Neurons communicate with neighboring neurons through synaptic connections. This neuronal 'wiring' can change and adapt over time. Many aspects of the mechanisms controlling the changes in neuronal connections remain enigmatic. Nevertheless, the ability to strengthen and weaken neuron connections is critically important because this rewiring is linked to learning and memory formation. Each synaptic connection hosts a large collection of proteins poised to interpret patterns of neuronal communication. This research project seeks to discover how one team of proteins responds to synaptic activity to signal for synaptic changes. The central hub of the signaling team is Pyk2, a protein kinase. Pyk2 activates by rearranging its structure to form a large signaling assembly with associated signaling factors. The goal of this research is to determine the architecture of the Pyk2 signaling assembly. Understanding the assembly and activation of the Pyk2 signaling team will yield insights into how neuronal rewiring is initiated at the molecular scale. This proposal will train graduate and undergraduate students by using a team-based concept where graduate and undergraduate students will have an advisor/advisee relationship.
The objectives of this project are to reveal the molecular mechanisms of Pyk2 signaling linking post-synaptic Ca2+ influx to the Src cascades that tune synaptic plasticity. Illuminating the architecture of scaffolded signaling complexes remains a frontier goal in understanding synaptic plasticity. However, multi-protein signaling complexes are typically large and highly dynamic, challenging targets for structural characterization. While structures have been determined for isolated pieces of the signaling effectors, a critical question remains: how do the pieces assemble into functional signaling complexes? This project employs several strategies to dissect the activational signaling complex of the Pyk2 non-receptor tyrosine kinase. By leveraging hydrogen/deuterium exchange mass spectrometry, protein interactions and conformational changes will be mapped. Single particle electron microscopy will illuminate the overall shape of the activation complex. These approaches are amenable to describing heterogeneous ensembles of accessible conformers and architectures. Structural restraints derived by the complementary biophysical approaches will be integrated to build a model of the higher-order architecture of the signaling complex responsible for activating Pyk2. This project is supported by the Molecular Biophysics Cluster of the Molecular and Cellular Biosciences Division in the Biological Sciences Directorate.
