Many hypotheses about how scaffolds coordinate positive and negative feedback come from engineered scaffolds and mathematical modeling.  In three-kinase signaling cascades, scaffolds bind all three kinases, enhancing kinase specificity and restricting signal amplification by limiting kinase phosphorylation to only one downstream target. These abilities may be related to stability of the interaction between the scaffold and the kinases, the basal phosphatase activity in the cell, scaffold location, and expression levels of the signaling components.