Figure 6. Unique interpretation of Ras•GTP signals by different effectors in multi-effector networks encodes multiple distinct temporal outputs in the system response.
(A) Depiction of the experimental design: a fixed step-input is applied to a particular network configurations in which more than one effector molecule is, resulting in multiple simultaneous system outputs that are measured. (B) Absolute and normalized responses to step-input of C-Raf RBD and B-Raf RBD in the absence of any GAP activity. (C) as in (B) but with 1 μM NF1-GAP present in the signaling network. (D) Absolute and normalized responses to step-input of C-Raf RBD and A-Raf RBD with 1 μM NF1-GAP present in the signaling network. (E) Absolute and normalized responses to step-input of C-Raf RBD and the C-RafN64A mutant RBD with 1 μM NF1-GAP present in the signaling network. RBD, Ras-binding domain
Figure 6—figure supplement 1. Additional examples of how the unique interpretation of Ras•GTP signals by different effectors in multi-effector networks encodes multiple distinct temporal outputs in the system response.
(A) Depiction of the experimental design: a fixed step-input is applied to a particular network configurations in which more than one effector molecule is, resulting in multiple simultaneous system outputs that are measured. (B) Absolute and normalized responses to step-input of C-Raf RBD and A-Raf RBD in the absence of any GAP activity. (C) Absolute and normalized responses to step-input of C-Raf RBD and C-RafN64A RBD in the absence of any GAP activity. RBD, Ras-binding domain
Figure 6—figure supplement 2. Kinetic modeling and simulations show that competition between effectors allows multiple temporal responses to be encoded in the system output.
(A) Output of Kintek simulation using a three-state GTPase model with competition between GAP and effectors as described in the main-text 'Materials and methods', in which two effectors (one c-Raf like (koff = 0.001 s-1), one B-Raf like (koff = 0.00025 s-1) are present in the system at 50 nM. Other initial conditions were 50 nM effector, 1 μM GEF, 1 μM GAP, and 'infinite' nucleotide (100000 nM). This simulation recovers the observation that B-Raf can respond in a sustained way while C-Raf can respond in a transient way. (B) Output of Kintek simulation using a three-state GTPase model with competition between GAP and effectors as described in the main-text Materials and methods, in which two effectors have very similar concentrations and parameters (as indicated on the figure). Other initial conditions were 50 nM effector, 1 μM GEF, 1 μM GAP, and 'infinite' nucleotide (100000 nM). This simulation recovers the observation that small parameter differences between effector can alter the timing and duration of transient signaling outputs. (C) Output of Kintek simulation using a three-state GTPase model with competition between GAP and effectors as described in the main-text 'Materials and methods', in which three effectors with different parameters and concentrations (as indicated in the figure) are present in the system. Other initial conditions were 50 nM effector, 1 μM GEF, 1 μM GAP, and 'infinite' nucleotide (100000 nM). This simulation shows that a complex sequence of effector outputs can be produced (3 THEN 2 THEN 1) in response to a step input simply by titration of levels and altering effector parameters. GAP, GTPase-activating protein; GEF, guanine exchange factor.