Pyk2 is a non-receptor tyrosine kinase that evolved from gene duplication of focal adhesion kinase (FAK) and subsequent functional specialization in the brain and hemopoietic cells. Pyk2 shares a domain organization with FAK, with an N-terminal regulatory FERM domain adjoining the kinase domain. FAK regulation involves integrin-mediated membrane clustering to relieve autoinhibitory interactions between FERM and kinase domains. Pyk2 regulation remains cryptic, involving Ca2+ influx and protein scaffolding. While the mechanism of the FAK FERM domain in autoinhibition is well-established, the regulatory role of the Pyk2 FERM is ambiguous. We probed the mechanisms of FERM-mediated autoinhibition of Pyk2 using hydrogen/deuterium exchange mass spectrometry and kinase activity profiling. The results reveal FERM–kinase interfaces that are responsible for autoinhibition. Pyk2 autoinhibition impacts the activation loop conformation. In addition, the autoinhibitory FERM–kinase interface exhibits allosteric linkage with the FERM basic patch conserved in both FAK and Pyk2.

Upon ligand binding, bone morphogenetic protein (BMP) receptors form active tetrameric complexes, comprised of two type I and two type II receptors, which then transmit signals to SMAD proteins. The link between receptor tetramerization and the mechanism of kinase activation, however, has not been elucidated. Here, using hydrogen deuterium exchange mass spectrometry (HDX-MS), small angle X-ray scattering (SAXS) and molecular dynamics (MD) simulations, combined with analysis of SMAD signaling, we show that the kinase domain of the type I receptor ALK2 and type II receptor BMPR2 form a heterodimeric complex via their C-terminal lobes. Formation of this dimer is essential for ligand-induced receptor signaling and is targeted by mutations in BMPR2 in patients with pulmonary arterial hypertension (PAH). We further show that the type I/type II kinase domain heterodimer serves as the scaffold for assembly of the active tetrameric receptor complexes to enable phosphorylation of the GS domain and activation of SMADs.

Pyk2 is a non-receptor tyrosine kinase that evolved from gene duplication of focal adhesion kinase (FAK) and subsequent functional specialization in the brain and hemopoietic cells. Pyk2 shares a domain organization with FAK, with an N-terminal regulatory FERM domain adjoining the kinase domain. FAK regulation involves integrin-mediated membrane clustering to relieve autoinhibitory interactions between FERM and kinase domains. Pyk2 regulation remains cryptic, involving Ca 2+ influx and protein scaffolding. While the mechanism of the FAK FERM domain in autoinhibition is well-established, the regulatory role of the Pyk2 FERM is ambiguous. We probed the mechanisms of FERM-mediated autoinhibition of Pyk2 using hydrogen/deuterium exchange mass spectrometry (HDX-MS) and kinase activity profiling. The results reveal FERM-kinase interfaces responsible for autoinhibition. Pyk2 autoinhibition impacts activation loop conformation. In addition, the autoinhibitory FERM-kinase interface exhibits allosteric linkage with the FERM basic patch conserved in both FAK and Pyk2.

The student author, whose presentation of the scholarship herein was approved by the program of study committee, is solely responsible for the content of this dissertation. The Graduate College will ensure this dissertation is globally accessible and will not permit alterations after a degree is conferred.

Pyk2 is a non‐receptor tyrosine kinase enriched at the post‐synaptic density of glutamatergic synapses. Activated by Ca2+influx, Pyk2 participates in the translation of synaptic activity into signaling cascades that tune synaptic strength. Pyk2 is a multi‐domain protein kinase with a regulatory FERM domain. Structures have been reported for isolated Pyk2 domains, yet the architectures of the auto‐inhibited Pyk2 and Pyk2 activation complex remain unclear. We employed a cysteine cross‐linking strategy to define the inter‐domain interactions responsible for kinase regulation. Cysteine residues were engineered at putative interfaces in both kinase and FERM domains. Rates of cysteine disulfide bond formation were measured using mass spectrometry (MS). Ultimately, cross‐linking results were integrated with hydrogen/deuterium exchange MS to develop a model of the auto‐inhibited architecture.Support or Funding InformationNSF MCB grant 1715411; Linder Family Undergraduate Research FellowshipThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.