Date of Award

Fall 10-1-2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Pharmacology

First Advisor

Turk, Benjamin

Abstract

AbstractProteome-wide screening for mitogen-activated protein kinase docking motifs and interactors Guangda Shi 2021 Kinases catalyze the transfer of phosphate to substrates, a reaction critical for many cellular events. Mitogen-activated protein kinases (MAPKs), including ERK, p38, and JNK, phosphorylate hundreds of substrates, and each plays a pivotal role in distinct processes such as cell growth, survival, differentiation, and apoptosis. To correctly respond to external stimuli, MAPKs use several mechanisms to achieve a high degree of selectivity for their target substrates. A critical aspect of MAPK specificity comes from docking interactions occurring between sites distal from the site of catalysis and short linear sequence motifs located in MAPK interactors. These docking sequences conform to a general motif of ψ(1-3)-X(2-6)-ΦΦ-X-Φ (ψ: basic residue, X: any residue, Φ: hydrophobic residue). However, sites conforming to this motif can nonetheless bind specifically to any of the distinct MAPK subfamilies. MAPK preference for docking sites is therefore coded within sub-motifs falling within the consensus. Here I present yeast-based genetic screens to identify docking sequences selectively binding to either JNK or p38 MAPKs. First, I comprehensively characterized all possible amino acid substitutions with two known docking sites, revealing new information about amino acid residues critical for MAPK binding. Second, I screened for functional p38α and JNK1 binding sequences from the human proteome. I subsequently picked 36 peptide sequences selected in these screens and determined their binding affinity to both p38 and JNK. Over 90% of the peptides showed the predicted binding specificity, thus validating the screen results. I have also validated that hit sequences can serve to recruit MAPKs to phosphorylate protein substrates. Systematic analysis of sequences selected by each MAPK revealed key features conferring MAPK specificity. This work has provided unbiased insights into MAPK substrate specificity, and also suggests new biological processes regulated by MAPKs.

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