Date of Award

Fall 2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor

Turk, Benjamin

Abstract

Mitogen-activated protein kinases (MAPKs) are critical components of complex signal transduction networks that control cell physiology in response to a broad range of stimuli. Alterations to MAPK signaling networks drive many diseases, including cancer, immunological, developmental, neurological, cardiovascular, and metabolic disorders. Despite their high sequence similarity, the three major mammalian MAPK subfamilies (ERK, JNK, and p38) are activated in response to different stimuli and mediate distinct cellular responses. Each MAPK must engage in highly specific interactions with unique substrates, regulators, and activators to achieve appropriate cellular responses. Short linear motifs (known as “D-sites”) found in MAPK interacting partners are thought to play critical roles in promoting MAPK specificity by mediating docking interactions. The first chapter overviews the importance of short linear motifs in MAPK signaling and discusses high throughput strategies used to discover SLiMs interaction. Many methods used to identify SLiMs have poor sensitivity in detecting very low-affinity interactions, including MAPK docking interactions. The second chapter describes the development of a Y2H screening system designed to identify SLiMs that dock MAPKs.D-sites interact with a conserved shallow pocket called the D-recruitment site (DRS) located outside the MAPK catalytic cleft. However, all known D-sites conform to the same general sequence consensus that all MAPKs target. Thus, the molecular features within D-sites encoding specificity towards a single MAPK are unknown. The third chapter focuses on applying the Y2H screen system to probe a library of SLiMs mined from the human proteome to define ERK2 and p38a docking interactomes. In addition to expanding the D-site interactomes for ERK and p38, we identify new substrates and a previously unappreciated sequence feature that mediates p38 specificity. Recurrent cancer-associated ERK2 hotspot mutants D321N, E322K, and R135K map to the same three-dimensional space in the DRS. Phenotypic characterization of these mutants reveals that instead of disrupting all docking interactions, these mutations rewire MAPK specificity allowing only specific docking interactions to occur. However, the scope of affected ERK2 interactions is unknown, and we are currently unable to predict what components of ERK2 signaling networks are affected by these mutations to promote cancer. The fourth chapter describes the application of a Y2H screen to identify D-sites that interact with ERK2 mutants E322K and D321N. The new docking site interactomes are harnessed to identify the molecular features that allow the prediction of docking interactions disrupted by the cancer-associated mutations in the ERK DRS.

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