Scaffolding proteins serve as sites of binding and organization for the multiple components involved in many signaling pathways. Scaffolding promotes key protein-protein interactions and can substantially amplify a signal; however, if proteins are bound too tightly to a scaffold, signal propagation may actually be hindered. The precise mechanisms that regulate the interactions between scaffolding proteins and signaling pathway components are poorly understood, leading Vanderbilt Basic Sciences investigators Tina Iverson, Carlos Lopez, and Vsevolod Gurevich to explore the role of arrestin-3 as a scaffold for the JNK3 signaling pathway. JNK3, a mitogen activated protein kinase (MAPK) that regulates apoptosis in neuronal cells, is activated when it is phosphorylated on Tyr-223 by MKK4 (MAPK kinase 4) and Thr-221 by MKK7 (MAPK kinase 7). MKK4 and MKK7, in turn, are activated when they are phosphorylated by ASK1 (apoptosis signal-related kinase1). From prior work, the researchers knew that all four of these proteins bind to arrestin-3, and that this interaction promotes activation of JNK3. In the current studies, they demonstrated that ASK1, MKK4, MKK7, and JNK3 all bind to the T1A peptide (amino acids 1-25) of arrestin-3. MKK4 and MKK7 appear to compete for the same binding site on the arrestin-3 peptide, and the binding affinities of MKK4 and JNK3 are reduced by phosphorylation. The researchers measured the rate of JNK3 phosphorylation by activated MKK4, MKK7, or both enzymes together in the presence and absence of arrestin-3. They used these data, along with binding constants they had measured for the interaction of these proteins with arrestin-3 and some literature values to construct a computational reaction model for the activation process. The results demonstrated that, under these in vitroconditions, arrestin-3 promotes the phosphorylation of Thr-221 over Tyr-223 by 2.2-fold and leads to an overall 1.2- to 1.8-fold (3-fold at early time points) increase in the rate of dual phosphorylation of JNK3. The kinetic constant for Thr-221 phosphorylation by MKK7 was two orders of magnitude higher than that of Tyr-223 phosphorylation by MKK4, suggesting that the MKK4-catalyzed reaction is rate limiting. A final set of experiments demonstrated that the expression of a fused arrestin-3-JNK3 protein in HEK293 cells led to reduced activation of free JNK3 when compared to the activation of JNK3 in cells expressing free arrestin-3. These findings suggest that release of JNK-3 from arrestin-3 is critical for arrestin-3-mediated promotion of JNK3 activation. In summary, the results confirm that JNK3 and the kinases that are responsible for its activation all bind to arrestin-3 and that this scaffolding promotes JNK3 activation. The researchers proposed a “conveyor belt” model for this process, proposing that scaffolding of ASK1, MKK4, and MKK7 to arrestin-3 produces a complex that can activate multiple JNK3 molecules, thereby facilitating signal propagation. Critically, the reduction in affinity for arrestin-3 that occurs following JNK3 phosphorylation promotes its dissociation, helping to maintain flexibility in the system. This model provides a critical understanding of arrestin-3-mediated scaffolding and could well apply to other signaling pathways in which scaffolding occurs. The work is published in the journal Proceedings of the National Academy of Sciences U.S.A[N. A. Perry et al., (2018) Proc. Natl. Acad. Sci. U.S.A., published December 27, DOI: 10.1073/pnas.1819230116