Empirical inference of circuitry and plasticity in a kinase signaling network

Abstract

Our understanding of physiology and disease is hampered by the difficulty of measuring the circuitry and plasticity of signaling networks that regulate cell biology, and how these relate to phenotypes. Here, using mass spectrometry-based phosphoproteomics, we systematically characterized the topology of a network comprising the PI3K/Akt/mTOR and MEK/ERK signaling axes and confirmed its biological relevance by assessing its dynamics upon EGF and IGF1 stimulation. Measuring the activity of this network in models of acquired drug resistance revealed that cells chronically treated with PI3K or mTORC1/2 inhibitors differed in the way their networks were remodeled. Unexpectedly, we also observed a degree of heterogeneity in the network state between cells resistant to the same inhibitor, indicating that even identical and carefully controlled experimental conditions can give rise to the evolution of distinct kinase network statuses. These data suggest that the initial conditions of the system do not necessarily determine the mechanism by which cancer cells become resistant to PI3K/mTOR targeted therapies. The patterns of signaling network activity observed in the resistant cells mirrored the patterns of response to several drug combination treatments, suggesting that the activity of the defined signaling network truly reflected the evolved phenotypic diversity.signaling network | phosphoproteomics | systems biology | PI3K | MEK

Disulfide-activated protein kinase G Iα regulates cardiac diastolic relaxation and fine-tunes the Frank–Starling response

“…18). Phosphopeptide abundance—directly relating to the amount of substrate phosphorylation—was determined by a label-free quantitative analysis1920 (Supplementary Dataset 1). Substrate phosphorylation was assessed when PKGIα was activated by the Cys42 disulfide bond or via the classical pathway with cGMP, and compared with phosphorylation when PKGIα was in its basal ‘unactivated' state (control; Fig.…”

“…The abundance of phosphopeptides in each of the kinase reaction mixtures described above was determined by a label-free quantitative phosphoproteomic analysis1920. LC–MS/MS analysis was performed on an Ultimate 3000, nLC system (ThermoFisher) connected to an LTQ Orbitrap XL instrument to.…”

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“…Samples were processed as described60, with the following modifications. Primary MEFs were lysed 48 or 96 h after 4-OHT treatment in 1 ml of urea buffer per 15 cm tissue culture dish.…”

Phosphotyrosine-based-phosphoproteomics scaled-down to biopsy level for analysis of individual tumor biology and treatment selection

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