Temporal regulation of EGF signalling networks by the scaffold protein Shc1

Abstract

Cell-surface receptors frequently employ scaffold proteins to recruit cytoplasmic targets, but the rationale for this is uncertain. Activated receptor tyrosine kinases, for example, engage scaffolds such as Shc1 that contain phosphotyrosine (pTyr) binding (PTB) domains. Using quantitative mass spectrometry, we find that Shc1 responds to epidermal growth factor (EGF) stimulation through multiple waves of distinct phosphorylation events and protein interactions. Following stimulation, Shc1 rapidly binds a group of proteins that activate pro-mitogenic/survival pathways dependent on recruitment of the Grb2 adaptor to Shc1 pTyr sites. Akt-mediated feedback phosphorylation of Shc1 Ser29 then recruits the Ptpn12 tyrosine phosphatase. This is followed by a sub-network of proteins involved in cytoskeletal reorganization, trafficking and signal termination that binds Shc1 with delayed kinetics, largely through the SgK269 pseudokinase/adaptor protein. Ptpn12 acts as a switch to convert Shc1 from pTyr/Grb2-based signaling to SgK269-mediated pathways that regulate cell invasion and morphogenesis. The Shc1 scaffold therefore directs the temporal flow of signaling information following EGF stimulation.

Early signaling dynamics of the epidermal growth factor receptor

“…Manipulating components within the network experimentally and computationally has uncovered many features of the system that influence behaviors such as proliferation and survival (4)(5)(6). With many phenotypic responses occurring on the order of hours to days, most phosphorylation measurements have been on the timescale of minutes to hours, when phosphorylation levels are highest.…”

A Hydraulic Model Is Compatible with Rapid Changes in Leaf Elongation under Fluctuating Evaporative Demand and Soil Water Status

“…A whole cell cycle takes about 1 d (Granier and Tardieu, 1998;Granier et al, 2000), changes in cell wall properties take minutes to hours (Chazen and Neumann, 1994), and hydraulic processes occur over seconds to minutes (Ye and Steudle, 2006;Tang and Boyer, 2008;Parent et al, 2009). While analysis of time constants is a common method to identify the most likely mechanisms affecting time courses in physics (Kim et al, 2008;Knowles et al, 2009) or enzymology (Schweizer et al, 1998;Wang et al, 2007;Zheng et al, 2013), it is less common in studies of growth or of genomics applied to responses to environmental conditions. The progress of phenotyping now allows one to obtain a large number of time courses of LER, transpiration, and environmental conditions with a time step of minutes (Sadok et al, 2007), thereby making possible the use of this method.…”

CoreFlow: A computational platform for integration, analysis and modeling of complex biological data

“…Our labs have used it in over fifty very diverse projects that include large-scale mass spectrometry, RNAi, next-generation sequencing, imaging and animal models [13][14][15][16]. We typically use it in conjunction with complementary tools targeted to a particular experimental application (e.g., MaxQuant [4] and Proteome Discoverer (ThermoScientific) for mass spectrometry quantitation).…”

“…In a recent paper involving MRM [16], CoreFlow was used to normalize all samples within a time course to the bait, examine reproducibility between samples, and cluster proteins with similar profiles (Fig. 2).…”

“…Trend smoothing was applied by using the spline function in R to identify the average trend (gray shaded line). Complete details of the Shc1 study are provided in [16]. …”