Sorting through the JuNK: Using Chemical and Genetic Tools to Probe c-Jun N-Terminal Kinase Allostery and Scaffolding, as well as a General Methodology for Studying Localized Kinase Biology
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This dissertation explores the complicated nature of protein kinase allostery and the use of ATP-competitive inhibitors as tools to study kinase function. Much of this work focuses on the c-Jun N-Terminal Kinases (JNKs), a subfamily of the Mitogen Activate Protein Kinases (MAPK), which are involved in a broad range of signaling processes, including those that are involved in both cell survival and cell death. The JNKs possess docking surfaces, distal to their ATP-binding sites, that engage with scaffold proteins, upstream activators, and downstream substrates. Prior work indicated that binding of certain regulatory proteins to a specific docking surface, termed the D-recruitment site (DRS), could allosterically modulate the conformation of the ATP-binding site of the JNKs. We used a diverse panel of ATP-competitive inhibitors to further investigate the allosteric relationship between the ATP-binding pockets and DRSs of JNKs. We showed that by changing the ATP-binding site occupancy, we could allosterically enhance or decrease binding of JNKs to the scaffold protein JIP1. Strikingly, the lowest and highest affinity JNK-inhibitor complexes showed a greater than 50-fold difference in dissociation constant. We also found that scaffolding of activated JNKs by JIP1 could prevent specific ATP-competitive inhibitors of JNKs, but not others, from binding. Additionally, we discovered that ATP-competitive inhibitors of JNKs could promote or attenuate activation loop phosphorylation by JNK’s upstream activators, MKK4 and MKK7. Given that JIP1, MKK4, and MKK7 all interact with JNK DRSs, these results demonstrate that there is functional allostery between the ATP-binding sites and DRSs of these kinases. Further, our data indicates that the type of JNK inhibitor used in cells may influence the resulting cellular phenotype, and also, that the cellular state of JNKs may affect their ability to bind to certain inhibitors. However, it remains difficult to study JNK regulation in vivo using ATP-competitive inhibitors, mainly due to their promiscuity. To achieve additional selectivity of kinase inhibitors in cells, our lab has designed orthogonal, conformation selective inhibitors that are mono-selective for mutant kinases. We have shown here that they can selectively interact with mutant JNKs, thus providing an attractive tool-set to selectively study these allosteric changes in vivo, in each of the individual JNK isoforms. Finally, we present the development of ATP-competitive inhibitor tools and methodology that will allow further exploration into the spatial regulation of these complex signaling mediators. We have functionalized ATP-competitive inhibitors such that they can be tethered to locally expressed SNAP-tag fusion proteins in vivo. By modifying the specific kinase inhibitor used, or by using different SNAP-fusions to alter the region of SNAP expression, this methodology could be used to study a broad range of kinase -or other protein- targets, and can be employed in any region of interest for which there is a known localization domain.
- Chemistry