Date of Award

2017

Degree Type

Dissertation

Degree Name

Doctor of Philosophy in Biological and Environmental Sciences

Specialization

Cell and Molecular Biology (CMB)

Department

Cell & Molecular Biology

First Advisor

Gongqin Sun

Abstract

Protein tyrosine kinases (PTK) play essential roles in cell signal transduction pathways. PTKs are associated with numerous diseases and promote oncogenic cell signal transduction in unregulated forms. Thus PTKs represent a large group of potential targets for anticancer drug discovery. Src was the first identified oncogene and has been intensively studied for decades. The work described in this dissertation studied the structure-function relationships of Src, to build a knowledge base for the function of conserved residues in the kinase domain. To accomplish this, the first focus of this dissertation was to develop a high throughput screening method to identify kinase functionalities regarding catalytic activity and substrate specificity. The developed method utilizes a bacterial screen system and detects phosphotyrosine in cell lysate by a high throughput manner. Combining the developed functional screening with a rational mutation library, functional mutants can be identified for further analysis. This in vivo strategy was validated by model PTKs and could be universally applied to study kinase catalytic activity and substrate specificity. A successful application of the developed method on the study of the conserved HRD motif in Src led to a more comprehensive understanding of the role of HRD in the kinase function.

The second focus of the dissertation was to study the regulatory function of the arginine (R385) in the HRD motif of Src, since R385 was found unnecessary for catalysis in the first part of this dissertation. The effect of R385 mutation on the key regulatory mechanisms of Src and binding affinity towards PTK inhibitors was examined. First, autophosphorylation of Tyr416 in Src activates this enzyme, and overrides the negative regulation by Csk that phosphorylates Src on Tyr527. However, mutation of R385 to other residues abolished this override. The presence of the arginine prevents the transition of the active Src to inactivated Src. Second, type II inhibitors bind to the inactive conformation of Abl but not to Src even through Src and Abl are structurally very similar. Mutation of R385 greatly increased the affinity of Src towards type II inhibitors, but had no effect on type I inhibitor binding, which targets the active conformation. All together, when induced by Csk phosphorylation or PTK inhibitors that target the inactive conformation of kinase domain, R385 behaves as an energy barrier in the conformational transition from active to inactive conformation. The replacement of the R385 by any other amino acid made the transition much easier. R385 is the first identified residue among protein tyrosine kinases, that controls conformational transitions. This study sheds light on the fundamental mechanism of regulatory conformational changes and provides more insight on design and development of PTK inhibitors.

Available for download on Sunday, November 10, 2019

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