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. 2012 Dec 21;3:475.
doi: 10.3389/fphys.2012.00475. eCollection 2012.

How Scaffolds Shape MAPK Signaling: What We Know and Opportunities for Systems Approaches

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Free PMC article

How Scaffolds Shape MAPK Signaling: What We Know and Opportunities for Systems Approaches

Franziska Witzel et al. Front Physiol. .
Free PMC article

Abstract

Scaffolding proteins add a new layer of complexity to the dynamics of cell signaling. Above their basic function to bring several components of a signaling pathway together, recent experimental research has found that scaffolds influence signaling in a much more complex way: scaffolds can exert some catalytic function, influence signaling by allosteric mechanisms, are feedback-regulated, localize signaling activity to distinct regions of the cell or increase pathway fidelity. Here we review experimental and theoretical approaches that address the function of two MAPK scaffolds, Ste5, a scaffold of the yeast mating pathway and KSR1/2, a scaffold of the classical mammalian MAPK signaling pathway. For the yeast scaffold Ste5, detailed mechanistic models have been valuable for the understanding of its function. For scaffolds in mammalian signaling, however, models have been rather generic and sketchy. For example, these models predicted narrow optimal scaffold concentrations, but when revisiting these models by assuming typical concentrations, rather a range of scaffold levels optimally supports signaling. Thus, more realistic models are needed to understand the role of scaffolds in mammalian signal transduction, which opens a big opportunity for systems biology.

Keywords: MAPK and ERK signaling; Ste5; mathematical modeling; scaffold; signaling; ultrasensitivity.

Figures

Figure 1
Figure 1
The role of scaffolds KSR and Ste5 in MAPK signaling. (A) In quiescent cells an inactive KSR/MEK complex exists in the cytosol. (B) Upon stimulation of the cell, KSR translocates to the cell membrane and forms an active complex with phosphorylated Raf, MEK, ERK. Activated ERK detaches from the scaffold with three outcomes; (1) ERK dimerizes in the cytoplasm where the dimer remains or translocates to the nucleus; (2) ERK translocates to the nucleus; (3) KSR acts as a platform where ERK dimers are assembled and the new complex can interact with substrates in the cytoplasm. (C) Schematic of the yeast mating pheromone response pathway.
Figure 2
Figure 2
Schematic of MEK phosphorylation by the allosteric transition of KSR induced by B-Raf binding. A KSR2-MEK side on side dimer is formed where the activation segments are facing each other and are thus inaccessible. A regulatory Raf interacts with KSR in cis to induce a conformational shift of KSR2 α C helix into the active position. Due to steric hindrance a separate catalytic Raf in trans can then phosphorylate MEK.
Figure 3
Figure 3
The optimal scaffold concentration range. (A) A generic scaffold molecule with three binding sites binds to a kinase X reversibly with the dissociation constant KD. The probability for kinase X to be bound to the scaffold is given by the concentration of the scaffold-kinase complex SX divided by the total amount of scaffold ST. Assuming that binding of all 3 kinases R (Raf), M (MEK), and E (ERK) is independent from the occupation of the other binding sites, single binding probabilities can be multiplied to obtain the probability for the situation where several kinases are bound at the same time. (B) The model presented in (A) was used to calculate the concentration of a full scaffold complex (all 3 kinases bound) vs. total scaffold concentration. We consider either an equal amount of ligands (2 Raf, 2 MEK, and 2 ERK, left panel) or different amounts of ligands (2 Raf, 10 MEK, and 20 ERK in the mid panel, 2 Raf, 100 MEK, and 200 ERK in the right panel) for high (KD = 0.01 left and mid panel, KD = 0.1 right panel) and low (KD = 1 left and mid panel, KD = 10 right panel) binding affinity. (C) Given a set of various amounts of ligands (here 2 Raf, 5 MEK, and 10 ERK) the optimal range of scaffolds can be explained as follows: Firstly, the least abundant ligand (Raf) determines the maximal number of full complexes that can be formed (two). Until an amount of scaffolds that equals the number of the second less abundant ligand, MEK (5), every scaffold can be occupied with MEK and ERK and so 2 out of 2 possible full complexes can be formed. Having one scaffold molecule more than MEK molecules (6 scaffolds), one scaffold will miss MEK and with a certain probability (p = 1/6) Raf will bind to the one that misses MEK and in this case, only 1 out of 2 full scaffold complexes can be formed.
Figure 4
Figure 4
Proposed effects for the action of scaffolds. (A) Schematic for high and low phosphatase activity with and without scaffold. (i) When there is low phosphatase activity and no scaffold present, the kinases can readily activate each other and the signal spreads exponentially along the cascade. (ii) When kinases are bound to scaffold under low phosphatase activity, each kinase is localized so it is only likely to interact with their neighboring substrate kinase and signal amplification is attenuated. (iii) When the level of phosphatase activity is high and there is no scaffold for kinases to complex with, the propogation of the signal is impeded as the kinase will likely encounter a phosphatase and become deactivated before it meets its downstream target. (iv) Under conditions of high phosphatase activity, the scaffold complex facilitates signal amplification due to the enhanced local concentration of kinases. This is because the likelihood for a kinase to successfully interact with its substrate kinase is much higher than for the encounter with a deactivating phosphatase which is not localized to the scaffold. Gray squares represent phosphatases. (B) Trans activation of incomplete scaffold complexes. Kinases bound to different partially occupied scaffolds may activate each other in trans.
Figure 5
Figure 5
Scaffold proteins and specificity in the yeast MAPK pathway. The mating response (A), filamentous growth response (B), and osmotic stress response (C) pathways all share the MAP3K Ste11. In the osmotic stress response, Pbs2 acts as a scaffold and also a MAP2K of the pathway at the same time. The mating response and filamentous growth pathways both share MAP2K Ste11 and MAP3K Ste7. Specificity occurs through the scaffold Ste5, which is required for the activation of Fus3 during the mating response. However there may be accidental activation of Kss1 during the mating response and further specification can be achieved by a negative feedback. The filamentous growth pathway possibly does not require a scaffold. Signal flow is shown by black arrows and red T shaped bar indicates inhibition.
Figure 6
Figure 6
Tissue-specificity of gene expression. Gene expression of scaffolds varies strongly between tissue types and each tissue has a specific pattern of expressed scaffolds. Expression was averaged across all samples, and log2-changes from this average are shown in the heatmap, with red indicating high expression and blue low expression. Data were obtained from the GNF mouse gene atlas V3, GEO accession number GSE10246, normalized using rma with standard parameters.

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