![]() Specifically, scaffolds enhance signal propagation when prevailing conditions would lead to attenuation. Results from our simulations indicate that, depending on cellular conditions, the spatial organization of kinases on scaffold proteins can either enhance or inhibit signal propagation through a kinase cascade. Yet, it provides enough relevant features to allow the mechanisms extracted from this model to be biologically meaningful. The simple model we studied (with a relatively small number of parameters) allows us to meticulously study these different scenarios in depth. Other important issues that we examined with this model are the consequences of whether kinases bound to the scaffold can phosphorylate downstream kinases that are in solution and whether phosphatases in solution can act on proteins bound to the scaffold. Schematics of how signaling occurs in solution and on a scaffold are shown in Fig. We studied how scaffolds influence signal propagation for different physiological conditions determined by values of these parameters. 1) can be characterized by the following small number of parameters: the rate at which an active kinase can phosphorylate a downstream kinase, the rate at which phosphatases can remove a phosphate group from a kinase, the binding affinity of kinases to the scaffold or exchange rate, the relative concentration of scaffolds and kinases, and the parameters characterizing the mobility of the various protein kinases. The signaling module that we studied ( Fig. We investigated how scaffolds can influence protein motion, phosphorylation of downstream kinases by an active kinase, and phosphatase-mediated deactivation of kinases. For these reasons, we carried out computer simulations to study whether, how, and under what conditions assembling a sequence of kinases on a scaffold affects signal propagation through a multitiered kinase cascade. It would therefore be very useful to identify the most influential variables on which experiments should focus. Furthermore, a systematic variation of the many factors that may influence mechanisms through which spatial localization of kinases on a scaffold may affect signal propagation is currently not tractable. Therefore, it is problematic to study this potentially more generic function of scaffolds. It is difficult to ascertain that specific effects (e.g., catalysis or feedback) are absent in an experimental system. More complexity is added by suggestions that some scaffolds may recruit phosphatases to their scaffold-bound substrates ( 8) or, in contrast, protect scaffold bound kinases from phosphatase-mediated deactivation ( 12, 14).Īlthough functions such as catalysis could be important for specific systems, the ubiquity of scaffolds suggests that the physical effects of tethering members of the cascade to a scaffold may have a functional role. Recent reports also indicate that certain scaffolding proteins, such as Ste5 involved in the MAPK pathway of the yeast mating response, can catalytically activate a MAPK upon binding by inducing autophosphorylation of the threonine residue in the TxY motif in the MAPK, Fus3 ( 13). Indeed, one signature of a scaffold protein is believed to be the appearance of a “bell-shaped” protein titration curve. For example, the relative concentration of scaffolding proteins has been shown to be a key variable that modulates signal output in many instances ( 11, 12). Scaffold proteins are believed to be involved in many regulatory processes such as intracellular trafficking and pathway sequestering, and several factors have been shown to influence their signaling function ( 8). The general principles underlying how scaffold proteins function to influence signaling in protein kinase cascades are still poorly understood. Excluding phosphatases from interacting with scaffold-bound proteins is also considered. Phosphatases are allowed to interact with active kinases that are bound to the scaffold. When assembled on a scaffold, active kinases need only overcome the thermal energy barrier to activate their downstream target. 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. ( B) Schematics are shown for the sequence of signaling events in solution and on a scaffold in our model. Phosphatases are present that can encounter and deactivate activated kinases. An active A (MAPKKK) in turn activates a B kinase (MAPKK), which then can activate kinase C (MAPK). ( 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). Computer simulations model the effects of scaffolding a kinase cascade.
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