Dr. Eric Underbakke, Biophysics and Molecular Biology, ISU
Host: Dr. Young-Jin Lee
Dynamic, multi-domain signaling proteins are hallmarks of sophisticated cellular signaling systems. The higher-order architecture of signaling complexes confers sensitivity, adaptability, control, and crosstalk. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a powerful tool for defining protein interactions and conformational changes associated with signaling control. We are applying HDX-MS to investigate the dynamics of signaling protein kinases, including the tyrosine kinase Pyk2. Pyk2 and paralog focal adhesion kinase (FAK) share an N-terminal regulatory FERM domain adjoining the kinase domain. FAK activation involves integrin-mediated membrane interactions and clustering to relieve auto-inhibitory interactions between FERM and kinase domains. Pyk2 activation remains far more cryptic, involving Ca2+ influx and protein scaffolding-mediated clustering. Given the striking structural similarities between FAK and Pyk2, it remains unclear how vastly different stimuli (e.g., integrin clustering vs. Ca2+ influx) trigger parallel downstream activation processes. Pyk2 activation involves a multi-stage higher-order assembly maturation process. We reconstituted the scaffolded activation complex in vitro. Scaffolding interactions and phosphorylation-induced conformational changes were probed using HDX-MS. Scaffolding stimulates phosphorylation of the Pyk2 interdomain linker, followed by activation loop phosphorylation. Phosphorylated Pyk2 exhibits changes in dynamics around the active site and N-lobe. Allosteric sites in the FERM domain link to regulatory interactions with the kinase domain activation loop.