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Signal transduction networks can be perturbed biochemically, genetically, and pharmacologically to unravel their functions. But at the systems level, it is not clear how such perturbations are best implemented to extract molecular mechanisms that underlie network function. Here, we combined pairwise perturbations with multiparameter phosphorylation measurements to reveal causal mechanisms within the signaling network response of cardiomyocytes to coxsackievirus B3 (CVB3) infection. Using all possible pairs of six kinasa inhibitors, we assembled a dynamic nine-protein phosphorylation signature of perturbed CVB3 infectivity. Cluster analysis of the resulting dataset showed repeatedly that paired inhibitor data were required for accurate data-driven predictions of kinase substrate links in the host network. With pairwise data, we also derived a highconfidence network based on partial correlations, which identified phospho-I[kappa]B[alpha] as a central 'hub' in the measured phosphorylation signature. The reconstructed network helped to connect phosphol[kappa]B[alpha] with an autocrine feedback circuit in host cells involving the proinflammatory cytokines, TNF and IL-1. Autocrine blockade substantially inhibited CVB3 progeny release and improved host cell viability, implicating TNF and IL-1 as cell autonomous components of CVB3-induced myocardial damage. We conclude that pairwise perturbations, when combined with network-level intracellular measurements, enrich for mechanisms that would be overlooked by single perturbants. pairwise perturbation | signaling network | systems biology | viral rnyocarditis | picornaviridae doi/ 10.1073/pnas.1006478107

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2008.08.034 Byline: Kevin A. Janes (1)(2), H. Christian Reinhardt (1), Michael B. Yaffe (1) Keywords: SYSBIO; SIGNALING Abstract: Signaling networks respond to diverse stimuli, but how the state of the signaling network is relayed to downstream cellular responses is unclear. We modeled how incremental activation of signaling molecules is transmitted to control apoptosis as a function of signal strength and dynamic range. A linear relationship between signal input and response output, with the dynamic range of signaling molecules uniformly distributed across activation states, most accurately predicted cellular responses. When nonlinearized signals with compressed dynamic range relay network activation to apoptosis, we observe catastrophic, stimulus-specific prediction failures. We develop a general computational technique, 'model-breakpoint analysis,' to analyze the mechanism of these failures, identifying new time- and stimulus-specific roles for Akt, ERK, and MK2 kinase activity in apoptosis, which were experimentally verified. Dynamic range is rarely measured in signal-transduction studies, but our experiments using model-breakpoint analysis suggest it may be a greater determinant of cell fate than measured signal strength. Author Affiliation: (1) Koch Institute for Integrative Cancer Research, Center for Cell Decision Processes, Department of Biology, and Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA (2) Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA Article History: Received 12 June 2007; Revised 12 June 2008; Accepted 16 August 2008 Article Note: (miscellaneous) Published: October 16, 2008

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2006.01.041 Byline: Kevin A. Janes (1)(2), Suzanne Gaudet (1)(3), John G. Albeck (1)(3), Ulrik B. Nielsen (4), Douglas A. Lauffenburger (1)(2)(3), Peter K. Sorger (1)(2)(3) Abstract: Tumor necrosis factor (TNF) is a proinflammatory cytokine that induces conflicting pro- and antiapoptotic signals whose relative strengths determine the extent of cell death. TNF receptor (TNFR) has been studied in considerable detail, but it is not known how crosstalk among antagonistic pro- and antiapoptotic signals is achieved. Here we report an experimental and computational analysis of crosstalk between prodeath TNF and prosurvival growth factors in human epithelial cells. By applying classifier-based regression to a cytokine-signaling compendium of [approximately equal to]8000 intracellular protein measurements, we demonstrate that cells respond to TNF both directly, via activated TNF receptor, and indirectly, via the sequential release of transforming growth factor-[alpha] (TGF-[alpha]), interleukin-1[alpha] (IL-1[alpha]), and IL-1 receptor antagonist (IL-1ra). We refer to the contingent and time-varying series of extracellular signals induced by TNF as an 'autocrine cascade.' Time-dependent crosstalk of synergistic and antagonistic autocrine circuits may serve to link cellular responses to the local environment. Author Affiliation: (1) Center for Cell Decision Processes, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA (2) Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA (3) Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA (4) Merrimack Pharmaceuticals, Cambridge, MA 02139, USA Article History: Received 26 May 2005; Revised 19 October 2005; Accepted 5 January 2006 Article Note: (miscellaneous) Published: March 23, 2006

Despite extensive molecular-level information on how external stimuli affect cell fate, there is minimal understanding of how such intracellular processing occurs at a systemwide level. Most extracellular 'inputs' initiate complex [...], Signal transduction pathways control cellular responses to stimuli, but it is unclear how molecular information is processed as a network. We constructed a systems model of 7980 intracellular signaling events that directly links measurements to 1440 response outputs associated with apoptosis. The model accurately predicted multiple time-dependent apoptotic responses induced by a combination of the death-inducing cytokine tumor necrosis factor with the prosurvival factors epidermal growth factor and insulin. By capturing the role of unsuspected autocrine circuits activated by transforming growth factor-α and interleukin-1α, the model revealed new molecular mechanisms connecting signaling to apoptosis. The model derived two groupings of intracellular signals that constitute fundamental dimensions (molecular 'basis axes') within the apoptotic signaling network. Projection along these axes captures the entire measured apoptotic network, suggesting that cell survival is determined by signaling through this canonical basis set.

Author(s): Kathryn Miller-Jensen [1, 2, 5]; Kevin A. Janes [1, 3, 5]; Joan S. Brugge [3]; Douglas A. Lauffenburger (corresponding author) [1, 2, 4] The fundamental components of many signalling [...]

An experimental and computational analysis of crosstalk between prodeath (Tumor necrosis factor) TNF and prosurvival growth factors in human epithelial cells are reported. The findings suggest that the extent of crosstalk between autocrine and intracellular networks and their relative physiological importance might depend on cell type and stimulus.