kinases (AKT) and translation initiation complexes (EIFs) to mTOR has been extensively modeled, but does not fully describe mTOR system behavior. Here, we use quantitative multiplex co-immunoprecipitation to monitor a protein interaction network (PIN) composed of 300+ binary interactions among mTOR-related proteins. Using a simple model system of serum deprived or fresh-media-fed mouse 3T3 fibroblasts, we observed extensive PIN remodeling involving 27+ individual protein interactions after one hour, despite phosphorylation changes observed after only five minutes. Using small molecule inhibitors of PI3K, AKT, mTOR, MEK and ERK, we define subsets of the PIN, termed 'modules', that respond differently to each inhibitor. Using primary fibroblasts from individuals with overgrowth disorders caused by pathogenic PIK3CA or MTOR variants, we find that hyperactivation of mTOR pathway components is reflected in a hyperactive PIN. Our data define a "modular" organization of the mTOR PIN in which coordinated groups of interactions respond to activation or inhibition of distinct nodes, and demonstrate that kinase inhibitors affect the modular network architecture in a complex manner, inconsistent with simple linear models of signal transduction.
Patients harboring mutations in the PI3K-AKT-MTOR pathway-encoding genes often develop a spectrum of neurodevelopmental disorders including epilepsy. A significant proportion remains unresponsive to conventional anti-seizure medications. Understanding mutation-specific pathophysiology is thus critical for molecularly targeted therapies. We previously determined that mouse models expressing a patient-related activating mutation in PIK3CA, encoding the p110α catalytic subunit of phosphoinositide-3-kinase (PI3K), are epileptic and acutely treatable by PI3K inhibition, irrespective of dysmorphology. Here we report the physiological mechanisms underlying this dysregulated neuronal excitability. In vivo, we demonstrate epileptiform events in the Pik3ca mutant hippocampus. By ex vivo analyses, we show that Pik3ca-driven hyperactivation of hippocampal pyramidal neurons is mediated by changes in multiple non-synaptic, cell-intrinsic properties. Finally, we report that acute inhibition of PI3K or AKT, but not MTOR activity, suppresses the intrinsic hyperactivity of the mutant neurons. These acute mechanisms are distinct from those causing neuronal hyperactivity in other AKT-MTOR epileptic models and define parameters to facilitate the development of new molecularly rational therapeutic interventions for intractable epilepsy.
