Regulation and Dysregulation via Docking Interactions in WNK and ERK1/2 MAPK Signaling




Taylor, Clinton A., IV

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Protein-protein interactions are essential for nearly every cellular process. Within signaling pathways, such interactions carry out numerous functions such as defining substrate specificity, inhibition of both other interactions and enzyme activity, localizing signaling partners, acting in a switch-like manner depending on post-translational modifications, etc... My work has focused primarily on protein-protein interactions, and in particular protein-peptide docking interactions, within two independent cellular signaling pathways, the WNK, and ERK1/2 MAPK pathways. The WNK pathway is an essential regulator of cellular ionic composition and volume. Initially discovered due to the genetic link between mutations in some of the WNK kinases and an inherited form of hypertension, the pathway is now well defined and known to regulate the activity of multiple SLC12 cation chloride coupled cotransporters. WNK kinases activate SPAK and OSR1 kinases, which then regulate the activity of cotransporters. My initial work within this pathway was focused on understanding the role of SPAK and OSR1 activation loop domain-swapping. I solved the structure of the inactive SPAK kinase domain, and utilized a previously identified dimerization blocking mutation to probe the role of SPAK dimerization on activation and activity. I determined that SPAK has multiple activation states and that the monomeric form is both active and can be activated by WNK1. I next shifted focus to the SPAK and OSR1 CCT domains, which mediate protein-peptide interactions with motifs found in WNKs, cotransporters, and other interaction partners. I further defined the specificity of the CCT domains for the motifs, and discovered a new motif variant. I used this information to predict new interaction partners. Although validation of the predictions is still in the early stages, my initial choice for validation was a group of inward rectifier potassium channels. Through collaboration, we now have evidence that OSR1 kinase activity regulates flux through one of the eight channels, and testing of more of the channels is underway. The ERK1/2 MAPK pathway is involved in numerous cellular processes, and is well known for its role in responding to extracellular signals that regulate cell growth and differentiation. As such, aberrant signaling within the pathway is found within approximately thirty percent of all cancers. The terminal kinases of the cascade, ERK1 and ERK2, phosphorylate hundreds of cytosolic and nuclear substrates, and in many cases protein-peptide docking interactions between ERK1/2 and substrates are required. One of the docking sites on ERK2 has been shown to be the site of a mutation, E322K, that is the most enriched ERK2 missense mutation in human cancers. Using a previously determined dataset, I was able to refine the structure of the mutant ERK2. As expected the site of the mutation was structurally disrupted, but the mutation also caused structural changes throughout the entire kinase domain, including solvent exposure of the activation loop. Docking induced solvent exposure of the activation loop had already been suggested to be important for MAPK activation and inactivation by presenting the sites of phosphorylation to modifying enzymes. Therefore, the structural data fit well with previous findings indicating that the mutant was able to be activated by upstream kinase MEK1, but could not be inactivated by phosphatase DUSP6. I also found that the mutation affected a distal docking site and disrupted interactions with at least some interaction partners at that site. The combined results of this project have shown that this mutation has multiple effects on ERK2 structure and function, and implies that this mutation is enriched primarily because it allows ERK2 to be activated similar to wild type, while having a diminished capacity to be deactivated.

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