Enhancing Signaling Flux via Light-Induced Assembly of the Mitogen-Activated Protein Kinase (MAPK) Pathway

Abstract

Liquid-liquid phase separation of macromolecules occurs in many cellular contexts. The functions of the resulting droplets, however, have been less well understood. One hypothesized function of protein droplets specifically is to locally increase the concentration of enzymes and substrates, enhancing reaction rates and allowing for greater flux through cellular pathways. This hypothesized function makes protein phase separation an appealing avenue for synthetic biology because it allows one to design stimulus-responsive droplets to concentrate proteins of interest and tune flux. However, the limits of this hypothesized function remain unexplored, making it difficult to determine which pathways would benefit from synthetic cluster formation. To explore the hypothesis that clustering could enhance flux through a signaling pathway, we set out to create light-responsive droplets containing model kinase-substrate pairs drawn from MAP kinase signaling pathways. Using the p38 pathway as a model system, we find that light-induced clustering and light-induced heterodimerization achieve similar levels of flux enhancement. Furthermore, in agreement with the idea that local concentration affects signal enhancement, we find that if components of the p38 pathway are already co-localized through a linker, clustering has no effect. Similarly, the JNK pathway, which contains an endogenous scaffold protein, does not show flux enhancement when clustered. We use the results from this study to suggest that scaffold proteins co-localize proteins in order to mediate flux in kinase pathways, though further testing is needed. Ultimately, the findings in this work contribute to establishing a better understanding of the effects of liquid-liquid phase separation and the role of scaffold proteins in the context of cellular pathways.