Scaffold proteins confer diverse regulatory properties to protein kinase cascades

Abstract

The assembly of multiple signaling proteins into a complex by a scaffold protein guides many cellular decisions. Despite recent advances, the overarching principles that govern scaffold function are not well understood. We carried out a computational study using kinetic Monte Carlo simulations to understand how spatial localization of kinases on a scaffold may regulate signaling under different physiological conditions. Our studies identify regulatory properties of scaffold proteins that allow them to both amplify and attenuate incoming signals in different biological contexts. These properties are not caused by the well established prozone or combinatorial inhibition effect. These results bring coherence to seemingly paradoxical observations and suggest that cells have evolved design rules that enable scaffold proteins to regulate widely disparate cellular functions.

Fig. 1.

Computer simulations model the effects of scaffolding a kinase cascade. (A) In a model kinase cascade such as the MAPK cascade, an initial stimulus, S* (e.g., Ras-GTP), is recruited to and activates kinase A (MAPKKK). An active A (MAPKKK) in turn activates a B kinase (MAPKK), which then can activate kinase C (MAPK). Phosphatases are present that can encounter and deactivate activated kinases. (B) Schematics are shown for the sequence of signaling events in solution and on a scaffold in our model. For a chemical reaction to occur in solution, the appropriate species must first come into contact with its substrate and then overcome a thermal energy barrier to model catalysis. When assembled on a scaffold, active kinases need only overcome the thermal energy barrier to activate their downstream target. Phosphatases are allowed to interact with active kinases that are bound to the scaffold. Excluding phosphatases from interacting with scaffold-bound proteins is also considered.

Fig. 2.

Scaffold proteins can amplify signals that would otherwise attenuate. The case of high basal phosphatase activity, E₄ = 0. Shown are calculated values of signal amplification, ϕ, φ ≡ 〈C*/A* − 1〉 (A) and signal magnitude, θ, θ ≡ 〈C*/[A]₀〉 (B) for increasing values of the kinase-scaffold binding affinity E. The A (MAPKKK) and B (MAPKK) concentrations equal that of the scaffold, whereas the concentration of C (MAPK) is five times larger (other situations are described in SI Text and SI Fig. 7). A strong stimulus, σ = 1 (σ ≡ [S*]/[A]₀), is used. Two cases are considered: a “constrained” case, where species bound to the scaffold cannot activate species in solution (diamonds in A and circles in B) and an “unconstrained” case, where species bound to the scaffold can activate species in solution (triangles in A and squares in B).

Fig. 3.

Scaffold proteins attenuate signals that would otherwise strongly amplify. Low basal phosphatase levels are considered (E₄ = 6). Signal amplification ϕ, φ ≡ 〈C*/A* − 1〉 (A and C), and signal magnitude θ, θ ≡ 〈C*/[A]₀〉 (B and D) are considered as a function of scaffold binding affinity, E. Two cases are shown: a weak stimulus (σ ≪ 1, σ ≡ [S*]/[A]₀) of small amplitude and low basal phosphatases levels (E₄ = 6) (A and B) and a stimulus of large amplitude (σ = 1, σ ≡ [S*]/[A]₀) (C and D). All other conditions are the same as those reported in Fig. 2. As shown in these plots, assembling signaling components onto a scaffold by increasing E results in significantly lower amplification and signal output. Again, in each panel, two cases are considered: a constrained case, where species bound to the scaffold cannot activate species in solution (diamonds in A and C and circles in B and D), and an unconstrained case, where species bound to the scaffold can activate species in solution (triangles in A and C and squares in B and D).

Fig. 4.

Serial engagement of the last kinase can greatly influence signal output in a MAPK cascade. We consider the scenario where the first two kinases, A and B (MAP3K, MAP2K) bind tightly (E₂ = 20) and the binding affinity C (MAPK) is varied as follows: weak (E₂ = 4), intermediate (E₂ = 8), and strong (E₂ = 20) disassociation energies. The ordinate represents the fraction of activated C (MAPK) proteins (a measure of signal output), and the abscissa is the scaled strength of signal, σ, σ ≡ [S*]/[A]₀. The signal output is largest when kinase C (MAPK) is allowed to rapidly disassociate (E₂ = 4) from the scaffold. Data presented are for a 1:1:10:1 ratio of A/B/C/scaffolds.

Fig. 5.

Summary of different regimes that characterize scaffold-mediated signal transduction. The characterization of four regimes of scaffold-mediated signal transduction: (i) high signal strength and high phosphatase activity, (ii) high signal strength and low phosphatase activity, (iii) low signal strength and high phosphatase activity, and (iv) low signal strength and low phosphatase activity. * indicates the case when kinases bound to a scaffold cannot phosphorylate their downstream substrates that remain in solution. ** indicates the case when kinases can interact with their downstream targets that are present in the solution. The absence of * or ** indicates that the effect occurs regardless of whether scaffold bound proteins can activate downstream targets in solution. These results summarize our findings when phosphatases are allowed to act on scaffold-bound kinases and reflect the balance between stoichiometric constraints and removal of transport limitations by scaffolds. As described in Results and SI Fig. 9, preventing phosphatases from acting on scaffold-bound kinases does not alter these results substantially.