During the past decade, our knowledge of molecular mechanisms involved in growth factor signaling has proliferated almost explosively. However, the kinetics and control of information transfer through signaling networks remain poorly understood. This paper combines experimental kinetic analysis and computational modeling of the short term pattern of cellular responses to epidermal growth factor (EGF) in isolated hepatocytes. The experimental data show transient tyrosine phosphorylation of the EGF receptor (EGFR) and transient or sustained response patterns in multiple signaling proteins targeted by EGFR. Transient responses exhibit pronounced maxima, reached within 15-30 s of EGF stimulation and followed by a decline to relatively low (quasi-steady-state) levels. In contrast to earlier suggestions, we demonstrate that the experimentally observed transients can be accounted for without requiring receptor-mediated activation of specific tyrosine phosphatases, following EGF stimulation. The kinetic model predicts how the cellular response is controlled by the relative levels and activity states of signaling proteins and under what conditions activation patterns are transient or sustained. EGFR signaling patterns appear to be robust with respect to variations in many elemental rate constants within the range of experimentally measured values. On the other hand, we specify which changes in the kinetic scheme, rate constants, and total amounts of molecular factors involved are incompatible with the experimentally observed kinetics of signal transfer. Quantitation of signaling network responses to growth factors allows us to assess how cells process information controlling their growth and differentiation.

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A systems biology approach is applied to gain a quantitative understanding of the integration of signalling by the small GTPase Ras. The Ras protein acts as a critical switch in response to signals that determine the cell's fate. In unstimulated cells, Ras switching between an inactive GDP-binding and active GTP-binding state is controlled by the intrinsic catalytic activities of Ras. The calculated high sensitivity of the basal Ras-GTP fraction to changes in the rate constant of GTP-hydrolysis by Ras can account for the carcinogenic potential of Ras mutants with decreased GTPase activities. Extracelluar stimuli initiate Ras interactions with GDP/GTP exchange factors such as SOS, and GTP-hydrolysis activating proteins such as RasGAP. Our data on freshly isolated hepatocytes stimulated with epidermal growth factor (EGF) show transient SOS activation and sustained Ras-GTP patterns. We demonstrate that these dose-response data can only be explained by transient RasGAP activitation, and not by merely switching off the SOS signal, e.g. by inhibitory phosphorylation of SOS. A transient RasGAP activity can be brought about by a number of mechanisms. A comprehensive kinetic model of the EGF receptor (EGFR) network was developed to explore feasible molecular scenarios, including the receptor-mediated recruitment of SOS and RasGAP to the plasma membrane, phosphorylation of RasGAP and p190 RhoGAP by soluble tyrosine kinases, and RasGAP interactions with phosphoinositides and p190 RhoGAP. We show that a transient RasGAP association with EGFR followed by the capture of RasGAP through the formation of complexes with p190 RhoGAP can account for data on hepatocytes. In summary, our results demonstrate that a combination of experimental monitoring and integrated dynamic analysis is capable of dissecting regulatory mechanisms that govern cellular signal transduction.

Stimulation of isolated hepatocytes with epidermal growth factor (EGF) causes rapid tyrosine phosphorylation of the EGF receptor (EGFR) and adapter/target proteins, which was monitored with 1 and 2 s resolution at 37, 20, and 4 degrees C. The temporal responses detected for multiple signaling proteins involve both transient and sustained phosphorylation patterns, which change dramatically at low temperatures. To account quantitatively for complex responses, we employed a mechanistic kinetic model of the EGFR pathway, formulated in molecular terms as cascades of protein interactions and phosphorylation and dephosphorylation reactions. Assuming differential temperature dependencies for different reaction groups, such as SH2 and PTB domain-mediated interactions, the EGFR kinase, and the phosphatases, good quantitative agreement was obtained between computer-simulated and measured responses. The kinetic model demonstrates that, for each protein-protein interaction, the dissociation rate constant, k(off), strongly decreases at low temperatures, whereas this decline may or may not be accompanied by a large decrease in the k(on) value. Temperature-induced changes in the maximal activities of the reactions catalyzed by the EGFR kinase were moderate, compared to such changes in the V(max) of the phosphatases. However, strong changes in both the V(max) and K(m) for phosphatases resulted in moderate changes in the V(max)/K(m) ratio, comparable to the corresponding changes in EGFR kinase activity, with a single exception for the receptor phosphatase at 4 degrees C. The model suggests a significant decrease in the rates of the EGF receptor dimerization and its dephosphorylation at 4 degrees C, which can be related to the phase transition in the membrane lipids. A combination of high-resolution experimental monitoring and molecular level kinetic modeling made it possible to quantitatively account for the temperature dependence of the integrative signaling responses.