PI3K‐dependent cross‐talk interactions converge with Ras as quantifiable inputs integrated by Erk

Abstract

Although it is appreciated that canonical signal‐transduction pathways represent dominant modes of regulation embedded in larger interaction networks, relatively little has been done to quantify pathway cross‐talk in such networks. Through quantitative measurements that systematically canvas an array of stimulation and molecular perturbation conditions, together with computational modeling and analysis, we have elucidated cross‐talk mechanisms in the platelet‐derived growth factor (PDGF) receptor signaling network, in which phosphoinositide 3‐kinase (PI3K) and Ras/extracellular signal‐regulated kinase (Erk) pathways are prominently activated. We show that, while PI3K signaling is insulated from cross‐talk, PI3K enhances Erk activation at points both upstream and downstream of Ras. The magnitudes of these effects depend strongly on the stimulation conditions, subject to saturation effects in the respective pathways and negative feedback loops. Motivated by those dynamics, a kinetic model of the network was formulated and used to precisely quantify the relative contributions of PI3K‐dependent and ‐independent modes of Ras/Erk activation. ### Synopsis Historically, intracellular signal transduction has been characterized in terms of distinct pathways, comprised of linear, sequential activation processes. This concept is exemplified by the canonical mitogen‐activated protein kinase (MAPK) cascades, such as the Ras→Raf→MEK→extracellular signal‐regulated kinase (Erk) pathway in mammals. Our current understanding of signal‐transduction networks includes more complex interactions, including those between the classically defined pathways (cross‐talk) and those responsible for feedback regulation/reinforcement. Although there is a great deal of qualitative information available as to how signaling networks are wired, an as yet unmet challenge is their systematic quantification; to understand cell regulation at the molecular level, we need to know the relative magnitudes of classical and cross‐talk interactions that converge and collaborate to activate key nodes in the network. In most signaling networks, Erk isoforms are both master integrators of upstream inputs and master controllers of transcription factors and other effectors ([Kolch, 2000][1]). In this study, we systematically combine quantitative biochemical measurements and computational modeling to quantify the magnitudes of cross‐talk and negative feedback interactions in a signaling network. We investigated signaling mediated by platelet‐derived growth factor (PDGF) receptors in fibroblasts, leading to activation of the Ras/Erk pathway and exceptionally robust activation of phosphoinositide 3‐kinases (PI3Ks), which control responses such as directed cell migration, survival, and proliferation through the production of specific lipid second messengers ([Vanhaesebroeck et al , 2001][2]; [Engelman et al , 2006][3]; [Hawkins et al , 2006][4]). Although cross‐talk between the PI3K and Ras/Erk pathways has been studied extensively, a definitive pattern of regulation has not yet emerged, because the two pathways apparently affect each other in various ways and in a context‐dependent manner. We measured Erk phosphorylation and PI3K‐dependent Akt phosphorylation in PDGF‐stimulated NIH 3T3 fibroblasts by quantitative immunoblotting for an array of 126 experimental conditions, sampling different combinations of ligand dose, stimulation time, and molecular manipulation; the Erk data set is shown in [Figure 1][5]. Considering biological replicates and parallel determination of total Erk and Akt levels, this data set comprises 2772 total measurements. As shown in [Figure 1B and C][5], blocking the activity of either Ras or PI3K, by the expression of dominant‐negative (S17N) H‐Ras or incubation with a pharmacological inhibitor, respectively, only partially reduces PDGF‐stimulated Erk phosphorylation; the degree of inhibition depends, in a complex manner, on both stimulation dose and time. By comparison, the Akt phosphorylation results showed that the PI3K pathway is not significantly affected by perturbations affecting Ras and Erk; cross‐talk is apparently unidirectional, from PI3K to Ras/Erk, in this network. We went on to show that simultaneous inhibition of Ras and PI3K almost completely abolished PDGF‐stimulated Erk phosphorylation, indicating that Ras and PI3K are responsible for all of the major pathways from PDGF receptors to Erk, and at least one mode of PI3K‐dependent cross‐talk to Erk is independent of Ras. We further refined this conceptual model by measuring Ras‐GTP, the active form of Ras, for selected experimental conditions using a quantitative enzymatic assay ([Scheele et al , 1995][6]; [Kaur et al , 2006][7]). These experiments showed that PI3K‐dependent cross‐talk affects the Erk pathway both downstream and upstream of Ras, and they also served to characterize a known negative feedback mechanism affecting Ras as a consequence of signaling through the Erk cascade. A second regulatory loop that we characterized for this pathway is the increased expression of MAPK phosphatase‐1 (MKP‐1), which responds to and antagonizes Erk activation. Motivated by the dynamics revealed in our unique data set, a kinetic model of the network was formulated and used to precisely quantify the relative contributions of PI3K‐dependent and ‐independent modes of Ras/Erk activation. In the context of the model, the magnitudes of the Ras‐ and PI3K‐dependent inputs converging on MEK/Erk determines the saturability of Erk phosphorylation with respect to PDGF dose and also the degree to which the response adapts, and conversely, the observed dynamics can be used to quantify the magnitudes of the inputs. Unspecified parameter...