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Date of Award

Spring 5-15-2018

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Immunology)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

The RAS-RAF-MEK-ERK (MAPK) pathway is a signaling pathway important for cell proliferation, survival and differentiation, and not surprisingly, dysregulation of this pathway is commonly found in many types of cancer. The mammalian RAF family includes three highly conserved serine-threonine kinases—ARAF, BRAF and CRAF—and two pseudokinases, KSR1 and KSR2. Recently, it was determined that a key step in the activation of the RAF kinases is homo- and heterodimerization between RAF family members. Dimerization then results in allosteric transactivation of the binding partner and can potentially explain the function of the pseudokinases.

Previous work in the lab showed that each isoform has distinct functions in the process of allosteric activation. This suggests that allosteric signaling is important and different compositions of RAF dimers may play different roles. In this work, I established a method to measure the stoichiometry of different RAF isoforms in different cells and then used mathematical modeling to predict what effects this would have on the signaling pathway. To study the structural determinants of dimerization, I developed a luciferase complementation assay to allow RAF dimerization to be quantitated. Our results confirmed that RAF inhibitors could differentially induce dimerization of some but not other forms of RAF and that certain mixed dimers were favored. Structurally, our data emphasizes the importance of phosphorylation of the N-terminal acidic motif for RAF dimerization.

Lastly, to better understand the role of KSR1 which has previously been shown in model organisms to enhance the signaling of constitutively active RAS, we bred KSR deficient animals to a well-characterized model of RAS-dependent pancreatic ductal adenocarcinoma. We found that the absence of KSR led to a modest decrease in morbidity but almost no detectable difference in tumor number or progression. Collectively, this work adds to our understanding of RAF family kinase dimerization and regulation of MAPK signaling. Our data provides some new insight into the evolution of a system that uses distinct dimerization patterns of closely related proteins to coordinate signal transduction.

Language

English (en)

Chair and Committee

Andrey A. Shaw, Marco Colonna

Committee Members

Daniel C. Link, Kenneth M. Murphy, Jason D. Weber,

Comments

Permanent URL: https://doi.org/10.7936/K7XK8F0W

Available for download on Sunday, May 15, 2118

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