FIGURE 6.
Simulations of RAF phosphorylation and dissociation. A, the reaction scheme and rate constants (s−1) used for the simulation. The values for the parameters were determined by globally fitting the kinetic models to the on-time distributions. The simulation does not include photobleaching. RCp, Cp, and φp are the phosphorylation states of the RC, C, and φ states, respectively. X is the state of the complex between RAF and its kinase on the plasma membrane when dissociated from RAS. B, probabilities of each association state of RAF with the membrane as a function of time after association with RAS-GTP. The inset is a magnification of the initial 1-s period of the reactions. C, assuming a steady state of RAF phosphorylation, the flow of RAF molecules through the reaction network was calculated according to the results of the kinetic analysis (see supplemental material). RAF in the inactive closed form recognizes RAS-GTP via RBD (step 1), and then RAS-GTP opens the RAF conformation in parallel with the association between CRD and RAS-GTP (step 2) (18). Most (∼95%) of the open-form RAF molecules on RAS-GTP interact with the kinases on the plasma membrane (step 3). The phosphorylation of RAF mainly occurs in the ternary complex formed by RAS-GTP, RAF, and its kinase (steps 4 and 5), but about 20% of RAF phosphorylation occurs after the dissociation of RAF from RAS (step 6).