Molecular mechanisms of Pyk2 regulation

Proline-rich tyrosine kinase 2 also known as Pyk2 is a non-receptor tyrosine kinase that functions various signaling pathways such as cell proliferation and synaptic plasticity. Pyk2 expression is predominant in hematopoietic, neuronal, and epithelial cells. Pyk2 is a FAK homolog and functions like FAK in many cell types. However, Pyk2 adopts unique Ca2+-responsiveness in neuronal cells. This research thesis will focus on the structural determination of how purified Pyk2 is auto inhibited and the role a scaffold plays in the activation of Pyk2 at the molecular level. In this thesis, I show that unstimulated Pyk2 is auto-inhibited, and this auto-inhibition is mediated by its N-terminal domain. The N-terminal FERM domain of Pyk2 interacts with the kinase domain resulting in inhibition of kinase activity. Kinase inhibition is due to steric restriction of active site access due to the conformation of the FERM domain. Using a combination of hydrogen/deuterium exchange mass spectrometry (HDX-MS) and site-directed mutagenesis, I mapped functionally important intra-domain interfaces that control the activity and allosteric landscape of Pyk2. Together, these studies underlie the first structural model of the auto-inhibited conformational of Pyk2. This thesis also explores the role of scaffold-induced clustering in Pyk2 activation. Although it is well-established that Ca2+ regulates Pyk2 activation, little is known regarding the molecular mechanisms. I determined that PSD-95 mediates Pyk2 activation through a clustering-based mechanism. PSD-95 binds to auto-inhibited Pyk2 via the first two PDZ domains. HDX-MS implicated the Pyk2 F3 FERM subdomain and kinase C-lobe as the primary scaffolding interfaces. Kinase activity profiling indicates that PSD-95 mediated activation is based on a clustering mechanism. Altogether, using biochemical and biophysical methods, the research reported here determined the auto-inhibited conformation of Pyk2 and elucidated the role of scaffolding in the activation mechanism