Cell Type–Specific Importance of Ras–c-Raf Complex Association Rate Constants for MAPK Signaling

Ras–c-Raf association rates affect downstream signaling in the absence of negative feedback but not its presence. Network Topology Dictates Robustness to Dynamical Changes Stimulation of the epidermal growth factor receptor (EGFR) leads to activation of a large complex signaling network that regulates numerous cellular functions, including cell proliferation, differentiation, and apoptosis. Activation of the kinase c-Raf by the small GTPase Ras is central to the subsequent activation of the extracellular signal–regulated kinase (ERK) mitogen-activated protein kinase (MAPK) branch of the EGFR signaling network. Here, Kiel and Serrano, who previously found that the rate of association between Ras and c-Raf is evolutionarily conserved, investigated the effects of various c-Raf mutations that affected the rate of Ras–c-Raf association, dissociation, or both. They found that association rates seemed to have more effect on ERK signaling than dissociation rates. Intriguingly, however, the effects depended on the specific details of the ERK pathway. In a cell line in which EGF elicits sustained ERK phosphorylation and signaling, c-Raf mutants with similar affinity for Ras, but lower association rates, were less effective at stimulating downstream ERK signaling than wild-type c-Raf, whereas the reverse was true for mutants with higher association rates. In contrast, these effects were not apparent in cell lines in which negative feedback from ERK led to its transient phosphorylation and downstream signaling. We generated 17 c-Raf (RAF proto-oncogene serine-threonine protein kinase) mutants with altered Ras-Raf association and dissociation rates to investigate the role of electrostatically driven Ras-Raf association rates on epidermal growth factor (EGF)–activated mitogen-activated protein kinase (MAPK) signal transduction. Some of these mutants had compensating changes in association and dissociation rates, enabling the effects of changes in association rate to be distinguished from those of changes in affinity. In rabbit kidney (RK13) cells, these mutants affected downstream signaling, with changes in Ras–c-Raf association rates having a greater effect on MAPK signaling than did similar changes in dissociation rates. Mutants with compensating decreases in both association and dissociation rates stimulated less extracellular signal–regulated kinase (ERK)–dependent reporter activity than did wild-type c-Raf, whereas the converse was true for mutants with increased association and dissociation rates. In marked contrast, the mutants had little or no effect on signaling in human embryonic kidney (HEK) 293 cells. These two cell lines also showed distinct patterns of EGF-dependent ERK phosphorylation and signaling: ERK activation and signaling were transient in HEK293 cells and sustained in RK13 cells, with the difference resulting from the lack of negative feedback from ERK to Sos (Son of Sevenless) in the latter. Computer simulation revealed that, in the presence of negative feedback, changes in the rate of Ras–c-Raf binding have little effect on ERK activation. Thus, EGF-MAPK activation kinetics and feedback regulation is cell type specific and depends on the network topology.