Mechanisms of regulation and scaffolding-induced activation of Pyk2 tyrosine kinase
Pyk2 is a non-receptor tyrosine kinase paralog of focal adhesion kinase (FAK), with 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, monitoring reciprocal site-specific phosphorylation between Pyk2 and the downstream kinase Src. Scaffolding interactions and phosphorylation-induced conformational changes were probed using hydrogen/deuterium exchange mass spectrometry (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.