Degree Type

Thesis

Date of Award

2020

Degree Name

Master of Science

Department

Biochemistry, Biophysics and Molecular Biology

Major

Biochemistry

First Advisor

Eric Underbakke

Abstract

Pyk2 is a non-receptor tyrosine kinase localized in the postsynaptic density of neurons. As a paralogue of focal adhesion kinase (FAK), Pyk2 shares the FAK domain organization. Both contain an N-terminal FERM (4.1 protein, ezrin, radixin, moesin) domain, linked to a central kinase domain, and a C-terminal FAT (focal adhesion targeting) domain. Despite the domain similarity between FAK and Pyk2, the mechanisms of activation differ strikingly. FAK is canonically activated by recruitment to membrane focal adhesions and phosphoinositide interactions, whereas Pyk2 has adopted sensitivity to Ca2+ flux. Understanding the activation mechanism differences will illuminate how gene duplication can generate new signaling responses. The activation of both FAK and Pyk2 involve clustering to promote autophosphorylation in trans. Pyk2 clustering has been reported to be caused by the postsynaptic density scaffolding protein PSD-95. While the regulation and higher-order architecture of the Pyk2—PSD-95 interaction remains unclear, the C-terminal proline-rich repeats of the Pyk2 kinase—FAT linker are implicated in the clustering. Indeed, this region may also be involved in stabilizing a FERM-mediated dimerization interface observed in FAK. The in vitro investigation of the scaffolding complex has been limited by protein insolubility, largely due to the large size and putative disorder of the kinase—FAT linker. To enable in vitro investigations into the higher-order architecture of the scaffolded Pyk2 activation complex, new Pyk2 expression constructs were developed to promote solubility while encompassing the C-terminal linker and FAT domain. Likewise, constructs for site-specific biotinylation were developed to facilitate binding studies. Together, the new constructs yield soluble proteins for controlled studies of scaffolding architecture and Pyk2 activation.

DOI

https://doi.org/10.31274/etd-20200902-145

Copyright Owner

Shivani Sharma

Language

en

File Format

application/pdf

File Size

53 pages

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